Determination of cinnamaldehyde, thymol and eugenol in essential oils by LC-MS/MS and antibacterial activity of them against bacteria

doi:10.21203/rs.3.rs-3609123/v1

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Determination of cinnamaldehyde, thymol and eugenol in essential oils by LC-MS/MS and antibacterial activity of them against bacteria

https://doi.org/10.21203/rs.3.rs-3609123/v1

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Based on Liquid chromatography-tandem mass spectrometry (LC-MS/MS), A qualitative and quantitative method for simultaneous detection of 3 bioactive compounds, including cinnamaldehyde (CNM), thymol (THY) and eugenol (EUG) in plant essential oil was established in this study. We tested the antibacterial activity of 3 bioactive compounds. Several necessary tests for 12 steroids, such as LLOQ, recovery and precision, have verified the repeatability and practicability of the method. Then, the antibacterial activity of 3 bioactive compounds against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was evaluated by minimal inhibitory concentration and the synergistic antimicrobial effect. The results indicated that the MIC of CNM, THY and EUG against E. coli were 1.0, 1.0 mg/mL, and 0.5 µg/mL, respectively. And the MIC of CNM, THY and EUG against S. aureus were 0.5, 1.0 mg/mL, and 0.5 µg/mL, respectively. According to the results of fractional inhibitory concentration index (FICI), it is considered that CNM + THY and CNM + THY + EUG has obvious synergistic inhibitory effect on E. coli, and CNM + THY and CNM + EUG has obvious synergistic inhibitory effect on S. aureus. In addition, the trace metals in the treated bacteria were analyzed.

LC-MS/MS

synergistic antimicrobial

trace metal

essential oils

As one of the most common causes responsible for infectious diseases and an important pathogen in clinical practice, Staphylococcus aureus (S. aureus) has contributed to a severe threat, involving mild skin infection, severe tissue infection, and sepsis to human health due to its widely distribution in human skin, especially in the nasopharynx. In addition, S. aureus can also cause a variety of nosocomial infections derived from medical instruments and equipment pollution (Wei et al. 2023). Escherichia coli (E. coli) is a diverse group of Gram-negative bacteria, with some strains being non-pathogenic. However, certain strains of E. coli are capable of producing toxins and have a low infectious dose, making them highly transmissible through various media. This bacterium can persist in the environment for extended periods, proliferate within food sources, and ultimately pose significant threats to human health and safety (Chen et al. 2023). Under certain conditions, E. coli can cause gastrointestinal tract infections, urinary tract infections and other local tissue and organ infections (Wang et al. 2020). At present, the treatment of bacterial infection mainly relies on antibiotics. However, repeated and excessive use of antibiotics has led to serious antibiotic residues and bacterial resistance, along with decreased therapeutic efficacy. Therefore, it has become an important and urgent task to explore natural products with low toxicity and high anti-microbial activity for treating the initial stage of infection, reducing the use of antibiotics, and limiting the development of drug resistance of pathogenic microorganisms (Wei et al. 2023).

