Chemical composition and biological activities of the essential oils from Lippia alba and Lippia origanoides

Abstract

There is an increasing interest in essential oils extracted from Verbenaceae plant species as potential sources of biologically active compounds that could provide a starting point for designing novel phyto-pharmaceuticals in aquaculture. The present study was aimed to investigate the chemical composition, antioxidant activity, acute toxicity and antimicrobial effects against Vibrio parahaemolyticus of essential oils extracted from Lippia alba and L. origanoides. Approximately 23 components were identified and quantified by gas chromatography-mass spectrometry and flame ionization detection in each species’ essential oil. The most predominant compounds were geranial (23.0%), limonene (17.0%) and neral (15.5%) in L. alba, and thymol (47.2%), p-cymene (16.0%) and E-caryophyllene (11.3%) in L. origanoides. The essential oils have antibacterial activity against Vibrio parahaemolyticus presenting Minimum Inhibitory Concentration (MIC) and Bactericidal Concentration (MBC) values between 156-625 µg mL^-1. The essential oils also show antioxidant potential estimated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assays, presenting IC₅₀ of 60.16 mg mL^-1 and 0.22 mg mL^-1 for L. alba and L. origanoides EO, respectively. Both oils were classified as toxic to Artemia salina nauplii. Therefore, these essential oils may be useful for controlling pathogenic bacteria important to the aquaculture industry.

Key words
Lippia alba ; Lippia origanoides ; minimum bactericidal concentration; minimum inhibitory concentration; oxygenated monoterpenes

INTRODUCTION

The bacteria of the genus Vibrio, which includes over 100 species, are predominantly associated with a variety of aquatic habitats, estuaries and coastal waters. In humans, these microorganisms are responsible for mild and inconvenient gastroenteritis to severe and life-threatening septicaemia and skin and soft tissue infections (Janda et al. 2015). The species V. parahaemolyticus is a leading cause of bacterial gastroenteritis transmitted by seafood worldwide (Ortiz-Jiménez 2018).

In the shrimp industry, infectious diseases caused by Vibrio species pose major challenges facing the world, causing considerable economic losses (Srinivasan & Ramasamy 2017). On Pacific white shrimp farms (Litopenaeus vannamei), for example, the main opportunistic pathogens belonging to the Vibrio genus are V. harveyi, V. alginolyticus and V. parahaemolyticus, known to cause serious outbreaks (Tepaamorndech et al. 2019).

The bacterium V. parahaemolyticus is the pathogen that causes acute hepatopancreatic necrosis syndrome, responsible for severely damaging shrimp production and consequently economic income (Wangman et al. 2018). The infection caused by this bacterium in shrimp can cause inactivity of the animals, slow growth, empty stomach and medium intestine, and atrophy associated with the pallor of the hepatopancreas. In the first 20 to 30 days after population with post-larvae, this disease can cause up to 100% mortality (Elshopakey et al. 2018).

The global emergence of bacterial resistance to antibiotics has become a major problem for healthcare, and therefore, new alternatives to antibiotics are needed to overcome this complication, especially natural compounds, herbs and phytochemicals (Prabu et al. 2018). In fact, herbal products may be more effective and economical than chemotherapeutic agents, and offer a viable solution to much pathogen control (Harikrishnan et al. 2011).

The family Verbenaceae consists of about 175 genera and 2,300 species of trees, shrubs, lianas and herbs distributed mainly in tropical and subtropical regions around the world (Cavalcanti et al. 2010). This taxon includes the species Lippia alba (Mill.) N.E.Br. ex Britton & P. Wilson and L. origanoides Kunth. The first is a medicinal small bush used as tranquilizer and gastroprotective agent (Gomes et al. 2018) and antispasmodic (Carvalho et al. 2018) in Central and South America. The species L. origanoides is a shrub with odorous leaves native from northeastern Brazil (Veras et al. 2017). The essential oil of the plant has potential use as an antiparasitic drug (Hashimoto et al. 2016) and antifungal and antibacterial agent (Pinto et al. 2016).

