Antinociceptive and anti-inflammatory effects of the essential oil of Lippia hermannioides, an endemic species of Brazil

Abstract

Lippia hermannioides (Verbenaceae) is a species endemic to Brazil with reported antioxidant and antimicrobial activity. The objective of this study was to determine the chemical composition of the essential oil of L. hermannioides leaves (EOLH), using chromatographic and spectrometric methods, as well as its antinociceptive and anti-inflammatory potential by implementing of chemical and thermal nociception models. The results revealed germacrene D (18.39%) as the major compound of the essential oil, followed by bicyclogermacrene (11.72%), 1,8-cineole (11.24%), sabinene (10.38%), E-caryophyllene (8.33%), β-pinene (7.37%), and α-pinene (6.18%). Intraperitoneal administration of EOLH at doses of 300 and 2,000 mg.kg-1 showed no signs of toxicity in mice. EOLH doses of 75 and 300 mg.kg-1 did not affect the animals’ motor coordination in the rotarod test but reduced the number of abdominal writhing induced by acetic acid and decreased paw licking time in both phases of the formalin test. There was also an increased latency time in the hot plate test. These results indicate that this essential oil has antinociceptive and anti-inflammatory activity, thus supporting further research on the use of this phytotherapeutic resource in the health field.

Key words:
chemical composition; inflammation; pain; toxicity; volatile oil

Resumo

Lippia hermannioides (Verbenaceae) é uma espécie endêmica do Brasil com indicativo de atividade antioxidante e antimicrobiana. O objetivo deste trabalho foi determinar a composição química do óleo essencial das folhas de Lippia hermannioides (OELH), através da utilização de métodos cromatográficos e espectrométricos, e o potencial antinociceptivo e anti-inflamatório pela realização de modelos químicos e térmicos de nocicepção. Os resultados revelaram o germacreno D (18,39%), biciclogermacreno (11,72%), 1,8-cineol (11,24%), sabineno (10,38%), E-cariofileno (8,33%), β-pineno (7,37%) e α-pineno (6,18%) como os compostos majoritários presentes no óleo. A administração intraperitoneal do OELH nas doses de 300 e 2.000 mg.kg-1 não mostrou sinais de toxicidade em camundongos. Nas doses de 75 e 300 mg.kg-1, o OELH não afetou a coordenação motora dos animais no teste do rota-rod, diminuiu o número de contorções abdominais induzidas por ácido acético, reduziu o tempo de lambida da pata em ambas as fases do teste da formalina, bem como aumentou o tempo de latência no teste da placa quente. Esses resultados indicam que o óleo essencial estudado apresenta atividade antinociceptiva e anti-inflamatória, fornecendo, dessa forma, subsídios para futuras pesquisas com a finalidade de utilizar este recurso fitogenético na saúde.

Palavras-chave:
composição química; inflamação; dor; toxicidade; óleo volátiltoxicidade

Introduction

The genus Lippia (Verbenaceae) comprises approximately 140 species distributed worldwide, predominately in South and Central America and Africa (Cardosoet al. 2021). Brazil is a center of diversity for this genus, hosting 87 species, including aromatic herbs, shrubs, and small trees, with a high level of endemism (62 species) (Salimena & Cardoso 2023).

Traditionally, some Lippia species have been used worldwide to treat pain, inflammation, fever, spasms, hypertension, and infections affecting the gastrointestinal and respiratory systems (Siqueira-Limaet al. 2019; Bautista-Hernándezet al. 2021; Macêdoet al.2022; Ladeiraet al. 2023). Both traditional knowledge and scientific studies have shown the therapeutic potential of species in this genus, mainly Lippia alba(Mill.) N.E.Br. ex Britton & P.Wilson,Lippia grataSchauer, andLippia origanoidesKunth, for treating central nervous system disorders and managing pain (Siqueira-Limaet al. 2019).

