Essential Oil Composition, Antimicrobial and Pharmacological Activities of Lippia sidoides Cham. (Verbenaceae) From São Gonçalo do Abaeté, Minas Gerais, Brazil

Plant material

Rooted cuttings of L. sidoides native to São Gonçalo do Abaeté, Minas Gerais were transplanted and grown in Hidrolândia, Goiás (16°54'1.3” S, 49°15'35.2” W, elevation of 835 m). Voucher specimens were deposited in the herbarium of Universidade Federal de Goiás (record number UFG-47319). Leaves were collected in April 2012, dried at room temperature, and ground in a knife mill.

Extraction, identification, and analysis of essential oil

Powdered leaves were grinded and hydrodistilled in a Clevenger-type apparatus. The essential oil was desiccated with anhydrous sodium sulfate and stored in a freezer at –10°C. The essential oil was subjected to gas chromatography coupled with mass spectrometry (GC/MS) using a Shimadzu QP5050A instrument with a fused silica capillary column (CBP-5; 30 m × 0.25 mm × 0.25 µm). Helium was used as the carrier gas, with a constant flow rate of 1 mL/min. The column was heated with programmed temperatures (60°C for 2 min, 3°C per min up to 240°C, 10°C per min up to 280°C, and 280°C for 10 min). The ionization energy was 70 eV. An injection volume of 1 µL of each sample diluted in dichloromethane (20% w/v) was used, with a split ratio of 1:50.

Essential oil components were identified by comparing mass spectra and retention indices with values found in the literature.[21] Retention indices were calculated by coinjecting a mixture of hydrocarbons (C8–C32) and applying the Van Den Dool and Kratz equation.[22]

Preparation of extracts and fractions

Powdered leaves of L. sidoides were macerated three times at room temperature with 95% EtOH (v/v) using a proportion of 1:5 (w/v), then filtered and concentrated in a rotary evaporator below 40°C to obtain the crude ethanol extract (EELS).

Fractions were obtained from the EELS. Fifty grams of EELS were dissolved in 100 mL of MeOH/H₂O (7:3) and subjected to liquid–liquid partitioning with solvents of increasing polarity (hexane, dichloromethane, and ethyl acetate).[23] The solvents from each fraction were evaporated in a rotary evaporator. For the final MeOH/H₂O fraction, methanol was eliminated in a rotary evaporator and the resulting AF was lyophilized. Four fractions were obtained and designated as follows: hexane fraction (HF), dichloromethane fraction (DF), ethyl-acetate fraction (EAF), and aqueous fraction (AF).

Antimicrobial activity

Essential oil, crude ethanol extract, and fractions (HF, DF, EAF, and AF) were subjected to a microdilution test in broth to determine the minimum inhibitory concentration (MIC) using sterile 96-well microplates with “U”-shaped wells, as recommended by the Clinical and Laboratory Standards Institute.[2425] Experiments were carried out in duplicate.

Microorganisms used in the assays were standard strains from the American Type Culture Collection (ATCC) and clinical isolates provided by the Bacteriology and Mycology Laboratory of the Tropical Pathology and Public Health Institute) of Universidade Federal de Goiás (UFG). The following strains were used: fungi: Candida parapsilosis (ATCC 22019), C. parapsilosis (86U), C. albicans (63U), Cryptococcus sp. (ATCC D), C. gatti (L48), and C. neoformans (L3); Gram-positive bacteria: Bacillus cereus (ATCC 14579), B. subtilis (ATCC 6633), Micrococcus roseus (1740), M. luteus (ATCC 9341), Staphylococcus epidermidis (ATCC 12229), S. aureus (6538), and S. aureus (ATCC 25923); Gram-negative bacteria: Enterobacter aerogenes (ATCC 13048), E. cloacae (HMA/FT502), E. coli (ATCC 11229), Pseudomonas aeruginosa (ATCC 9027), P. aeruginosa (SPM1), Salmonella spp. (19430), and Serratia marcenscens (ATCC 14756).

Fungi were cultivated in Sabourad dextrose agar plates and incubated at room temperature for either 24 h (Candida spp.) or 48 h (Cryptococcus spp.). The bacteria were incubated in Casoy broth at 35°C for 24 h, transferred to an inclined Casoy agar, and incubated at 35°C for an additional 24 h to reactivate the cultures.