Plant essential oils (EOs) is a substance originally derived from plants, also known as aromatic oil or volatile oil (Cai et al. 2022). These contain a wide variety of plant secondary metabolites that can inhibit or slow the growth microorganisms. The main constituents of EOs are mono- and sesquiterpenes, along with carbohydrates, phenols, alcohols, ethers, aldehydes, and ketones, which are responsible for the biological activity of aromatic and medicinal plants as well as for their fragrance. Plant essential oils have multiple pharmacological effects, such as antioxidant effects, action as analgesic, antipyretic, hypoglycemic, antiulcer, anticonvulsant, anti-pathological aging, and anticancer agents, and other potential therapeutic effects (Semeniuc et al. 2015). Many essential oils and their volatile components have been found to have strong antibacterial activity, which inhibit the growth of some bacteria, fungi, and other microorganisms, and suppress the production or accumulation of mycotoxins. The antibacterial activity of essential oils is mainly caused by its complex active components (mainly including alcohols, aldehydes, ketones, and phenols). Essential oils can use its own hydrophobicity to penetrate lipids, thus destroying the structure of the cell wall and changing the permeability of the cell membrane: this leads to the outflow of ions and matter within the cyst, causing cell death. Combining two or more compounds could be more effective for improving antioxidant and antimicrobial activity and could offer a synergistic potential (Jaradat et al. 2017). The use of plant EOs as antibacterial agents not only implies greater safety for humans and a more environmentally friendly option due to their natural origin, but also represents a low risk of pathogenic microorganisms developing resistance (Wang et al. 2023). However, the use of phytochemicals to prevent foodborne microbes is poorly studied. In this study, a novel LC-MS/MS method was constructed to determine bioactive compounds levels in chili essential oils. Following these results, this work was designed to validate a highly sensitive and selective method for the qualitative and quantitative determination of these components in the chili essential oils. The purpose of this study was to extend the functional potential of CEOs by investigating their antibacterial activity against two major food-borne bacteria and also to examine the possible underlying antibacterial mechanisms.

2.1 Chemicals and reagents

All solvents used in the current experiment were of HPLC grade. All chemicals and reference powders were of AR grade. Cinnamaldehyde (CNM), cinnamic acid (CA), thymol (THY) and eugenol (EUG) reference powder were procured from Solarbio (Beijing, China). Formic acid, methanol and acetonitrile were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). In-house Milli-Q Plus purification equipment, purchased from Millipore Company (Billerica, MA, USA), generates the required water at HPLC grade (Attwa et al. 2023) .

2.2 Microorganisms and essential oil

The Gram-positive strain: S. aureus and Gram-negative strains: E. coli were procured from our laboratory depository. These two bacteria were revived in the Luria Broth medium (Beijing Land Bridge Technology Co., Ltd., Beijing, China) at 27 ^◦C for 16 h for activation of the test cultures. Chili essential oil was purchased from Jiangxi Jinyuan Natural Flavor Co., Ltd. (Jiangxi, China) (Wang et al. 2020) .

2.3 Extraction of essential oils

In total, 1 g chili essential oil were added to 10 ml volumetric bottle and fill with acetonitrile to 10 ml, then CNM, THY and EUG were extracted by ultrasonic-assisted extraction (UAE) method. The extracts were filtered through a filter paper after 60 min of extraction time. The filtrate was filtered through a 0.22 µm organic phase filter membrane for subsequent HPLC analysis. Three operation replicates were performed (Li et al. 2018).

2.4 LC-MS/MS Instrumentation and Conditions

Separation and quantification were achieved by using a Nexera XR HPLC from Shimadzu Corporation coupled to a QTRAP 5500 mass spectrometer from ABSciex operated at unit resolution for both quadrupoles. The LC-MS/MS system was used for chromatographic separation of analytes peaks and mass spectrometric detection of separated ion peaks. HPLC and electrospray ionization source (ESI) were managed by Analyst software. The column used was an XSelect® HSS T3 3.5 µm, 2.1 mm×100 mm Column from Waters Corporation maintained at 40°C. The mobile phase composition was A (water) and B (methanol) both containing 0.1% formic acid (Table 1) (Farré-Segura et al. 2022). The injection volume was 5 µl. Quantification was performed in an MRM mode using ESI in the positive ion mode (Table 2 and Table 3) (Liu et al. 2021).