Lippia hermannioidesCham. is an aromatic shrub endemic to Brazil (Salimena & Cardoso 2023) with limited documentation in the literature. The methanol extract of this species shows indications of antimicrobial and antioxidant activities (Fabriet al. 2011), but no pharmacological studies have been conducted on its essential oils. Thus, considering the importance of further investigation to elucidate and ensure the effectiveness of this species as a therapeutic resource and provide foundation for further research focused on herbal medicine development, the objective of this study was to analyze the chemical composition and antinociceptive and anti-inflammatory potential of essential oil of L. hermannioidesleaves.

Material and Methods

Plant material

Leaves of Lippia hermannioides Cham were collected from plants grown along the Mucugê-Ibicoara road, in Mucugê, Bahia, Brazil (13°0’46”S, 41°29’5”W), in April 2018. A voucher specimen was deposited at the Herbarium of the State University of Feira de Santana (HUEFS), under number 243182. Tânia Regina Santos Silva, Ph.D., identified the botanical material. The accession was recorded in the National System for Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under number 8EE138.

Essential oil extraction

The essential oil was extracted from air-dried and ground leaves (100 g), in triplicate, by hydrodistillation in a modified Clevenger-type apparatus, using 1 L of distilled water for 3 hours, following the method described by Silva et al. (2018), with modifications. The resulting pure essential oil was dried over anhydrous sodium sulfate and stored in an amber bottle at -22 °C until analysis. Oil content was determined by calculating the ratio between oil volume and dry plant biomass.

Chemical analysis

The chemical composition of the essential oil was determined by gas chromatography with flame ionization detection (GC/FID) and gas chromatography-mass spectrometry (GC/MS) (Adams 2007). An aliquot of 20 mg of essential oil was diluted in 1 mL of dichloromethane, and 1 µL of the volume was injected. The GC used was a Shimadzu GC-2010 model, equipped with a Palm Combi autosampler and FID detector. The analysis was performed with a DB-5 capillary column (30 m × 0.25 mm × 0.25 μm). The FID and injector temperatures were 240 °C and 220 °C, respectively. The oven temperature was set at 60 °C, increased by 3 °C per minute until 240 °C, and held for 20 minutes. Helium was used as the carrier gas at a flow rate of 1 mL min^-1. The analysis was performed in split mode with a ratio of 1:20. In the GC/MS analysis, the GC used was a Shimadzu GC-2010 model equipped with a Palm Combi autosampler coupled to a GC QP-2010 Shimadzu mass detector model. A BPX-5 capillary column (30 m × 0.25 mm × 0.25 μm) was used. The injector port was heated to 220 °C, and injections were carried out in split mode at a ratio of 1:100. The oven temperature was as described above for GC/FID. Helium was used as the carrier gas at a flow rate of 1 mL min^-1. The ionization source temperature was maintained at 240 °C, with ionization energy at 70 eV and ionization current at 0.7 kV. A mixture of linear hydrocarbons (C₈H₁₈-C₂₄H₅₀) was injected under the same experimental conditions. The constituents of the essential oil were identified by comparing the obtained spectra with those from the equipment database (NIST 21 and NIST107) and using the Kovats Index, which was calculated for each constituent as described by Adams (2007). Data were acquired and processed using the Shimadzu GC-MS Solution software. Results were expressed as the relative percentage of each compound (%), calculated by normalizing the chromatographic peak area.

Pharmacological tests

Animals

The experiments were conducted using adult male Swiss mice (26-31 g each), except for the acute toxicity test, which was performed using nulliparous and non-pregnant females obtained from the Central Animal Facility at the State University of Feira de Santana. The animals were housed at a temperature of 22±2 °C and relative air humidity of 50±10%, with a 12-hour photoperiod, and provided with ad libitum access to water and food until taken to the experimental room.

Acute toxicity

Female mice (n = 5) fasted for 4 hours before the test and were randomly divided into three groups based on treatments. After fasting, they were weighed and treated intraperitoneally with essential oil of L. hermannioides leaves (EOLH) at doses of 300 and 2,000 mg kg^-1 (in saline solution + 5% Tween 80 v.v^-1) or vehicle (saline solution + 5% Tween 80 v.v^-1, 0.01 mL.g^-1). The mice fasted for 2 hours after treatment administration and were observed for the first 24 hours (mainly at the first 30, 60, 120, 180, and 240 minutes) to monitor general behavioral signs, such as changes in skin, fur, and eyes, asl well as alterations in the respiratory and nervous systems (central and autonomic), following a protocol used for behavioral pharmacological screening (Almeida et al. 1999). The animals were kept under observation for 14 consecutive days to check mortality, and they were weighed on the first, seventh, and fourteenth days to assess possible weight changes compared to the control group.