The culture medium used in the antifungal activity test was RPMI 1640 and the medium for the antibacterial activity test was 2× Müller Hinton broth (MH). Samples (essential oil, EELS, HF, DF, EAF, and AF) were solubilized in 10% dimethyl sulfoxide (DMSO) and 0.02% Tween 80® and diluted in either RPMI (antifungal activity) to obtain a concentration of 1000 µg/mL or in MH broth (antibacterial activity) to obtain a concentration of 2000 µg/mL.

For the antifungal assays, we added 100 µL of RPMI to all microplate wells in columns 2–12 and 200 µL of each sample to wells A through G in column 1. Using a multichannel pipette, serial dilutions were performed up to column 11, where 100 µL were discarded. Fungal inoculants were prepared in a sterile saline solution (NaCl 0.85%) to obtain a turbidity equivalent to half of the McFarland 1.0 scale (83.2–85% transmittance, measured with a spectrophotometer at 530 nm). Two dilutions (first 1:50, then 1:20) were prepared in RMPI to obtain cellular concentrations between 1 and 5 × 10⁵ UFC/mL. The final dilution of the inoculants in the wells ranged from 0.50 to 0.25 × 10³ UFC/mL. One hundred µL of each inoculate were added to each well to obtain sample concentrations between 1000 µg/mL and 0.97 µg/mL. No microorganisms were added to wells in line G, since they were used as controls of the sterility of the medium and of the samples tested. The positive control consisted of itraconazole (Sigma) at a concentration of 16 µg/mL. After inoculation, microplates were covered and incubated at room temperature for 48 h for Candida species, and 72 h for Cryptococcus species. The occurrence of fungal growth was checked visually.

For the antibacterial assays using microplates, 100 µL of MH were added to all wells from columns 2–12 and 200 µL of each sample were added to wells A through G in column 1. A multichannel pipette was used to obtain serial dilutions up to column 11, where the final 100 μL were discarded, for concentrations ranging from 2000 µg/mL to 1.95 µg/mL. Two hundred µL of MH broth containing 10% DMSO and 0.02% Tween 80® were added to line H as a negative control, and pure MH broth was added to column 12 to control for microbial growth. In addition, vancomycin (Sigma) at 32 µg/mL and gentamicin (Sigma) at 128 µg/mL were used as positive controls for Gram-positive and Gram-negative bacteria, respectively.

Bacterial inoculants were prepared in sterile saline solution (NaCl 0.85%) to obtain a turbidity equivalent to half of the McFarland 1.0 scale (79.4–83.2% transmittance, measured with a spectrophotometer at 625 nm). These inoculants were diluted 1:10 in saline to obtain a cell concentration of 10⁷ UFC/mL. Five μL of the inoculants were placed in the wells, for a final concentration of 5 × 10⁵ UFC/mL. No microorganisms were added to wells in line G, which were used as controls of the sterility of the medium and the samples tested. After bacterial inoculation, microplates were sealed and incubated at 35°C ± 2°C for 18–24 h. After the incubation period, 20 µL of 0.5% triphenyltetrazolium chloride (TTC) were added to all wells, and the microplates were reincubated for approximately half an hour. The appearance of a reddish color was considered proof of bacterial growth.

The MIC was defined as the lowest concentration of the sample (in µg/mL) fully capable of inhibiting bacterial or fungal growth. The classification proposed by Holetz et al.[26] was used to interpret the results of the tests of antimicrobial activity. Using this classification, MIC values below 100 µg/mL indicate good inhibitory activity; values between 100 and 500 µg/mL, moderate inhibitory activity; values between 500 and 1000 µg/mL, weak inhibitory activity; and values above 1000 µg/mL characterize an inactive substance.

Pharmacological tests

Experiments were performed using female Swiss albino mice (25–35 g) from the Central Animal House of Universidade Federal de Goiás (UFG). Animals were kept in plastic cages at 22 ± 2°C and on a 12 h light/dark cycle, with free access to pellet food and water, in compliance with the International Guiding Principles for Biomedical Research Involving Animals. All experimental protocols were developed according to the principles of ethics and animal welfare designated by the Brazilian College of Animal Experiments (COBEA). The experimental protocols were approved by the Ethics Commission of UFG (number: 104/08).