Table 1

The liquid phase gradient parameter
Time (min)	% B
1	50
1.5	50
2	50
5	100
10	100
11	50
15	50

Table 2

MRM parameters for the quantification of CNM, THY and EUG and CA (IS).
Compounds	Tr (min)	Transition		MS Parameters (V)
Compounds	Tr (min)	Q1 Mass (Da)	Q3 Mass (Da)	DP	EP	CXP	CE
cinnamaldehyde	2.58	132.90	55.00	104.07	11.96	8.11	15.09
thymol	5.45	151.10	91.00	100.83	11.64	10.87	19.17
eugenol	4.29	165.30	124.00	19.47	3.83	16.40	17.95
cinnamic acid	2.92	149.00	77.10	153.44	4.22	11.01	33.92

Table 3

Ion source parameters
Parameter	Setting
Scan Type	MRM
Ionization Mode	ESI +
Curtain Gas	20
Collision Gas	Medium
Ion Source Gas 1	27
Ion Source Gas 2	60
Source Voltage	5500
Temperature	600

2.5 Method Validation

2.5.1 Linearity

CNM, THY, EUG and CA standards were prepared and diluted in acetonitrile that was prepared at a 7:3 ratio in acetonitrile/water, v/v. The three standard solutions generate an external standard calibration line by three repeated injections of the standard solution at six concentration levels (S1-S6). A plot of peak area with respect to the corresponding concentration was used to demonstrate linearity. The linear regression equation and correlation coefficient were calculated by weighted (1/x²) least-squares linear regression analysis. Linearity was considered to be acceptable when correlation coefficients were 0.99 or better and calibrators had accuracies of 85–115% and precisions within ± 15% RSD (relative standard deviation) (Tine et al. 2017).

2.5.2 Lower Limit of the Measuring Interval

The LOQ (signal-to-noise > 10) was the lowest concentration point of calibration curve at which accuracy (relative error, RE) within 20% and precision below 20% can be considered acceptable (Tine et al. 2017).

2.5.3 Precision and Accuracy

The intra-assay precision and accuracy were tested. In this work, quality control (QC) samples at high, middle, and low concentrations (three parallels at each concentration) in six days were studied. The variability of determination was expressed as the RSD%, and the accuracy was expressed as the relative error (RE%). Herein, the accuracy data within ± 15% RE from the nominal values and a precision data within ± 15% RSD were acceptable. However, the precision and accuracy data of the LLOQ should be within ± 20% (Liu et al. 2021).

2.5.4 Carryover

Carryover was evaluated by three independent experiments each consisted of running two extractions in the sequence of low₁–high–low₂, where low₂ is a re-injection of low₁. A passing test meant that low₁ is within 20% of low₂ and that low₂ is within 3 standard deviations of the low₁ value. The standard deviation was determined using low₁ values (El-Khoury et al. 2012).

2.6 Colony Formation Assay

The antibacterial activity of CNM, THY and EUG were also examined by colony formation assay (Sun et al. 2020). Aliquots of 50 µl spore suspension (approximately 10⁴ cells/ml) of S. aureus and E. coli were mixed with 50 µl of different concentrations of CNM, THY and EUG solution. The mixtures were incubated at 27°C for 4 h, and then were each plated onto 3 agar plates (20 µl/plate). After incubation at 27°C for 24 h, the resulting fungal colonies in each plate were counted. The percent of fungicidal activity was calculated as follows: [number of colonies (control-test)/number of colonies (control)]×100 (n = 3) (Li et al. 2022).

2.7 Antimicrobial synergy assay

The synergistic antimicrobial effect of the combination of CNM, THY and EUG were assessed by the same as colony formation assay. Bacterial cells are grown and aliquoted into new microcentrifuge tube, and then mixed with 50 µl volume of different combinations of CNM, THY and EUG mixture (Li et al. 2022). The plates were incubated at 27°C. The fractional inhibitory concentration (FIC) index is a quantitative criterion for assessing the synergism of tested antimicrobial agents. The FIC index was calculated based on the following formula:

FIC_A = (MIC_A in combination) / (MIC_A alone)

FIC_B = (MIC_B in combination) / (MIC_B alone)

FIC index = FIC_A + FIC_B

A FIC index of < 0.5 indicates synergistic activity, > 0.5-1 indicates additive effects, > 1 to < 2 indifference, and ≥ 2 is considered to be antagonism (Kaur et al. 2022).