Natural products derived from plants have been of great interest in traditional medicine, as sources of potential alternative agents in the prevention and treatment of many infectious diseases (Khiya et al. 2018) and in the scavenging of free radicals (El Euch et al. 2019). Although many natural products have been reported to exhibit relevant biological activities, they may also cause some neurotoxic, hepatotoxic or other damage effects (Shirmohammadli et al. 2018). Therefore, tests on toxicity are also important, given that exposure to toxic agents can result in health impairment (Kampke et al. 2018).

This study, therefore, aimed to assess the chemical composition of L. alba and L. origanoides essential oils, evaluate their antioxidant activity, acute toxicity in Artemia salina and antibacterial effects against Vibrio parahaemolyticus were also investigated.

MATERIALS AND METHODS

Plant material

Accessions of L. alba and L. origanoides were collected in Parnaíba, Piauí, Brazil (03° 05’ 12.5”S; 41° 47’ 01.2”W), in March 2018. Both species were identified using the keys provided by Salimena & Múlgura (2015), and vouchers were deposited in the Herbarium of the Federal University of the Delta of Parnaíba - UFDPar, under numbers HDELTA5466 and HDELTA5469, respectively. All chemicals were of analytical grade and purchased from Sigma-Aldrich Chemical Co. Ltd. (St. Louis, MO. USA) unless otherwise specified. This study was approved by the Brazilian Genetic Heritage Management Council (CGEN) (Process number A00721D).

Extraction of essential oils

The fresh leaves of L. alba and L. origanoides were collected separately and dried at room temperature before being further processed. After the drying process, 600 g of dry leaves were mixed with 3.5 L of distilled water, in a hydrodistillation in a Clevenger-type apparatus. After the water started boiling, the process was maintained for four hours. At the end of the process, 3 g of L. alba EO (0.5% yield) and 9.6 g of L. origanoides EO (1.6% yield) were obtained. Then the EO obtained by the hydrodistillation process was stored in an amber glass bottle at 4 ° C until later use (adapted from Yen & Lin 2017).

A stock solution of each EO was prepared by dilution with 50% ethanol P.A., 1% Tween 80 USP and 49% distilled water, obtaining the final concentration of 100 mg.mL^-1.

Chemical characterization of essential oils

Chemical characterization was carried out by gas chromatography-mass spectrometry (GC-MS) analysis performed on an Agilent GC-7890B/MSD-5977A (quadrupole) instrument with electron impact at 70 eV HP-5MS methylpolysiloxane column (30 m × 0.25 mm × 0.25 μm; Agilent, Santa Clara, CA, USA), helium carrier gas with flow rate 1.00 mL min^-1 (8.8 psi) and constant linear velocity of 36.8 cm s^-1, injector temperature of 250°C, detector temperature of 150°C, transfer line temperature 280°C. Chromatographic furnace was set to an initial temperature of 70°C, with a heating ramp of 4°C min^-1 to 180°C and an increase of 10°C min^-1 to 250°C at the end of the run (34.5 min). The same parameters described above were employed for gas chromatography-flame ionization detection (GC-FID) analysis, using a Shimadzu GC-2010 Plus chromatograph.

The retention indices (RI) of the chemical components in L. alba and L. origanoides essential oils were calculated by injecting a mixture of standards containing a homologous series of C7-C30 alkanes in an HP-5MS column (Hu et al. 2019). The analytes were identified by comparing their mass spectra data with those from the National Institute of Standards and Technology (NIST) mass spectral library (Naumkin et al. 2012), and MS data from the literature (Adams 2007). The relative concentration of each compound in the essential oil was quantified based on the peak area integrated in the analysis program.

Antibacterial activity

Plant essential oils were tested for antimicrobial activity against bacterial strains of Vibrio parahaemolyticus provided by the Oswaldo Cruz Institute (OCI18950) and V. parahaemolyticus isolated from the hemolymph of farm-reared Litopenaeus vannamei shrimps from northeastern Brazil, after a high mortality event. The hemolymph of farmed shrimp samples were collected from ventral sinus by a puncture of the first abdominal segment with a 1-mL syringe containing ice-cold anticoagulant solution (citrate-EDTA) (Vargas-Albores et al. 1993). Then, the hemolymph was inoculated on agar plates with thiosulphate-citrate-bile salts-sucrose (TCBS) at 37°C for 24 h. Green or blue-green colonies were identified as V. parahaemolyticus-positive and transferred to tryptic soybean agar (TSA) plates containing 2% NaCl (Vieira et al. 2009).

