Extraction, Chemical Composition and <i>in vitro</i> Antibacterial Activity of the Essential Oil of <i>Lippia origanoides</i> Kunth from Northeast Brazil

Silva, Monique Ellen Torres da; Monte, Antônia Lucivânia de Sousa; Damaceno, Marlene Nunes; Braga, Renata Chastinet; Coimbra, Jane Sélia dos Reis; Ramanathan, Srinivasan; Almeida, Felipe Alves de

doi:10.1590/1678-4324-2022210480

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Article - Biological and Applied Sciences • Braz. arch. biol. technol. 65 • 2022 • https://doi.org/10.1590/1678-4324-2022210480 linkcopy

Extraction, Chemical Composition and in vitro Antibacterial Activity of the Essential Oil of Lippia origanoides Kunth from Northeast Brazil

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Lippia origanoides Kunth has among its synonyms Lippia sidoides Cham. and is popularly known as ‘alecrim-pimenta’ or ‘rosemary pepper’. Essential oil of L. origanoides (EOLO) has been previously reported for several biological activities without significant acute toxicity, even at higher doses. In this study, essential oil (EO) was extracted from dried leaves, inflorescences, and branches of L. origanoides from Limoeiro do Norte, Ceará, Brazil by hydrodistillation at 3.63% (w/w) yield. Posteriorly, the presence of chemical compounds in the EOLO was identified by gas chromatography coupled to mass spectrometry (GC-MS). The analysis allowed to identify the thymol as the main metabolite (83.53%), followed by p-cymene (5.92%), caryophyllene (4.31%), γ-terpinene (3.27%), thymol methyl ether (1.18%), β-myrcene (1.00%), and α-terpinene (0.79%). The minimal inhibitory concentration (MIC) of EOLO was assessed against Gram-negative and Gram-positive bacterial pathogens such as Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. At 10 µL/mL concentration, the EOLO exhibited antibacterial activity against all the tested bacterial pathogens, and this concentration was considered as the MIC. The minimum bactericidal concentration (MBC) of EOLO was assessed at the MIC concentration, which inhibited the complete growth of all tested bacterial pathogens after 3 h. Therefore, concentrations less than 10 µL/mL were set as MBC. Thus, our findings suggest that the EOLO can be used as a promising antibacterial agent in the industries.

Keywords:
Gram-negative bacterium; Gram-positive bacterium; monoterpene; pathogen; thymol.

HIGHLIGHTS

Thymol was identified as the main metabolite in the EOLO.
EOLO has antibacterial activity against Gram-negative and Gram-positive bacterial pathogens.
The antibacterial effect of EOLO may be associated with the presence of thymol.

GRAPHICAL ABSTRACT

Peak*	RT (min)	[M-H] *(m/z)*	Percentage of similarity (%)	Chemical compound	Relative percentage (%)
6	20.805	135.15	95	thymol	83.53	Aromatic monoterpene
3	10.715	119.15	96	p-cymene	5.92	Aromatic monoterpene
7	24.860	93.10	96	caryophyllene	4.31	Sesquiterpene
4	11.953	93.10	97	γ-terpinene	3.27	Monoterpene
5	18.410	149.15	97	thymol methyl ether	1.18	Aromatic monoterpene
1	9.548	41.10	96	β-myrcene	1.00	Acyclic monoterpene
2	10.416	121.15	94	α-terpinene	0.79	Monoterpene
				TOTAL	100.00

Concentration of EOLO (µL/mL)	Mean of the diameter of the inhibition zone + standard deviation (mm)
Concentration of EOLO (µL/mL)	*E. aerogenes*	*K. pneumoniae*	*P. aeruginosa*	Salmonella Typhimurium	*S. aureus*
0.00	0.0 + 0.0^e	0.0 + 0.0^f	0.0 + 0.0^f	0.0 + 0.0^f	0.0 + 0.0^e
1.25	0.0 + 0.0^e	0.0 + 0.0^f	0.0 + 0.0^f	0.0 + 0.0^f	0.0 + 0.0^e
2.50	7.2 + 0.8^d	6.7 + 1.0^e	8.2 + 0.8^e	7.9 + 0.8^e	7.3 + 0.3^d
5.00	8.5 + 0.5^d	9.1 + 0.5^d	9.7 + 0.3^de	8.2 + 0.8^e	8.6 + 0.4^cd
10.00	11.7 + 0.3 ^c	10.6 + 0.4 ^d	10.3 + 0.7 ^d	11.8 + 0.5 ^de	10.2 + 0.8 ^cd
20.00	13.0 + 0.5^bc	14.2 + 0.5^c	14.1 + 0.2^c	14.3 + 1.2^cd	15.4 + 0.4^bc
40.00	14.5 + 0.7^b	16.3 + 0.5^b	15.3 + 0.6^bc	16.4 + 1.1^bc	18.4 + 1.1^b
80.00	15.0 + 1.0^b	17.0 + 2.1^b	16.0 + 0.7^b	19.8 + 2.5^b	22.2 + 2.6^ab
160.00	20.6 + 1.5^a	19.4 + 0.5^a	21.4 + 1.7^a	24.8 + 2.9^a	26.6 + 6.2^a