2.8 Trace metals analysis

In this study, we chose 1/6 MIC (CNM + THY) to treat the bacteria. Bacteria cells were grown to log phase and resuspended to a cell density of 3 × 10⁸ cells/ml in LB medium. The medium containing the bacteria cells was centrifugated at 10000 g at room temperature for 5 min, and the pellet collected and washed with sterile PBS. Bacterial samples were crushed using an agate mortar and a pestle to approximately < 10 µm. Approximately, 20 mg of crushed sample was weighed out and suspended in 12 ml of 2% nitric acid (trace element grade). Next, the sample was sonicated for 2 h to ensure the dis-aggregation of the spores and a fairly consistent suspension. The suspension was then centrifuged for 5 min in order to reduce the number and average size of spores in suspension. This is an important step in order to avoid clogging of the sample introduction system. The supernatant was then separated and was ready for ICP-MS analysis (Gikunju et al. 2004).

All ICP-MS experiments in this study were carried out with a ICAP TQ Inductively-Coupled Plasma Mass Spectrometry system (Thermo, USA), using the operating conditions described in Table 4.

Table 4

ICP-MS instrument and plasma conditions for operation
Instrument operating conditions	Mode of operation
Instrument operating conditions	Ca, Mg, Fe, Cu, Zn, Se, Pb	V, Cr, Mn, Co, Ni, As, Mo, Ag, Cd, Sn, Ba, Au, Hg, Tl
Ar nebulizer gas flow	1.13 l/min	1.17 l/min
Auxilliary gas flow	0.80 l/min
Cool gas flow	14.0 l/min
Plasma power	1550 W
Measurement units	Cps (counts per second)
Curve type	linear
Sample units	µg/l
Replicates	12
Sweeps	10
Dwell time	100 ms
Channels	3
Spacing(u)	0.1
He gas flow	0 ml/min	4.5 ml/min
Measurement Mode	STD	KED
Internal Standard	¹⁰³Rh, ²⁰⁹Bi	¹⁰³Rh, ²⁰⁹Bi, ¹¹⁵In

2.9 Statistical Analysis

All experimental data are shown as means ± SEM, and statistically analyzed by SPSS 22.0 (IBM Inc., Chicago, IL, USA). And GraphPad Prism 8.0.2 was applied for graphical plotting and analysis. Statistical differences between concentration points were assessed using unpaired t-test, *p < 0.05; **p < 0.01; ***p < 0.001

3.1 LC-MS/MS Method

The cinnamic acid (CA) was selected as the internal calibration standard (IS) in the established LC-MS/MS method for quantifying CNM, THY and EUG due to two reasons: first, the chromatographic peaks of CA, CNM, THY and EUG were eluted with a good separation, and second, none of four ingredients were found together in chili essential oil.

Typical multiple reaction monitoring (MRM) chromatograms of CNM, CA, EUG and THY are displayed in Fig. 1. The concentrations of CNM, EUG and THY in chili essential oil were 1190, 77700 and 18.4 ng/ml, respectively.

3.2 Method Validation

The method was validated comprehensively following < < Consensus of method development and validation of liquid chromatography–tandem mass spectrometry in clinical laboratories> >

3.2.1 Calibration curve and lower limit of quantification (LLOQ)

Calibration curves were prepared using calibration samples. Linear regression analysis was performed by plotting the analyte/IS peak area ratio versus the concentration, and correlation studies were conducted using the Pearson correlation coefficient (r²). Table 5 shows the concentration ranges evaluated using 6 calibration points. The calibration curves for three analytes were best fitted with a quadratic regression weighted 1/c². CNM, THY and EUG yielded good linearity over the concentration ranges of 500–10000 ng/mL, 2000–100000 ng/mL and 10–400 ng/mL respectively, with mean correlation coefficients (r²) of 0.9970, 0.9976 and 0.9957, respectively.