Rotarod test

Motor performance of the animals was assessed using a rotarod apparatus to investigate whether the treatments affected their motor activity and consequently impaired the assessment of the nociceptive behavior in the experimental models. Initially, mice that were able to remain on the rotarod apparatus longer than 180 s (7 rpm) were selected 24 hours before the test. On the following day, the eligible animals were randomly divided into three groups: control (saline solution + 5% Tween 80 v.v^-1, 0.01 mL.g^-1), standard (Diazepam at 1.5 mg.kg^-1), and experimental (EOLH at doses of 75 or 300 mg.kg^-1 in saline solution + 5% Tween 80 v.v^-1). The time each animal remained on the rotating bar was recorded for up to 180 seconds at 30, 60, 90, and 120 minutes after intraperitoneal administration of the treatments.

Acetic acid test

The acetic acid-induced writhing test was used to evaluate analgesic activity. Mice were randomly distributed into three groups: control (saline solution + 5% Tween 80 v v^-1, 0.01 mL.g^-1), standard (indomethacin 10 mg.kg^-1), and experimental (EOLH at doses of 75 or 300 mg.kg^-1 in saline solution + 5% Tween 80 v.v^-1). Each animal received intraperitoneal administration of 0.85% acetic acid solution (0.01 mL.g^-1) 30 minutes after intraperitoneal administration of its respective treatment. The animals were observed for 15 minutes, starting 5 minutes after the acetic acid injection; the number of abdominal writhing movements exhibited by each animal was recorded during this period.

Formalin test

The animals were divided into three groups for the formalin test, following the same procedure as the acetic acid-induced writhing test.

A 20 µL injection of 2.5% formalin was administered to the animals in the subplantar region of the right hind paw 30 minutes after the intraperitoneal administration of the treatments. Mice were placed in a triangular observation box (25 × 25 × 25 cm), where the licking time (seconds) of the injected paw or leg was recorded for 5 minutes (phase 1 or initial phase). This parameter was recorded for 15 minutes after a 10-minute interval (phase 2 or late phase).

Hot plate test

Twenty-four hours before the tests, mice were subjected to screening with a cutoff time of 15 seconds, without treatment administration, to prevent misinterpretation of results due to possible resistance to thermal stimulation. The hot plate device was kept at a temperature of 50±1 °C, and the reaction time (lifting, licking hind paws, kicking, or jumping) was evaluated; those manifesting a reaction before the 15-second cutoff time were considered suitable for the study. On the following day, the three groups (control: saline solution + 5% Tween 80 v.v^-1, 0.01 mL.g^-1; standard: 10 mg.kg^-1 morphine; and experimental: 75 or 300 mg.kg^-1 of EOLH in saline solution + 5% Tween 80 v.v^-1) were exposed to thermal stimulation (50±1 °C) at 30, 60, and 120 minutes after treatment administration, with individual observations to record the time (seconds) between placing the animal on the hot plate and manifestation of acute discomfort behavior; the maximum duration of exposure to the hot plate was 30 seconds to prevent tissue damage to individuals.

Statistical analysis

Data, expressed as mean ± standard error of the mean, were evaluated using analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s test. Results were considered significant when p < 0.05.

Results and Discussion

Chemical composition

The content of essential oil of Lippia hermannioides leaves (EOLH) obtained from dry biomass was 1.23±0.21% (v.w^-1). Twenty-nine compounds were identified in the essential oil analysis, representing 93.18% of the total volume, divided into monoterpenes (43.72%) and sesquiterpenes (49.45%). Germacrene D (18.39%) was the major constituent, followed by bicyclogermacrene (11.72%), 1,8-cineole (11.24%), sabinene (10.38%), E-caryophyllene (8.33%), β-pinene (7.37 %), and α-pinene (6.18%) (Montanari et al. 2011), and Lippia javanica (Tab. 1).