Bacteria	Inhibition zone diameter (mm)				MIC	MBC	MID	Main metabolite	Reference
	10 µL	20 µL	10 µL/mL	Undefined				Main metabolite
Gram-negative
Aeromonas hydrophila ATCC 7966	-	-	-	-	1.25 mg/mL	1.25 mg/mL	-	thymol	Majolo et al. [⁴⁰]
Aeromonas hydrophila ATCC 7966	-	-	-	-	1.25 mg/mL	1.25 mg/mL	-	-	Majolo et al. [³⁹]
Enterobacter aerogenes ATCC 13048	-	-	-	-	2.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Enterobacter aerogenes ATCC 13048	-	-	11.7	-	-	< 10.0 µL/mL	-	thymol	This study
Enterobacter cloacae	-	-	-	-	0.256 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Enterobacter cloacae HMA/FT502	-	-	-	-	2.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Escherichia coli	13.5	23.0	-	-	< 50.0 µL/mL	-	-	thymol	Bertini et al. [¹⁷]
Escherichia coli	-	-	-	-	0.00125 mg/mL	-	-	carvacrol	Pereira et al. [⁵⁸]
Escherichia coli	-	-	-	-	0.512 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Escherichia coli ATCC 11229	-	-	-	-	2.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Escherichia coli ATCC 25753	-	-	-	-	< 3.0 µL/mL	-	-	-	Castro et al. [²³]
Escherichia coli ATCC 25922	-	-	-	5.0	< 0.015 mg/mL	-	-	thymol	Linden et al. [³⁸]
Pool of 30 samples of Escherichia coli isolated of homemade Minas cheese	-	23.0	-	-	13.0 µL/mL	25.0 µL/mL	-	-	Castro et al. [²³]
Klebsiella pneumoniae	-	-	-	-	0.256 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Klebsiella pneumoniae ATCC 10031	-	-	10.6	-	-	< 10.0 µL/mL	-	thymol	This study
Providencia rettgeri	-	-	-	-	0.256 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Pseudomonas aeruginosa	0.0	0.0	-	-	-	-	-	thymol	Bertini et al. [¹⁷]
Pseudomonas aeruginosa	-	-	-	-	1.25 mg/mL	1.25 mg/mL	-	-	Melo et al. [⁴³]
Pseudomonas aeruginosa	-	-	-	-	0.00037 mg/mL	-	-	carvacrol	Pereira et al. [⁵⁸]
Pseudomonas aeruginosa	-	-	-	-	0.512 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Pseudomonas aeruginosa ATCC 25619	-	-	10.3	-	-	< 10.0 µL/mL	-	thymol	This study
Pseudomonas aeruginosa ATCC 9027	-	-	-	-	2.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Pseudomonas aeruginosa ATCC15442	-	-	-	-	-	-	< 0.001 mg/mL air	thymol	Veras et al. [⁷³]
Pseudomonas aeruginosa SPM1	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Pseudomonas syringae pv.	4.7	5.9	-	-	-	-	-	carvacrol	Guimarães et al. [³⁴]
Salmonella enterica serovar Enteritidis ATCC 13076	34.7	-	-	-	-	-	-	-	Ambrosio et al. [¹²]
Salmonella enterica serovar Typhi ATCC 19430	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Salmonella enterica serovar Typhimurium ATCC 14028	-	-	11.8	-	-	< 10.0 µL/mL	-	thymol	This study
Serratia marcenscens ATCC 14756	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Xanthomonas campestris pv. vesicatoria	3.9	4.9	-	-	-	-	-	carvacrol	Guimarães et al. [³⁴]
Cont. Table 3
Gram-positive
Bacillus cereus ATCC 14579	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Bacillus subtilis ATCC 6633	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Clavibacter michiganensis subsp. Michiganensis	3.0	5.6	-	-	-	-	-	carvacrol	Guimarães et al. [³⁴]
Enterococcus faecalis	-	-	-	-	0.512 mg/mL	-	-	thymol	Veras et al [⁷⁴]
Lactobacillus plantarum ATCC 8014	37.8	-	-	-	-	-	-	-	Ambrosio et al. [¹²]
Listeria monocytogenes L2	-	-	-	-	-	1.0 µL/mL	-	thymol	Vázquez-Sánchez et al. [⁷⁰]
Listeria monocytogenes L8	-	-	-	-	-	5.0 µL/mL	-	thymol	Vázquez-Sánchez et al. [⁷⁰]