Table 5

Linear range, correlation coefficient (r²) and LLOQ
Compounds	Regression Equation	r²	Linear Range (ng/mL)	LOQ(n = 10)
Compounds	Regression Equation	r²	Linear Range (ng/mL)	Nominal (ng/mL)	Found (ng/mL)	Accuracy (%)	CV (%)
CNM	y = 0.9735x + 74.878	0.9970	500 -10000	500	439.73	87.95	9.73
THY	y = 0.9108x + 1543.2	0.9976	2000-100000	2000	2022.00	101.10	3.00
EUG	y = 0.8983x + 7.9582	0.9957	10–400	10	8.43	85.30	3.92

To determine the LLOQ, 10 replicates of samples with set concentrations were measured, the accuracy of results was distributed in the range of 85.30 − 101.10%, and the CV < 9.73%. The proven LLOQ is lower than the clinical reference interval and fully matches the requirement of medical detection. The lower limit of quantification (LLOQ) of CNM, THY and EUG were determined to be 500, 2000 and 10 ng/ml.

3.2.2 Precision and accuracy

Precision refers to the proximity of independent measurement results obtained under specified conditions, which reflects the repeatability of detection methods. Intra-assay precision and accuracy results (n = 3) for low, medium, and high concentration levels are reported in Table 6. The mean intra-assay accuracies ranged from 85.17–115.00%, whereas the intra-assay precision and inter-assay precision, described as the relative standard deviation (RSD) of the calculated concentrations, were below 14.21% and 6.16%, respectively. These results demonstrated the method for quantitative determination of CNM, THY and EUG was reliable and repeatable. The CV% values of within-run, between-run and overall meet the criteria of method validation, indicating the extreme reproducibility of methods.

Table 6

Accuracy and precision results of CNM, EUG and THY
Analyte	Concentration (ng/mL)	Day	mean (ng/mL)	Recovery	Intraday (CV)	Interday (CV)
CNM	500	1	461.67	92.33%	4.85%	2.33%
		2	429.67	85.93%	9.08%
		3	444.33	88.87%	1.31%
		4	449.67	89.93%	8.50%
		5	448.00	89.60%	6.75%
		6	450.33	90.07%	10.15%
	2000	1	2170.00	108.50%	3.23%	4.09%
		2	2053.33	102.67%	4.73%
		3	2003.33	100.17%	8.49%
		4	2116.67	105.83%	5.31%
		5	1930.00	96.50%	4.61%
		6	2053.33	102.67%	6.36%
	8000	1	7026.67	87.83%	4.09%	4.72%
		2	7073.33	88.42%	3.63%
		3	7040.00	88.00%	3.49%
		4	7770.00	97.13%	0.53%
		5	7610.00	95.13%	6.78%
		6	7636.67	95.46%	1.00%
THY	2000	1	2076.67	103.83%	4.85%	2.94%
		2	1930.00	96.50%	9.08%
		3	2020.00	101.00%	1.31%
		4	1980.00	99.00%	8.50%
		5	1960.00	98.00%	6.75%
		6	2066.67	103.33%	10.15%
	10000	1	10966.67	109.67%	8.22%	3.14%
		2	11266.67	112.67%	4.00%
		3	11500.00	115.00%	0.87%
		4	11233.33	112.33%	2.86%
		5	10566.67	105.67%	7.71%
		6	11466.67	114.67%	3.52%
	50000	1	48833.33	97.67%	2.48%	1.55%
		2	50000.00	100.00%	1.40%
		3	50100.00	100.20%	3.60%
		4	48666.67	97.33%	1.54%
		5	48266.67	96.53%	5.63%
		6	48733.33	97.47%	2.21%
EUG	10	1	9.36	93.57%	12.56%	4.21%
		2	9.18	91.80%	13.17%
		3	8.59	85.87%	12.18%
		4	9.52	95.20%	13.14%
		5	9.74	97.37%	12.47%
		6	9.29	92.90%	0.73%
	50	1	49.80	99.60%	6.18%	3.72%
		2	53.07	106.13%	2.12%
		3	54.73	109.47%	7.22%
		4	53.73	107.47%	6.66%
		5	50.60	101.20%	7.51%
		6	53.70	107.40%	4.55%
	200	1	170.33	85.17%	6.00%	5.38%
		2	193.33	96.67%	14.21%
		3	194.67	97.33%	3.26%
		4	194.33	97.17%	5.85%
		5	195.67	97.83%	5.25%
		6	182.33	91.17%	4.43%

3.2.3 Carryover

Table 7 shows After multiple injections of standards and injections of blank samples, no carry-over effect was detected in the negative or positive control chromatograms.