This is the first study on the essential oil from leaves of Lippia hermannioides. The detected constituents were similar to those found in other essential oils of plants from the same genus, such as Lippia aff. microphylla, L. aristata, L. martiana, L. salviifolia (Silva et al. 2010), Lippia sericea, Lippia brasiliensis (Montanari et al. 2011), and Lippia javanica (Ngassapa et al. 2003), which showed, among other compounds, 23 of the 29 compounds identified in the present study.

Despite interspecific variability and the influence of biotic and abiotic stimuli (Almeida et al. 2018) on the presence and concentration of compounds, the prominent presence of germacrene-D was also been identified in oils from aerial parts of Lippia lupulina (Zoghbi et al. 2002) and in leaf oils of Lippia aff. microphylla (Silva et al. 2010). Germacrene-D has also been identified among the major constituents of the essential oil of Lippia alba (Marques et al. 2018), Lippia javanica (Ngassapa et al. 2003; Kamanula et al. 2017), and Lippia brasiliensis (Montanari et al. 2011).

The other components of EOLH (bicyclogermacrene, 1,8-cineole, sabinene, E-caryophyllene, β-pinene, and α-pinene) have also been identified among predominant constituents of essential oils of Lippia species. Bicyclogermacrene was identified in L. sericea (Montanari et al. 2011), as well as 1,8-cineole in L. turbinata (Quiroga et al. 2013), sabinene in L. sericea and L. brasiliensis (Montanari et al. 2011), β-pinene in L. turbinata (Quiroga et al. 2013), and α-pinene in L. aff. microphylla, L. aristate, and L. martiana (Silva et al. 2010).

Pharmacological tests

No relevant behavioral changes indicating toxicity or lethality were found during the fourteen-day observation period following administration of EOLH (300 and 2,000 mg.kg^-1). The animals’ body mass, assessed on the first, seventh, and last day, showed no statistically significant changes when compared to the control group.

In the rotarod test, animals treated with EOLH (75 or 300 mg.kg^-1) did not exhibit significant alterations in motor performance compared to the control group at 30 minutes (180.0±0.0, 180.0±0.0), 60 minutes (180.0±0.0, 180±0.0), 90 minutes (180.0±0.0, 180±0.0), and 120 minutes (180.0±0.0, 180.0±0.0) (Fig. 1).

As expected, the central nervous system depressant Diazepam (1.5 mg.kg^-1, intraperitoneal, standard drug) significantly reduced the time that treated animals could stay on the rotarod at all periods: at 30 minutes (5.33±2.6), 60 minutes (3.5±1.6), 90 minutes (10.0±4.0), and 120 minutes (13.0±8.0) compared to the control group.

The results obtained for the parameters evaluated in the acute toxicity test indicated absence of toxicity of EOLH up to the tested dose. This was also found in the rotarod test, as any change in motor activity diagnosed in this test could indicate toxicity (Dunham & Miya 1957; Lopes et al. 2014). The first test performed to assess the antinociceptive and anti-inflammatory activity of EOLH was the acetic acid-induced abdominal writhing test. Intraperitoneal application of acetic acid caused pain through direct or indirect mechanisms involving the production of some mediators at the free terminals of sensory polymodal neurons (Ikeda et al. 2001; Bahamonde et al. 2013), releasing considerable amounts of algogenic substances such as prostaglandins (Bahamonde et al. 2013) and cytokines (TNF-α, IL-1β and IL-8), which act synergistically, inducing writhing released by resident peritoneal macrophages and mast cells (Ribeiro et al. 2000; Pavao-de-Souza et al. 2012).

Table 1
Chemical constituents of Lippia hermannioides leaves essential oil.