Micrococcus luteus ATCC 9341	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Micrococcus roseus ATCC 1740	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Staphylococcus aureus	28.0	33.5	-	-	< 50.0 µL/mL	-	-	thymol	Bertini et al. [¹⁷]
Staphylococcus aureus	-	-	-	-	0.039 mg/mL	0.039 mg/mL	-	-	Melo et al. [⁴³]
Staphylococcus aureus	-	-	-	-	0.00063 mg/mL	-	-	carvacrol	Pereira et al. [⁵⁸]
Staphylococcus aureus	-	-	-	-	0.128 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Staphylococcus aureus ATCC 15656	-	-	-	20.0	< 0.015 mg/mL	-	-	thymol	Linden et al. [³⁸]
Staphylococcus aureus ATCC 25923	-	74.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus ATCC 25923	-	-	-	-	0.5 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Staphylococcus aureus ATCC 25923	-	-	10.2	-	-	< 10.0 µL/mL	-	thymol	This study
Staphylococcus aureus ATCC 29213	-	-	-	-	< 3.0 µL/mL	-	-	-	Castro et al. [²³]
Staphylococcus aureus ATCC 6538	-	-	-	-	0.5 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Staphylococcus aureus ATCC12624	-	-	-	-	-	-	0.000125 mg/mL air	thymol	Veras et al. [⁷³]
Staphylococcus aureus BH1	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH10	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH13	-	15.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH14	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH15	-	15.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH17	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH18	-	16.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH19	-	21.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH2	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH20	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH5	-	18.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus BH8	-	15.0	-	-	0.4 µL/mL	-	-	-	Oliveira et al. [⁵¹]
Staphylococcus aureus S8	-	-	-	-	-	0.25 µL/mL	-	thymol	Vázquez-Sánchez et al. [⁷¹]
Staphylococcus aureus S10	-	-	-	-	-	0.5 µL/mL	-	thymol	Vázquez-Sánchez et al. [⁷¹]
Staphylococcus aureus S102	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S14	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S16	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S26	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S27	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S46	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S81	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S82	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S83	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Staphylococcus aureus S86	-	29.0 - 60.4	-	-	-	-	-	thymol	Costa et al. [²⁴]
Pool of 30 samples of Staphylococcus aureus isolated of homemade Minas cheese	-	26.0	-	-	13.0 µL/mL	25.0 µL/mL	-	-	Castro et al. [²³]
Staphylococcus epidermidis	-	-	-	-	0.00063 mg/mL	-	-	carvacrol	Pereira et al. [⁵⁸]
Staphylococcus epidermidis ATCC 12229	-	-	-	-	1.0 mg/mL	-	-	isoborneol	Morais et al. [⁴⁷]
Streptococcus agalactiae KJ561060	-	-	-	-	0.3125 mg/mL	0.4167 mg/mL	-	thymol	Majolo et al. [⁴¹]
Streptococcus mitis	10.0	-	-	-	10.0 mg/mL	40.0 mg/mL	-	thymol	Botelho et al. [¹⁹]
Streptococcus mutans	-	-	-	-	0.256 mg/mL	-	-	thymol	Veras et al. [⁷⁴]
Streptococcus mutans ss-980	18.7	-	-	-	5.0 mg/mL	20.00 mg/mL	-	thymol	Botelho et al. [¹⁹]
Streptococcus salivarius	8.5	-	-	-	10.0 mg/mL	40.0 mg/mL	-	thymol	Botelho et al. [¹⁹]
Streptococcus sanguis	12.0	-	-	-	10.0 mg/mL	40.0 mg/mL	-	thymol	Botelho et al. [¹⁹]

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[TFN1] ^*
Peaks identified in the chromatogram in Figure 1; RT = Retention time; [M-H] = Molecular ion.