The bacteria had their identification confirmed using the OMNILOG GEN III system (Biolog Inc., USA) according to the manufacturer’s instructions. Presumptive V. parahaemolyticus colonies were inoculated in BUG^TM Agar (Biolog Inc., USA) with 2% NaCl and incubated at 34°C for 24 h. After incubation, a single colony was picked from a plate and transferred into a 10 mL-inoculation fluid (IF-B) (Biolog Inc., USA). The inoculated IF-B was dispensed into a GEN III microplate. The microplate was incubated at 33°C for 24 h. The readings were carried out by the OmniLog® Data Collection software using a semiautomated Biolog MicroStation™ system microplate reader.

The antibacterial assay was performed using the microdilution method in nutrient broth, adapted from Clinical and Laboratory Standards Institute (CLSI 2012). The strains were maintained on Mueller Hinton agar (Merck) and incubated at 34 ± 2°C. The bacterial suspensions were adjusted with sterile saline solution until the concentration of 1.5 × 10⁸ CFU mL^-1. Then, the bacterial suspension broth was dispensed into a 96-well microplate for test with different EOs concentrations (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 and 0.078 mg.mL^-1), with a final volume of 100 μL. Said concentrations were obtained in the 96-well plate as follows: 180 µL of the nutrient broth with bacteria were pipetted into the well in the first row (A) of the 96-well microplate, and in the other wells (B to H) 100 µL were dispensed on each. Subsequently, 20 μL of the stock solution (100 mg.mL−¹) of OE was pipetted into well A (totaling 200 µL in the well). Then, from well “A” to well “H”, the solutions were diluted in series (dilutions by 2) with nutrient broth, pipetting 100 µL from one well to another. At the end, 100 µL of solution from well H was discarded, and the final volume of all wells was maintained at 100 µL.

A comparison between both plant essential oils and positive control antibiotics (oxytetracycline, enrofloxacin and sodium salt of ampicillin) was performed. Sterility control (nutrient broth without addition of bacterial inoculum) and growth controls (nutrient broth with bacterial inoculum and EO dilution solution) were included on each microtiter plate. All measurements were taken in triplicate. All microdilution plates were incubated at 34 ± 2°C for 24 hours under aerobiose conditions.

Bacterial growth was confirmed by adding 20 μL of 3% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC) aqueous solution and incubating for 1 hour at the same temperature. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of essential oil that completely inhibited bacterial growth.

The minimum bactericidal concentration (MBC) was determined from the MIC by plating 10 µL-aliquots from those wells with no growth onto Petri dishes containing Mueller Hinton agar (2% NaCl) incubated at 34 ± 2°C for 24 h. The MBC is the lowest concentration of each essential oil that shows no bacterial growth on the agar. The bactericidal and bacteriostatic effect of the essential oils were determined using the ratio MBC/MIC (Marmonier 1990).

Antioxidant activity

Antioxidant activity of essential oils were determined by the radical scavenging activity method using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as free radical (Brand-Williams et al. 1995). A stock solution of each EO was prepared by diluting 100 mg of oil in 1 mL of P.A. ethanol. Then, aliquots of 30 μL of each essential oil ethanolic solutions at different concentrations, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.0312 mg.mL^-1, were added to 200 μL ethanolic DPPH solution (100 μM) in 96-well microplates. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as reference synthetic antioxidant compound at molar concentrations of 15.63, 7.81, 3.91, 1.95 and 0.98 μM. The absorbance was measured at 517 nm immediately after the addition of the compounds to be tested (time 0) and after incubation at room temperature for 30 min under dark conditions. Readings for each sample on the xMark™ Microplate Absorbance Spectrophotometer, using the software Microplate Manager®.