Table 7

Carryover results of CNM, EUG and THY
Sample	CNM	L-L	H-L	THY	L-L	H-L	EUG	L-L	H-L
L1	1150			6460			23.3
L2	1210	1210		5980	5980		23.6	23.6
L3	1170	1170		6440	6440		20.4	20.4
H1	4340			25400			96.1
H2	3810			24100			94.5
L4	1120		1120	6530		6530	21.8		21.8
H3	4390			23600			109
H4	4310			23200			109
L5	1130		1130	6170		6170	23.8		23.8
L6	1040	1040		6420	6420		20.8	20.8
L7	1090	1090		6760	6760		22.3	22.3
L8	1110	1110		6490	6490		21.3	21.3
H5	3880			21400			89.1
H6	4120			22700			103
H7	3920			22100			95.6
H8	4070			23900			99.7
L9	1090		1090	6710		6710	18.9		18.9
H10	3780			22000			94.4
H11	3890			22200			93.6
L10	984		984	6320		6320	19.8		19.8
Mean		1124.00	1081.00		6418.00	6432.50		21.68	21.08
Carryover		-3.83%			0.23%			-2.79%

Carryover (%) = (mean_L−L – mean_H−L)/mean_L−L*100%

where mean_L−L and mean_L−L represented the measured concentrations of low-concentration samples injected after the low concentration and the high-concentration samples, respectively. The calculated carryover is within ± 15%, indicating the accuracy of low-concentration samples will not be affected by former injecting.

3.3 Minimal inhibitory concentration (MIC) of CNM, THY and EUG

Colony formation assay demonstrated that CNM, THY and EUG were able to kill S. aureus and E. coli at all the concentrations tested (Fig. 2A and 2B). The MIC values of CNM killing S. aureus and E. coli were 1000 and 500µg/ml, respectively. The MIC values of THY killing S. aureus and E. coli were 1000 µg/ml. The MIC values of EUG killing S. aureus and E. coli were 500 ng/ml.

3.4 Synergistic effect of CNM, THY and EUG

Figure 2C and Table 8 shows that a complete synergistic effect was recorded in the case of CNM + THY and CNM + THY + EUG (FICI ≤ 0.5), whereas no synergistic inhibitory effect was observed at low concentrations of CNM + EUG and THY + EUG on E. coli (0.5 ＜ FICI ＜ 1). Among the S. aureus assays (Fig. 2D), synergism was observed for CNM + THY and CNM + EUG but no synergism was observed for THY + EUG and CNM + THY + EUG.

Table 8

FIC index of each component to *E. coil* and *S. aureus*
	CNM + THY	CNM + THY	EUG + THY	CNM + EUG + THY
E. coil	0.17	0.50	0.50	0.17
S. aureus	0.17	0.17	0.50	0.50

3.5 Elemental abundance of microbial cells

As shown in Fig. 3, the elevated concentration of Fe was observed in E. coli but Cu, V, Cr, Co, Ba and Tl concentrations were lower in cells treated with CNM + THY than control group. Among the S. aureus assays, the elevated concentration of Mg, Ca, Fe, Cu, Pb, V, Cr, Mn, Co, As, Ba and Tl was observed in S. aureus but Mo and Cd concentrations were lower in cells treated with CNM + THY than control group.

For other trace metals, i.e Zn, Se, Ni, As, Ag, Au and Hg, there was no significant differences between experimental group and control group.