Compounda		KIlit b	KIcalc c	Mean±SDd (%)
Monoterpenes hydrocarbons					29,17±0,32
	α-thujene	930	929	1,23±0,14
	α-pinene	939	937	6,18±0,60
	sabinene	975	976	10,38±1,01
	β-pinene	979	980	7,37±0,80
	β-myrcene	990	990	0,79±0,09
	α-terpinene	1017	1018	0,45±0,09
	p-cymene	1024	1026	0,38±0,06
	limonene	1029	1031	1,19±0,12
	γ-terpinene	1059	1061	0,79±0,15
	terpinolene	1088	1093	0,41±0,18
Oxygenated monoterpenes				14,55±0,44
	1,8-cineol	1031	1034	11,24±1,57
	cis-sabinene hydrate	1070	1075	0,24±0,05
	linalool	1098	1098	0,67±0,05
	terpinen-4-ol	1177	1178	1,77±0,43
	α-terpineol	1188	1191	0,63±0,08
Sesquiterpenes hydrocarbons				46,74±0,44
	δ-elemene	1338	1341	0,34±0,06
	α-copaene	1376	1382	0,67±0,14
	β-bourbonene	1388	1394	1,57±0,20
	β-cubebene	1388	1395	0,49±0,09
	β-elemene	1390	1395	0,61±0,08
	E-caryophyllene	1419	1423	8,33±0,91
	α-humulene	1454	1457	3,15±0,19
	allo-aromadendrene	1460	1470	0,90±0,06
	γ-muurolene	1479	1484	0,56±0,04
	germacrene D	1485	1491	18,39±2,09
	bicyclogermacrene	1500	1505	11,72±1,00
Oxygenated sesquiterpenes				2,71±0,08
	spathulenol	1578	1580	1,98±0,13
	caryophyllene oxide	1583	1586	0,52±0,04
	viridiflorol	1592	1595	0,22±0,07
Total identified compounds				93,18±0,36

• ^Notes:
  ^a = Compounds are listed in order of elution on the DB-5 column. ^bKI_lit = Kovats indices from literature. ^cKI_calc = Kovats indices calculated on BPX-5 column (relative to n-alkanes). ^dSD = standard deviation.

Figure 1
Effect of essential oil of Lippia hermannioides leaves (EOLH) (75 or 300 mg.kg^-1) on motor performance (rotarod test). Each column represents mean ± standard error of the mean (n = 6 per group). **** = p < 0.0001 versus control (two-way ANOVA followed by the Tukey’s test).

Treatment with EOLH (75 and 300 mg.kg^-1) significantly reduced the number of abdominal writhes compared to the control group (Fig. 2). The control group animals exhibited a mean of 39.13±3.35 abdominal writhing movements, whereas mice treated with EOLH at doses of 75 and 300 mg.kg^-1 exhibited 24.75±3.51 and 17.38±3.55, respectively. The inhibition percentage of abdominal writhes was 36.75% for the lowest EOLH dose and 55.58% for the highest dose. As expected, indomethacin significantly reduced the analyzed parameter, showing an inhibition of 90.74% (3.625±2.17).

The results obtained in this EOLH test denoted its antinociceptive activity; however, although the acetic acid-induced writhing test is a valuable tool for discovering new drugs, one of its disadvantages is the difficulty in distinguishing antinociceptive from anti-inflammatory effects (Collier et al. 1968). Thus, a formalin test was conducted to better assess the activity of EOLH. This test is configured as a biphasic nociception model, having an initial phase resulting from direct chemical stimulation of nociceptors (Shibata et al. 1989), followed by the release of substance P and bradykinin (Santos et al. 2014), which is an interphase resulting from the activation of nociception inhibitory mechanisms, and then a late phase resulting from an inflammatory response (Hunskaar & Hole 1987) mediated by histamine, serotonin, prostaglandin, and bradykinin (Shibata et al. 1989).

Figure 2
Effect of essential oil of Lippia hermannioides leaves (EOLH) (75 or 300 mg.kg^-1) on the acetic acid-induced writhing in mice. Each column represents mean ± standard error of the mean (n = 8 per group). ** = p < 0.01, *** = p < 0.001 or **** = p < 0.0001 versus control (one-way ANOVA followed by the Dunnett’s test).