The radical sequestering activity was calculated by the following equation: I (%) = [1-(A_sample / A_control)] × 100, where A_control is the absorbance of the control reaction (ethanolic DPPH solution) and A_sample is the absorbance in the presence of the tested compound at different concentrations. Based on the calculated I (%) values, the sample concentration providing 50% inhibition of free DPPH radicals (IC₅₀) calculated graphically using a calibration curve in the linear range, representing the EO concentration against the corresponding elimination effect. Tests were carried out in triplicate.

Acute toxicity

Artemia salina cysts (INVE Aquaculture, Belgium) were kept at 5°C until further analysis. The toxicity of EOs was determined according to an adapted method from Meyer et al. (1982) using freshly hatched nauplii. Dried cysts were incubated in natural seawater (salinity 35 g.L^-1; dissolved oxygen 5.3 mg L^-1; pH 8.2; and temperature 27.5°C) previously filtered through a 0.22 μm porosity filter under light conditions and continuous aeration. After 24 hours the A. salina nauplii were separated from the eggshells using a Pasteur pipette. All water used in the experiment was also prefiltered in a 0.22 μm porosity filter.

A stock solution of each EO was prepared in prefiltered sea water with 0.1% Tween 80 U.S.P., obtaining the final concentration of 100 mg.mL^-1. Toxicity assays were performed in 96-well microplates with 200 µL of EO solution in seawater at different concentrations and 10 nauplii to each well, in order to determine dose-response relationship. The EOs were diluted in seawater with 0.1% Tween 80 U.S.P. Each horizontal row of the 96-well plate corresponded to the EO tested concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.2, and 15.6 µg mL^-1. Each vertical row contained quadruplicate samples and controls. In addition to the tests performed with EO of L. alba and L. origanoides, 2 control groups were also used: control A - containing only nauplii and filtered seawater (to demonstrate that the volume of seawater used in the experiment allowed nauplii to survive for 24 hours); control B - containing nauplii and Tween 0.1% filtered seawater (to demonstrate that the concentration of Tween used did not influence the mortality of nauplii during the experiment).

After 24 hours of incubation, under normal photoperiod conditions and ambient temperature (27 to 30°C), the number of live and dead larvae in each well was counted using a stereoscopic microscope. Larvae that showed no internal or external movement during 30 seconds of observation were considered dead. In both control groups the survival of nauplii of A. salina was 100% after 24 hours of incubation.

Statistical analysis

The software Statistical Package for the Social Sciences (SPSS) 20.0 was used for statistical analysis by applying the analysis of unidirectional variance (ANOVA) followed by Tukey’s HSD (honestly significant difference) test with α<0.05 to determine statistical differences. Stundet t-test was used for independent samples (α<0.05). The determination of the average lethal concentration (LC₅₀) was performed by the Probit regression analysis method with 95% confidence limits (Finney 1971).

RESULTS AND DISCUSSION

Chemical characterization of essential oils

The chemical composition of the essential oils extracted from two Lippia species are described in Table I. Major compounds in EO samples from L. alba and L. origanoides were oxygenated monoterpenes, 56.6% and 52.9%, respectively, and by a low content of sesquiterpene hydrocarbons, 6.8% and 12.4%, and oxygenated sesquiterpenes, 8.0% and 1.4%, respectively.

Table I
Chemical composition of essential oils from Lippia alba and L. origanoides.

