Effect of Lavender (Lavandula angustifolia) – Essential Oil on Acute Inflammatory Response
Lavandula angustifolia is a plant of Lamiaceae family, with many therapeutic properties and biological activities, such as anticonvulsant, anxiolytic, antioxidant, anti-inflammatory, and antimicrobial activities. The aim of this study was to evaluate the effect of Lavandula angustifolia Mill. essential oil (LEO) on acute inflammatory response. LEO was analyzed using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) methods and showed predominance of 1,8-cineole (39.83%), borneol (22.63%), and camphor (22.12%). LEO at concentrations of 0.5, 1, 3, and 10 μg/ml did not present in vitro cytotoxicity. Additionally, LEO did not stimulate the leukocyte chemotaxis in vitro. The LEO topical application at concentrations of 0.25, 0.5, and 1 mg/ear reduced edema formation, myeloperoxidase (MPO) activity, and nitric oxide (NO) production in croton oil-induced ear edema model. In carrageenan-induced paw edema model, LEO treatment at doses of 75, 100, and 250 mg/kg reduced edema formation, MPO activity, and NO production. In dextran-induced paw edema model, LEO at doses of 75 and 100 mg/kg reduced paw edema and MPO activity. In conclusion, LEO presented anti-inflammatory activity, and the mechanism proposed of LEO seems to be, at least in part, involving the participation of prostanoids, NO, proinflammatory cytokines, and histamine.
The Lamiaceae family of plants is a major source of polyphenols and pharmacological properties described in the literature. Belonging to the Lamiaceae family, Lavandula angustifolia is indigenous to the mountainous regions of the Mediterranean, with many therapeutic properties and biological activities .
Phytochemical studies revealed that the major constituents of Lavandula angustifolia essential oil (LEO) are 1,8-cineole, camphor, and endo-borneol. Other components can also be found in minor quantities, such as α-pinene, camphene, α-pinene, β-pinene, p-cymene, limonene, terpinen-4-ol, and cryptone [2, 3]. However, the LEO composition may vary depending on the geographical origin of the plant material and environmental factors, such as geographical conditions, climate and seasonal variations, and the stage of the plant growth, and the extraction and detection methods also influence the LEO composition .
The extracts and Lavandula angustifolia essential oil have various pharmacological effects described in the literature, such as anticonvulsant , anxiolytic , antioxidant, anticholinesterase [7, 8], antimicrobial , and antifungal activities . Additionally, various constituents in the oil also have valuable pharmacological properties, such as anti-inflammatory, antioxidant, and antimicrobial [11–14].
Inflammation is a complex biological process involving vascular, cellular components and a variety of soluble substances, presenting as characteristic clinical signs: redness, heat, swelling, pain, and function loss . The purpose of the inflammatory process is the elimination of the aggressive agent and consequences of tissue injury . The leukocytes recruitment is essential in the acute inflammatory response, where cells act as the first line of defense in the initiation of the inflammatory process, and involves the participation of several inflammatory mediators , produced by inflammatory cells that play an important role in maintaining the inflammatory response .
Natural products and their essential oils have been popularly used for the treatment of various inflammatory diseases and the development of new therapeutic strategies. Studies suggest that the use of natural products may be safer and more effective since they have low toxicity and few side effects . Thus, the objective of this research was to investigate the LEO activity in acute inflammation, using different experimental models.
2. Materials and Methods
Zymosan, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], croton oil, dextran, celecoxib, promethazine, indomethacin, and λ-carrageenan were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Plant Material and Extraction of Essential Oil
The leaves and stem of the Lavandula angustifolia were commercially purchased from Cercopa Guarapuava, PR, Brazil. The essential oil was extracted by conventional steam distillation using a Clevenger-type apparatus for 3 h.
2.3. Analysis of the Essential Oil and Compound Identification
The LEO was analyzed using gas chromatography- (GC-) mass spectrometry (MS). GC was performed with a Thermo Electron Corporation Focus GC model under the following conditions: DB-5 capillary column (30 m × 0.32 mm, 0.25 mm); column temperature, 60°C (1 min) to 180°C at 3°C/min; injector temperature, 220°C; detector temperature, 220°C; split ratio, 1 : 10; carrier gas, He; flow rate, 1.0 mL/min. An injection volume of 1 μL was diluted in acetone (1 : 10). The retention index (RI) was calculated relative to a series of the n-alkanes (C8–C40, Sigma-Aldrich, St. Louis, MO, USA) on DB-5 column, using the Van den Dool and Kratz equation [20, 21].
Male Swiss mice (weighing 20–30 g) were provided by the Central Animal House of the State University of Maringá, Paraná, Brazil. The animals were housed at 22 ± 2°C under a 12/12 h light/dark cycle with free access to food and water. All of the protocols were approved by the Ethical Committee in Animal Experimentation of the State University of Maringá (CEEA/UEM number 3024210315).