EOLH was able to reduce the nociceptive behavior in both phases of the formalin test when compared to the control group (Fig. 3a-b). In the initial phase, the inhibition percentage of the parameter for animals treated with oil at doses of 75 and 300 mg.kg^-1 was 42.40% and 59.91%, respectively. In the late phase, the reduction percentage was 36.52% for the dose of 75 mg.kg^-1 and 54.95% for the dose of 300 mg kg^-1. Indomethacin, a peripherally acting drug, showed action in both phases but had a more pronounced result in the late phase (initial phase: 50.9% inhibition; late phase: 87.6% inhibition).

The data obtained indicate the antinociceptive and anti-inflammatory potential of the oil, and that its action can be mediated by peripheral and central mechanisms through inhibitory response in both phases (Hunskaar & Hole 1987; Guilhon et al. 2011).

The hot plate test was performed to better assess the action of this oil on the central nervous system. In this test, similar to other thermal stimulation tests, the application of the stimulus activates high-threshold sensory fibers present in the skin, which then activate neurons in the superficial dorsal horn of the spinal cord that continue to activate neurons in the medulla, mesencephalon, and thalamus (Allen & Yaksh 2004). When a treatment has no effect on motor function and there is an increase in latency in the hot plate test, it is considered an antinociceptive.

The results also showed that, like morphine (10 mg.kg^-1), the tested oil increased the onset time of response to thermal stimulation in the hot plate test after 30, 60, and 120 minutes of its administration compared to the control group (Fig. 4). EOLH at a dose of 75 mg.kg^-1 increased the time mice could stay on the plate by 85.92%, 75.77%, and 90.50% after 30, 60, and 120 minutes of its administration, respectively. The increases in this parameter for the oil dose of 300 mg.kg^-1 was 150.70%, 138.63%, and 117.95% at the respective observation times. Considering that there was no effect on motor function in the rotarod test, EOLH results in the hot plate test indicate a central antinociceptive action, which reduces over time when compared to morphine.

Figure 3
a-b. Effect of essential oil of Lippia hermannioides leaves (EOLH) (75 or 300 mg.kg^-1) of the formalin test in mice - a. on the initial phase; b. on the late phase. Each column represents mean ± standard error of the mean (n = 6 per group). ** = p < 0.01 or **** = p < 0.0001 versus control (one-way ANOVA followed by the Dunnett’s test).

Figure 4
Effect of essential oil of Lippia hermannioides leaves (EOLH) (75 or 300 mg.kg^-1) on the hot plate test in mice. Each column represents mean ± standard error of the mean (n = 8 per group). * = p < 0.05, ** = p < 0.01 or **** = p < 0.0001 versus control (two-way ANOVA followed by the Tukey’s test).

Some of the main compounds identified in this essential oil, such as α-pinene, β-pinene, 1,8-cineole, germacrene D, and E-caryophyllene, have been reported for their antinociceptive activity, with a diversity of mechanisms ranging from opioid agonists to modulators of NO synthesis (Guimarães et al. 2012; Fidyt et al. 2016). The opioid and endocannabinoid pathways of E-caryophyllene may be involved in the action of this sesquiterpene (Fidyt et al. 2016). The analgesic effect promoted by the monoterpene 1,8-cineole may be related to the TRP channel family (Guimarães et al. 2012). An anti-inflammatory activity can complement the analgesic effect, and some monoterpenes, including 1,8-cineole, suppress the expression or inhibit the activity of COX. Furthermore, a-terpineol, α-pinene, E-caryophyllene, and a-humulene regulate the NF-kB and TNF-a pathways (Guimarães et al. 2012; Viveiros et al. 2022).

In summary, the antinociceptive and/or anti-inflammatory effects reported for some of the main constituents found in EOLH may indicate the role of these metabolites in the observed effects in the tests. However, further studies are needed to elucidate which active principles exert the observed pharmacological effects, establishing their mechanism of action, as well as assessing whether there is a synergistic contribution.

Acknowledgements

This work was supported by the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES); and by the Foundation for Research Support of the state of Bahia (Fapesb).

Data availability statement

In accordance with Open Science communication practices, the authors inform that all data are available within the manuscript

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Edited by

Area Editor:

Dr. Leopoldo Baratto

Publication Dates

• Publication in this collection
  22 July 2024
• Date of issue
  2024

History

• Received
  10 Dec 2023
• Accepted
  28 Mar 2024