Compounds	Composition (%)
	IK*	L. alba	L. origanoides
α-Thujene	938	-	1.3
α-Pinene	947	-	0.6
β-Pinene	981	-	0.3
Sabinene	986	1.2	-
Myrcene	996	5.5	2.6
δ-3-Carene	1013	-	0.2
α-Terpinene	1019	-	1.8
p-Cymene	1026	2.7	16.0
Limonene	1041	17.0	0.9
1,8-Cineole	1044	-	2.1
β-(Z)-Ocimene	1047	0.7	-
γ-Terpinene	1059	5.1	5.6
Cis-Sabinene Hydrate	1069	0.2	0.3
Linalool	1102	1.2	-
trans-Sabinyl acetate	1103	-	0.4
Ipsdienol	1147	-	1.6
Terpinene-4-ol	1186	-	0.4
α-Terpineol	1199	-	0.5
Citronellol	1234	5.5	-
Thymol, methyl ether	1236	-	2.4
Neral	1243	15.5	-
Carvone	1246	1.3	-
Geraniol	1256	8.7	-
Geranial	1272	23.0	-
Thymol	1300	-	47.2
Thymol acetate	1357	-	0.2
β-Cubeben	1389	0.4	-
β-Elemene	1400	0.5	-
E-Caryophyllene	1422	0.2	11.3
Aromadendrene	1442	-	0.6
α-Humulene	1456	-	0.5
γ-Muurolene	1490	3.5	-
α-Zingiberene	1501	0.4	-
γ-Cadinene	1516	0.1	-
δ-Cadinene	1525	0.4	-
Elemol	1549	5.8	-
E-Nerolidol	1565	0.5	-
Spatulenol	1577	-	0.3
Caryophyllene oxide	1586	-	1.1
Guaiol	1600	0.4	-
TOTAL		99.8	98.2
Monoterpene hydrocarbons		28.4	31.5
Oxygenated monoterpenes		56.6	52.9
Sesquiterpenes hydrocarbons		6.8	12.4
Oxygenated sesquiterpenes		8.0	1.4

• *IK: Kovats Indices obtained in column RTX-5.

In both EOs twenty-three compounds were identified, being geranial (23.0%), limonene (17.0%) and neral (15.5%) the most abundant compounds in L. alba, and thymol (47.2%), p-cymene (16.0%) and E-caryophyllene (11.3%) the most predominant components in L. origanoides. The remaining compounds are listed in Table I.

Previous studies report the same major compounds in EOs extracted from L. alba and L. origanoides (Batista et al. 2018, Damasceno et al. 2018). However, variations on the concentration of major compounds in EOs from these two Lippia species can also be observed in the literature. For example, carvone and limonene are the major components of the EO obtained from L. alba (Teles et al. 2012), whereas carvacrol and thymol are the major components in the EO of L. origanoides, both collected at Bahia region, Brazil (Menezes et al. 2018). The individual variation in essential oil composition among plant species can also be influenced by other factors such as climatic variations, altitude, soil, crop area, harvest, processing and genetic varieties (Vaičiulytė et al. 2017, Farhat et al. 2019).

Antibacterial activity

The antibacterial activities of the essential oils are summarized in Table II. Both essential oils exhibited antimicrobial activity against Vibrio parahaemolyticus strains. Minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) ranged from 156 to 625 µg mL^-1 for these bacterial strains. The essential oil from L. alba showed the best antibacterial activity against V. parahaemolyticus strains. These results have also showed that both essential oils had a bactericidal effect against V. parahaemolyticus strains (OCI 18950 and isolated from farmed shrimp) as the MBC/MIC ≤ 4. According to Marmonier (1990) plant fractions are considered as bactericidal agents when the ratio MBC/MIC ≤ 4 and bacteriostatic agents when the ratio MBC/MIC > 4. Moreover, according to the classification proposed by Aligiannis et al. (2001) for plant materials, MIC values of ≤ 500 µg mL^-1 are considered strongly inhibitory. Therefore, the essential oils extracted from these Lippia species against V. parahaemolyticus strains show strong antimicrobial potential.

Table II
Antimicrobial activity of essential oils from Lippia alba and L. origanoides against Vibrio parahaemolyticus (OCI 18950) and V. parahaemolyticus isolated from farmed shrimp hemolymph.

Species	V. parahaemolyticus (OCI 18950)			V. parahaemolyticus in shrimp
	MIC	MBC	MBC/MIC	MIC	MBC	MBC/MCI
	Essential oils (µg mL-1)
L. alba	156	313	2.00	313	313	1.00
L. origanoides	313	313	1.00	313	625	2.00
	Antibiotics (µg mL-1)
Ampicillin	625	625	1.00	10000	-	-
Enrofloxacin	0.15	0.31	2.07	0.15	0.31	2.07
Oxytetracycline	0.00060	0.0012	2.00	1.22	2.44	2.00

• MIC = minimum inhibitory concentration; MBC = minimum bactericidal concentration.