We developed a novel LC-MS/MS method for CNM, THY and EUG determination in biological samples. CA is used as an IS. The method was demonstrated to be no carryover, linear, precise, and accurate for the determination of CNM, THY and EUG. Good linearity was reached in the range of 500–10000 ng/mL, 2000–100000 ng/mL and 10–400 ng/mL respectively, and the LLOQ of CNM, THY and EUG was determined to be 500, 2000 and 10 ng/ml, respectively. The mean intra-assay accuracy ranged from 85.17–115.00%, whereas the intra-assay precision (RSD) was below 14.21% (Liu et al. 2021). Then, the results of this study imply that CNM, THY and EUG could be efficiently employed to treat gram-negative and gram-positive infections. The European Committee for Antimicrobial Susceptibility Testing (EUCAST) has proposed (EUCAST, 2000) a classification of the FICI occurring from a combination of antibiotics according to which any FICI ≤ 0.5 denotes synergy, additive when 0.5 < FICI ≤ 1, indifference when 1 < FICI < 2, and antagonism when FICI ≥ 2. In E. coli experiments which exhibited synergism, the mean reduction in the effective drug doses were 6-fold for CNM + THY and CNM + THY + EUG; The corresponding mean reductions for the drugs assayed in S. aureus strains were 6-fold for CNM + THY and CNM + EUG (Alexopoulos et al. 2019) .

In this study, 21 metal elements were Fig. 3 measured by ICP-MS in bacteria. Changes in the concentration of metal elements adversely affect bacteria. Membrane stability is attributed to the binding attributed to alkaline earth metals. While the interior of the membrane structure is lipophilic, the inner and outer surfaces contain hydrophilic phosphate molecules (Barton et al. 2007). Metals stabilize cell membranes and principally, Mg²⁺ and Ca²⁺ are found to neutralize the negative charges on ionized phosphate molecules on the cell membrane surface, and magnesium ions play important roles in stabilizing both tertiary and quaternary structure (Hori et al. 2021), with low and high ion concentrations impacting the function of ribosomes. Copper in biological systems presents a formidable problem: it is essential for life, yet highly reactive and a potential source of cell damage. Free intracellular iron would be expected to be low because this metal is bound into specific sites on the proteins and the toxicity of free cationic iron is well known (Galera-Laporta et al. 2021). At higher concentrations, heavy-metal ions form unspecific complex compounds in the cell, which leads to toxic effects. Compared with the control group, the concentrations of Ca and Mg in the bacteria treated with CNM + THY changed, which may have caused damage to the cell membrane (Nies et al. 1999). Then, the concentration of some elements in the cell changes, resulting in cell damage.

Acknowledge

This work was supported by the Key Technologies R&D Program of Guangdong Province (2022B0303030002) and Tianjin Key R&D Program (22YFYSHZ00140).

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.

Ethics statement

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. No ethical approval was required as the research in this article related to microorganisms.

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Determination of cinnamaldehyde, thymol and eugenol in essential oils by LC-MS/MS and antibacterial activity of them against bacteria

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Chemicals and reagents

2.2 Microorganisms and essential oil

2.3 Extraction of essential oils

2.4 LC-MS/MS Instrumentation and Conditions

2.5 Method Validation

2.5.1 Linearity

2.5.2 Lower Limit of the Measuring Interval

2.5.3 Precision and Accuracy

2.5.4 Carryover

2.6 Colony Formation Assay

2.7 Antimicrobial synergy assay

2.8 Trace metals analysis

2.9 Statistical Analysis

3. Results

3.1 LC-MS/MS Method

3.2 Method Validation

3.2.1 Calibration curve and lower limit of quantification (LLOQ)

3.2.2 Precision and accuracy

3.2.3 Carryover

3.3 Minimal inhibitory concentration (MIC) of CNM, THY and EUG

3.4 Synergistic effect of CNM, THY and EUG

3.5 Elemental abundance of microbial cells

4. Discussion

Declarations

References

Additional Declarations

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