Effect on gross behavior or Irwin test

Experimental groups of mice (n = 5 per group) were treated orally (p.o.), intraperitoneally (i.p.), or subcutaneously (s.c.) with increasing doses of 50, 150, 450, and 1350 mg/kg crude ethanol extract solubilized (EELS) in 10% DMSO, whereas control groups received 10 mL/kg of the vehicle (10% DMSO). Animals were observed for 3 min in free ambulation on a flat surface at increasing intervals after treatment (5, 10, 20, 30, and 60 min, 4, 8, 24, and 48 h, and 4 and 7 days). The observed effects were compiled using a standard pharmacological screening approach, adapted from that described by Irwin.[27]

Antinociceptive activity

The antinociceptive effect of the EELS was tested using three different models of analgesia: the acetic acid-induced writhing test, the formalin-induced paw licking test, and the tail flick test.

Acetic acid-induced abdominal writhing test

Acetic acid-induced writhing is commonly used as a screening method for compounds with potential antinociceptive and/or anti-inflammatory activity. Nociception may be caused directly by the stimulation of nerve endings, or indirectly through the release of endogenous mediators involved in pain modulation.[28] The evaluation of abdominal contortions induced by acetic acid was carried out according to Hendershote and Forsaith[29] and Koster et al.[30] Groups of mice (n = 8) were treated by gavage with 100, 300, or 1000 mg/kg EELS (solubilized in 10% DMSO), vehicle (10% DMSO, 10 mL/kg – control group), or indomethacin (10 mg/kg – positive control for antinociceptive activity) 1 h before the application of an acetic acid solution (1.2% v/v; 10 m/kg, i.p.). The writhing response consisted of contraction of the abdominal wall and pelvic rotation followed by hind limb extension. The number of writhing movements made by each animal after the acetic acid injection was counted for 30 min immediately after acid administration. Results were expressed as mean number of writhes ± standard error of the mean (SEM) in 30 min for each experimental group. The median inhibitory dose was estimated on the basis of the dose-effect observed and used in other models.

Formalin-induced paw-licking test

Groups of mice (n = 10) were treated by gavage with 300 mg/kg EELS (solubilized in 10% DMSO), vehicle (10% DMSO, 10 mL/kg – control group), or 10 mg/kg indomethacin (positive control) 60 min before the administration of formalin 3% (formaldehyde 1.2% v/v) in the right hind paw, or s.c. with 5 mg/kg morphine (positive control) 30 min before the formalin application.[31] After the injection of the phlogistic agent, mice were individually placed in a transparent box with a mirror underneath to enable unhindered observation of the formalin-injected paw for 30 min. The duration of the reaction response (paw-licking time) was assessed in two periods. The first phase, from 0 to 5 min, corresponds to neurogenic pain caused by direct stimulation of the nociceptors, whereas in the second phase, between 15 and 30 min, the inflammatory pain is caused by the release of anti-inflammatory mediators.[3233] Pain reaction was defined as vigorous licking and biting of the formalin-injected paw. The duration of the response was measured in seconds.

Tail flick test

Groups of mice (n = 8) were treated p.o. with 300 mg/kg EELS (solubilized in 10% DMSO) or 10 mL/kg vehicle (10% DMSO, 10 mL/kg – control group), or s.c. with 10 mg/kg morphine (positive control). The distal third of the tail of each animal was exposed to thermal stimulation using an analgesymeter. The number of seconds before a tail flick was observed (latency period) was used as an index of nociception. The maximum allowed time of exposure to the stimulus was 20 s. Latency was measured – 30, 0, 30, 60, 120, 150, and 180 min from treatment.[34]

Anti-inflammatory activity

Central nervous system activities

Chimney test

Test groups of mice (n = 8) were treated p.o. with 100, 300, or 1000 mg/kg EELS (solubilized in 10% DMSO), vehicle (10% DMSO, 10 mL/kg – control group), or 5 mg/kg diazepam (positive control). After 60 min of treatment, animals were individually placed at a mark 10 cm from the end wall of a 25-cm-long glass tube with an inner diameter of 2.5 cm and in a horizontal position. Once the animals reached the opposite end, the tube was moved to a vertical position, and we recorded the amount of time for an animal moving backwards to reach the mark. The motor coordination of animals that did not perform this task within 90 s was considered impaired.[3940]

Pentobarbital-induced sleeping time

Groups of mice (n = 8) were treated p.o. with 100, 300, or 1000 mg/kg EELS (solubilized in 10% DMSO), vehicle (10% DMSO, 10 mL/kg – control), or 5 mg/kg diazepam (positive control for depressant effect). After 60 min, all animals were treated i.p. with 50 mg/kg sodium pentobarbital, a hypnosis inducer. The latency to sleep induction (loss of the righting reflex) and the duration of the hypnosis (time required to recover the righting reflex) were recorded for each animal.[41]

Statistical analysis

All results are expressed as means ± standard error of the mean (SEM). Data were analyzed statistically using either the Student-Newman-Keuls' to compare two means, or one-way analyses of variance (ANOVAs), followed by Tukey tests, when more than two means were involved.