2.5. Cell Viability Analysis (MTT Assay)
Leukocytes were obtained from the peritoneal cavity of mice 4 hours after injection of zymosan solutions (1 mg/cavity, i.p.). Briefly, the cells (5 × 105 cells/well) were exposed to LEO at concentrations of 0.5, 1, 3, 10, 30, or 90 μg/mL for 90 min (37°C/CO2 5%). A volume of 10 μL of MTT (5 mg/mL, Sigma) was added to each well. After 2 h, 150 μL of supernatant was removed, and 100 μl of dimethyl sulfoxide was added to each well, and absorbance was measured using a Biochrom Asys Expert plus microplate reader (Asys®) at a wavelength of 540 nm, as previously described . The percentage of viability was determined by the following formula:
2.6. In Vitro Leukocytes Chemotaxis
Leukocytes were obtained by the method described above. The chemotaxis assay was performed using a 48-well microchemotaxis plate (Neuro Probe), in which the chambers were separated by a polyvinylpyrrolidone-free polycarbonate membrane (5 μm pore size). LEO (used as chemoattractant) at concentrations of 2, 15, or 150 μg/mL or RPMI 1640 medium (control) was placed in the lower chamber. A leukocyte suspension (1 × 106 cells/mL, in RPMI 1640 medium) was placed in the upper chamber. The chambers were incubated (37°C/CO2 5%) for 1 h and the membrane was stained with Instant Prov. Cells present in the membrane were counted in five random fields from each well, using light microscopy, as previously described . The results are expressed as the mean number of leukocytes per field.
2.7. Evaluation of Topical Anti-Inflammatory Effect
Ear edema was induced by topical application of croton oil (200 μg 20/ear) diluted in 20 μl of acetone/water solution (vehicle) in the inner surface of the mouse right ear. The left ear received an equal volume of vehicle (n = 5–7 animals/group). LEO (0.125, 0.25, 0.5, 1, and 2.5 mg/ear), dexamethasone (reference drug, 0.1 mg/ear), or vehicle was applied topically to the right ear 1 h before croton oil application. Six hours after application of the inflammatory stimulus, the mice were euthanized, and a 6 mm diameter plug was removed from both the treated and untreated ears. Edema was measured as the weight difference between the two plugs. The data are expressed as the mean ± SEM weight of the ears.
2.8. Evaluation of Systemic Anti-Inflammatory Effect
To provide additional evidence supporting the potential anti-inflammatory effects produced by LEO, we also carried out a carrageenan or dextran-induced mice paw edema in mice (n = 5–7 animals/group). The negative control group received only subplantar injection of sterile saline. The positive control group received subplantar injection of carrageenan or dextran (500 μg/paw) and only treatment orally with LEO (50, 75, 100, and 250 mg/kg). The paw volume was measured by digital plethysmometer (Ugo Basile®, Italy) prior, 1, 2, and 4 hours after carrageenan injection, or 30, 60, 120, and 240 minutes after dextran injection. Indomethacin (5 mg/kg, p.o.) and celecoxib (10 mg/kg, p.o.) were used as the reference drug in carrageenan-induced foot paw edema, and promethazine (10 mg/kg, p.o.) was used as the reference drug in dextran-induced paw edema. The paw edema, in μL, was calculated by the difference in the paw volume prior and after carrageenan or dextran injection. After the last measurement, the animals were euthanized and the inflamed hind paws tissues were collected.
2.9. Determination of Myeloperoxidase (MPO) Activity
The plugs obtained from the right and left ears and paw sections were used to analyze myeloperoxidase (MPO) activity. The ear and paws sections were placed in 50 mM potassium phosphate buffer (pH 6.0) that contained 0.5% hexadecyl trimethyl ammonium bromide (Sigma, St. Louis, MO, USA) in a Potter homogenizer. The homogenate was shaken and centrifuged for 5 min. A 10 μL aliquot of the supernatant was added in triplicate to each well of microplate, in triplicate. The supernatant solution was then mixed with 200 μL of the buffer solution that contained O-dianisidine dihydrochloride (16.7 mg, Sigma), double distilled water (90 mL), potassium phosphate buffer (10 mL), and 1% H2O2 (50 μL). The enzyme reaction was stopped by addition of sodium acetate. MPO activity was determined by the absorbance measured at 460 nm using a microplate spectrophotometer (Spectra Max Plus).
2.10. Determination of Nitric Oxide (NO) Production
The NO production was determined by measuring the nitrite level by Griess reaction. Nitrite level was determined in ears and paws sections, obtained as above described. The samples were centrifuged at 1000 rpm for 10 min at 4°C. The supernatant was separated (50 μL) and incubated with equal volumes of Griess reagent mixtures (1% sulfanilamide in 5% phosphoric acid and 0,1% N-1-naphthylethylenediamine dihydrochloride in water) at room temperature for 10 min. The absorbance was measured in a microplate reader at 550 nm. NO concentrations were calculated from a sodium nitrite standard curve. Data were presented as the μM concentration of NO2-.
2.11. Statistical Analysis
Data are expressed as the mean ± SEM for each experimental group. The results were statistically analyzed by using one-way variance analysis (ANOVA) followed by Tukey’s test. Differences were considered significant when p < 0.05.
3.1. Analysis of LEO
The obtained pale yellow essential oil was dried over sodium sulfate and stored at 4°C in dark vials until tested. The yield of LEO was 0,14% v/w. The chemical composition of LEO was investigated by gas chromatography-mass spectrometry (GC-MS). The results of the GC-MS analysis (Figure 1) showed a predominance of 1,8-cineole (39,8%), endo-borneol (22,6%), and camphor (22,1%). A complete list of the components and their relative abundances are presented in Table 1.