The major components of these EOs are described in the literature as antibacterial compounds, such as E-caryophyllene (Yoo & Jwa 2019), geranial (Espina et al. 2017), neral (Liao et al. 2015), limonene (Costa et al. 2019), p-cymene (Miladi et al. 2017) and thymol (Cai et al. 2019). However, minority constituents in their composition should not be neglected, since possible synergistic or antagonistic interactions between components could influence their antibacterial potential and, as a result, their biological activities would vary accordingly to their overall composition (Chaturvedi et al. 2018).

Possible mechanisms of antimicrobial action of Lippia EOs are associated with high hydrophobicity of monoterpenes, and include cell wall degradation, increased membrane fluidity and permeability, cytoplasmic membrane damage, disruption of membrane-incorporated proteins, respiration inhibition, alteration of ion transport processes and leakage of intracellular materials (Badawy et al. 2019).

The antibacterial activity of these essential oils was of similar magnitude to that of ampicillin against V. parahaemolyticus (strain OCI 18950). However, an augmented antibacterial effect of all essential oils against the V. parahaemolyticus isolated from farmed shrimp hemolymph was noticed when compared with ampicillin. It is noteworthy that the strain isolated from the hemolymph of farmed shrimp showed higher resistance to ampicillin and oxytetracycline when compared with the strain of V. parahaemolyticus provided by the Oswaldo Cruz Institute (OCI18950). In several countries, antibiotic resistance of environmentally isolated Vibrio involve ampicillin, penicillin and tetracycline, which is a major concern in shellfish farming (Elmahdi et al. 2016).

Antioxidant activity

The antioxidant potential of Lippia alba and L. origanoides essential oils were evaluated and results are presented in Table III. The lower the IC₅₀ value, the greater the antioxidant potential of the tested substance.

Table III
Antioxidant activity of the essential oils against the DPPH solution (100 µM). IC₅₀ = concentration that provides 50% inhibition of the free radical DPPH (mg mL^-1).

Species	IC50
Trolox	2.75 ± 0.14 (× 10-3)c
Lippia alba	60.16 ± 0.28a
Lippia origanoides	0.22 ± 0.02b

• Means followed by different letters in the same column indicate significant differences using the Tukey test (P<0.05).

Antioxidant activity of L. alba EO, rich in limonene (27-77%), is reported in the literature to be similar to that of vitamin E, a product widely used as a natural and synthetic additive (Stashenko et al. 2004). Different L. alba chemotypes with high amounts of polyphenols, especially phenylpropanoids, may exhibit high antioxidant activities (Timóteo et al. 2015). Limonene and geranial, presented in major quantities in L. alba EO, as observed in this study, are common non-phenolic terpenoids of essential oils, whose kinetics of their antioxidant activity occur by co-oxidation with the substrate due to self-termination and cross-termination of the oxidative chain (Baschieri et al. 2017).

Also in our study, the antioxidant activity of L. origanoides essential oil was significantly higher (P < 0.05) than L. alba EO, which may be explained by differences in the chemical compositions between the two Lippia species. The higher antioxidant potential of L. origanoides EO is associated with large amounts of thymol in its composition (Damasceno et al. 2018). Moreover, p-cymene, the second most abundant compound in L. origanoides EO, has been described as having relevant antioxidant activity, and enhancing the antioxidant potential of other constituents (Milos & Makota 2012). Both thymol and p-cymene were present in L. origanoides EO in major amounts (>63 %; Table I).

The high antioxidant activity presented by compounds such as thymol and essential oils rich in phenolic monoterpenes are due to the hydrogen donation capacity of the phenolic hydroxyl, forming a phenolic radical (Teles et al. 2014, Baschieri et al. 2017). Natural antioxidants can protect cells against reactive oxygen species and therefore can help neutralize tissue damage mediated by oxidative stress (Ghorbani & Esmaeilizadeh 2017), which is very common during the management of captive shrimp and during infections.

Acute Toxicity

The brine shrimp lethality bioassay is considered a useful tool for testing biologically active compounds toxicity (Valadbeigi 2016). In our study, the estimated median lethal concentration (LC₅₀) of the essential oils required to kill 50% of the brine shrimp was lower than 1000 µg/mL (Table IV). According to the Meyer’s toxicity index (Meyer et al. 1982), plant essential oils with an LC₅₀ value < 1000 µg mL–¹ are considered cytotoxic. Therefore, Lippia alba and L. origanoides essential oils exhibited toxicity against A. salina indicating that samples are biologically active. No mortality was found in control group and differences between groups were not statistically significant (P<0.05).