Chemical analysis of the essential oil

The results of the quantitative and qualitative analyses of the essential oil, with volatile components listed in order of elution, are reported in [Table 1]. Total ion chromatograms of the essential oil can be seen in [Fig. 1].

Table 1

Chemical compounds (%) and retention indices (RI) of the essential oils of leaves of Lippia sidoides Cham. (Verbenaceae) from São Gonçalo do Abaeté, Minas Gerais, Brazil.

Figure 1

Total ion chromatogram of the chemical constituents of the essential oil from leaves of Lippia sidoides Cham. (Verbenaceae) from São Gonçalo do Abaeté, Minas Gerais, Brazil. (Arrows: major components).

The essential oil had a yield of 0.8%. Identified compounds were 22 in number (87.99% of the components), including 28.49% oxygenated monoterpenes, 27.10% monoterpene hydrocarbons and 32.40% sesquiterpene hydrocarbons. The major components were isoborneol (14.66%), bornyl acetate (11.86%), α-humulene (11.23%), α-fenchene (9.32%), and 1.8-cineole (7.05%) [Table 1].

Antimicrobial assays

The essential oil showed weak inhibitory activity (MIC = 500 µg/mL) against S. aureus 6538, S. aureus ATCC 25923, C. parapsilosis ATCC 22019, C. parapsilosis 86U, Cryptococcus sp. ATCC D and C. gatti L48, and was inactive toward the remaining microorganisms [Table 2].

Table 2

Minimum inhibitory concentration (μg/mL) of the essential oil, extracts and fractions from leaves of Lippia sidoides Cham. (Verbenaceae) from São Gonçalo do Abaeté, Minas Gerais, Brazil.

Moderate inhibitory activity (MIC = 125–250 µg/mL) was observed for EELS against Cryptococcus sp. ATCC D, C. gatti L48, C. neoformans L3 and C. parapsilosis 86U, and weak inhibitory activity against C. parapsilosis ATCC 22019 and C. albicans 63U (MIC = 250 µg/mL). Moderate antibacterial activity was observed against M. luteus ATCC9341 and S. aureus 6538, and the activity was weak or nonexistent for the other bacteria [Table 2].

The HF had good antifungal activity against Cryptococcus sp. ATCC D (MIC = 31.25 µg/mL) and C. gatti L48 (MIC = 62.5 µg/mL), and moderate inhibitory activity (MIC = 250 µg/mL) against C. neoformans L3, C. parapsilosis 86U, and C. parapsilosis ATCC 22019. This fraction had moderate inhibitory activity (MIC = 250 µg/mL) against Bacillus cereus ATCC 14579, S. aureus 6538, and S. aureus ATCC 25923, and weak inhibitory activity (MIC = 500 µg/mL) toward M. roseus 1740, M. luteus ATCC 9341, S. epidermidis ATCC 12229, E. aerogenes ATCC 13048, E. cloacae AMA FTA502, E. coli ATCC 11229, and P. aeruginosa ATCC 9027 [Table 2].

The DF had moderate inhibitory activity (MIC = 125–250 µg/mL) for all strains of Cryptococcus and weak activity (MIC = 500 µg/mL) against Candida. It had moderate inhibitory activity (MIC = 125–250 µg/mL) against Bacillus subtilis ATCC 6633, Micrococcus roseus 1740, M. luteus ATCC 9341, and the Gram-negative bacterium Enterobacter aerogenes ATCC 13048, and weak inhibitory activity (MIC = 500 µg/mL) toward S. aureus 6538, S. aureus ATCC 25923, E. cloacae AMA FTA502, and E. coli ATCC 11229. The EAF and AF were inactive against all bacterial and fungal strains [Table 2].

Pharmacological activities of the crude ethanol extract

Analgesic activity

For the acetic-acid-induced abdominal writhing test, the number of abdominal writhes in the groups treated with EELS at doses of 100, 300, or 1000 mg/kg (55.55 ± 6.28, 47.28 ± 9.89, and 42.25 ± 7.12, respectively) were significantly lower than for the control group (88.85 ± 5.90), but there was no significant difference in relation to indomethacin (51.00 ± 13.06) [Fig. 2].

Figure 2

Analgesic effect of the crude ethanolic extract from leaves of Lippia sidoides (EELS), as indicated by the number of acetic-acid-induced abdominal writhes in mice (n = 8). Indomethacin was used as a positive control. Bar heights and error bars express mean ± SEM of the number of writhing movements (%) in 30 min for each experimental group. ***P ≤ 0.001 versus the control group (vehicle).

In the first phase of the formalin test, the animals treated with vehicle had a paw-licking of 62.90 ± 3.97 s. EELS (300 mg/kg) or morphine (5 mg/kg) led a significant reduction of time to reactivity to pain to 46.77 ± 2.78 s and 1.16 ± 0.79 s, respectively, whereas indomethacin (10 mg/kg) did not alter the reaction time (52.45 ± 5.14 s). During the second phase of the test, paw-licking time was decreased by EELS from 205.20 ± 15.67 s (control group) to 50.66 ± 4.08 s, by indomethacin to 105.16 ± 11.69 s, and by morphine to 0 ± 0.00 s [Fig. 3].

Figure 3

Effect of the ethanolic extract from leaves of Lippia sidoides (EELS) on formalin-induced paw-licking test in mice (n = 8) during the neurogenic (1st phase; 0–5 min) and inflammatory phases (2nd phase; 15-30 min). Bar heights and error bars express means ± SEM of paw-licking time, in seconds. *P ≤ 0.05 and ***P ≤ 0.001 versus the control group (vehicle).

There was no significant difference in the latency to thermal stimulus in the tail of animals treated with EELS 300 mg/kg and those belonging to the control group. Treatment with 10 mg/kg of morphine increased latency to 30 min after the treatment [Fig. 4].

Figure 4

Effect of the ethanolic extract from leaves of Lippia sidoides (EELS) on pain reaction time in mice (n = 8). Symbols and error bars indicate means and SEM, respectively, expressed in seconds relative to the control group. Morphine (10 mg/kg) was used as a positive control. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 versus the control group (vehicle).

Evaluation of anti-inflammatory activity

In the croton oil-induced ear edema test, 300 mg/kg EELS or 2 mg/kg dexamethasone reduced the formation of ear edema from 15.99 ± 1.56 to 6.85 ± 0.85 and 2.02 ± 0.53, respectively [Fig. 5].

Figure 5

Effect of the ethanol extract from leaves of Lippia sidoides (EELS) on croton oil-induced ear edema in mice (n = 10). Bar heights and error bars show means ± SEM of differences in weight between right and left ear plugs. Dexamethasone (2 mg/kg) was used as a positive control. ***P ≤ 0.001 versus the control group (vehicle).

Central nervous system activities

In open field test, treatment with EELS (100, 300, or 1000 mg/kg) did not affect the number of squares crossed, squares invaded in the central region, time spent in the peripheral and central region, and rearing, parameters that were modified by diazepam at a dose of 5 mg/kg [Table 3].

Table 3

Effects of the ethanol extract from leaves of Lippia sidoides (EELS) in the open field test with mice (n = 8). Diazepam was used as a positive control.

In the chimney test, treatment with EELS (100, 300, or 1000 mg/kg) did not increase time spent by animals moving backwards to reach the chimney mark when compared to the control group. Treatment with diazepam (5 mg/kg) increased this time significantly in relation to the control group [Table 4].

Table 4

Effects of treatment with the ethanol extract of leaves of Lippia sidoides (EELS) on the chimney test and on the pentobarbital-induced sleeping time test using mice (n = 8). Diazepam was used as a positive control.

In the pentobarbital-induced sleeping time test, treatment with EELS (100, 300, or 1000 mg/kg) did not alter sleep latency or sleeping time induced by pentobarbital. Animals treated with diazepam (5 mg/kg), used as a positive control for this test, had decreased sleep latency and increased sleep time [Table 4].

Financial support and sponsorship

Nil

Conflict of interest

There is no conflict of interest.