Table IV
LC₅₀ (µg mL^-1) values of Lippia alba and L. origanoides essential oils in Artemia salina nauplii.

Species	LC50 (µg mL-1)	Classification
Lippia alba	307.95 ± 17.18a	Toxic
Lippia origanoides	215.73 ± 22.13a	Toxic

• Different letters in the same column indicate statistically significant differences by the T-student test (P<0.05).

Previous reports of acute toxicity (Olivero-Verbel et al. 2009) corroborates the toxicological property of essential oils from different species of the genus Lippia, using Artemia franciscana. However, for L. alba and L. origanoides essential oils, their findings indicate LC₅₀ values ranging from 8.87-20.13 μg mL^-1 and 10.29-34.90 μg mL^-1, respectively. That is, higher toxic potential compared with our results. Highier toxicity was also recorded for L. alba EO against A. salina with an LC₅₀ of 41.56 μg mL^-1 (Queiroga et al. 2019). This discrepancy suggests that the difference in the toxicity of essential oils may be related to the chemical composition of the oils, which in turn is determined by genetic factors and varies qualitatively depending on climate, soil type, time and method of extraction, etc. (Fernandes et al. 2011, Oliveira et al. 2012).

Lippia alba essential oil, containing predominantly linalool, eucalyptol, γ-muurolene and E-caryophyllene in its composition, has also shown genotoxic effects in fish (Oreochromis niloticus) and mammals (Mus musculus) (Kampke et al. 2018). However, L. origanides EO, containing thymol as one of its major constituents, was used in a mouse peritoneal macrophage toxicity assay, and showed no toxicity against mammalian cells (cytotoxic concentration - CC₅₀ > 100 μg mL^-1) (Borges et al. 2012). The toxicological property of thymol, major constituent in L. origanides EO, has already been observed (LC₅₀ = 514 µg mL^-1) in the brine shrimp lethality assay (Meyer et al. 1982).

On the whole, this toxicity of Lippia essential oils is mostly attributed to the presence of phenols such as thymol, in L. origanides EO, aldehydes such as the two isomeric acyclic monoterpenes geranial and neral, in L. alba EO, and acyclic alcohols such as geraniol, linalool, and citronellol, in L. alba EO exclusively (Bruni et al. 2004, Sacchetti et al. 2005).

CONCLUSIONS

Essential oils extracted from L. alba and L. origanoides shared several components and exhibited similar bioactivities when examined for their antimicrobial and toxicity effects. L. origanoides EO showed higher potential to free radical scavenging activity than L. alba EO. These essential oils also showed promising antibacterial activity against V. parahaemolyticus strains. To our knowledge, this is the first report of the antibacterial activity of L. origanoides essential oil against V. parahaemolyticus. Thereby, the studied EOs may be useful in controlling the pathogen V. parahaemolyticus, however, due to its exhibited toxicity against A. salina, further research is warranted into the usage of these EOs as candidates in the control of vibriosis infection. The major compounds identified in the essential oils appear to be directly involved in biological activity of these plants. In future research, it is important to isolate key components of essential oils to confirm their usefulness and to evaluate their antimicrobial, antioxidant and toxic potential separately, as well as conducting in vivo tests to attest the potential for use and safe dose in marine shrimp.

ACKNOWLEDGMENTS

This work was financially supported by the Banco Nacional de Desenvolvimento Econômico e Social (BNDES), Ministério da Agricultura, Pecuária e Abastecimento (MAPA), The Brazilian Agricultural Research Corporation (Embrapa) and Fundação Eliseu Alves (project number 11.17.02.001.03.10). The authors are also grateful to Mr. Francisco dos Santos Carvalho for laboratory technical assistance. The authors declare that they have no conflict of interest.

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Publication Dates

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

History

• Received
  25 Apr 2022
• Accepted
  08 July 2022