About this Journal Submit a Manuscript Table of Contents
Evidence-Based Complementary and Alternative Medicine
Volume 2012 (2012), Article ID 420715, 15 pages
http://dx.doi.org/10.1155/2012/420715
Research Article

Phytochemical Analysis and Antimicrobial, Antinociceptive, and Anti-Inflammatory Activities of Two Chemotypes of Pimenta pseudocaryophyllus (Myrtaceae)

1Unit of Exact and Technologic Sciences, Goiás State University, Anápolis, Brazil
2Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Brazil
3Faculty of Pharmacy, Federal University of Goiás, Goiânia, Brazil
4Department of Physiological Sciences, Institute of Biological Sciences, Federal University of Goiás, Goiânia, Brazil

Received 9 June 2012; Accepted 4 August 2012

Academic Editor: Vincenzo De Feo

Copyright © 2012 Joelma Abadia Marciano de Paula et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Preparations from Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) have been widely used in Brazilian folk medicine. This study aims to evaluate the antimicrobial activity of the crude ethanol extracts, fractions, semipurified substances, and essential oils obtained from leaves of two chemotypes of P. pseudocaryophyllus and to perform the antinociceptive and anti-inflammatory screening. The ethanol extracts were purified by column chromatography and main compounds were spectrally characterised (1D and 2D 1H and 13C NMR). The essential oils constituents were identified by GC/MS. The broth microdilution method was used for testing the antimicrobial activity. The abdominal contortions induced by acetic acid and the ear oedema induced by croton oil were used for screening of antinociceptive and anti-inflammatory activities, respectively. The phytochemical analysis resulted in the isolation of pentacyclic triterpenes, flavonoids, and phenol acids. The oleanolic acid showed the best profile of antibacterial activity for Gram-positive bacteria ( ), followed by the essential oil of the citral chemotype ( ). Among the semipurified substances, Ppm5, which contained gallic acid, was the most active for Candida spp. ( ) and Cryptococcus spp. ( ). The crude ethanol extract and fractions from citral chemotype showed antinociceptive and anti-inflammatory effects.

1. Introduction

Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) is a plant popularly known in Brazil as pau-cravo, louro-cravo, louro, craveiro, craveiro-do-mato, chá-de-bugre, and cataia [16]. In folk medicine, the leaves have been used to produce a refreshing drink with calming, diuretic, and aphrodisiac properties, as well as to treat colds and their complications and digestive and menstrual problems [2, 46]. It is the only Pimenta species native to Brazil [1, 3], and recent studies have shown the occurrence of different chemotypes for this species; these are characterised, for example, by the predominance of citral or ( )-methyl isoeugenol in the essential oils [7].

Invasive infections caused by Candida spp. and Cryptococcus neoformans have increased significantly in recent years [811]. The cause of this rise is often related to immunodeficiency associated with transplantation [11] and acquired immunodeficiency syndrome (AIDS) [9], as well as the use of intravascular catheters [10], dialysis, and abusive use of glucocorticoids and broad-spectrum antibiotics [8]. The drugs available to treat these infections are often not selective, are toxic, or have narrow action spectra [12]; moreover, some species are resistant to antifungal agents [13].

In the pharmacotherapy of bacterial diseases, the use of antibiotics in recent decades has significantly reduced the incidence of many infectious diseases. On the other hand, the severe side effects from many of these substances and the emergence of multiresistant microorganisms have stimulated research on the development of new antibacterial agents that are more specific, effective, and safe [24, 25].

There is thus a consensus on the need for further research on new alternative treatments for bacterial and fungal infections. Efforts have been focused on investigating the antimicrobial properties of products from plants [12, 25]. In addition to extensive use in folk medicine in diseases related to the common cold, which often involve microbial and/or inflammatory processes, experimental data show that plants of the genus Pimenta (Myrtaceae) have antimicrobial potential [26, 27]. The essential oil of P. pseudocaryophyllus leaves collected in two geographical areas in the state of São Paulo was active against strains of C. albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus [4]. In a previous study, we described the antimicrobial activity of the crude ethanol extract of leaves of this species collected in the Brazilian Cerrado against Gram-positive bacteria and Candida albicans [28], but we have not performed phytochemical studies for the isolation and identification of substances accountable for this activity.

Moreover, scientific studies have shown that Pimenta species, widely used in folk medicine, have analgesic and anti-inflammatory activities and are nontoxic in typical dosages [2932]. Thus, the aims of this work were to carry out the phytochemical study, evaluate the antimicrobial activity of the crude ethanol extracts, fractions, semipurified substances, and essential oils obtained from leaves of two chemotypes of P. pseudocaryophyllus, and perform the screening of the antinociceptive and anti-inflammatory activities.

2. Material and Methods

2.1. General Experimental Procedures

The 1H and 13C one-dimensional and two-dimensional NMR spectra (Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple Bond Correlation (HMBC)) were obtained using a Bruker Avance III-500 spectrometer running at 500 MHz (1H) and 125 MHz (13C), using deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (DMSO-d6) as solvents for nonpolar and polar samples, respectively. Tetramethylsilane (TMS) was used as an internal reference standard for chemical shifts ( , ppm), and in some cases, the peak from the solvent was used for reference.

Essential oil samples were analysed by GC/MS using a QP5050A instrument (Shimadzu, Kyoto, Japan) with a capillary column of CBP-5 melted silica (30 m × 0.25 mm × 0.25  m; 5% phenyl-methylpolysiloxane film) (Shimadzu, Kyoto, Japan). Additionally, helium was used with a flow rate of 1 mL min−1 as a carrier gas, and a thermal profile of 60°C to 240°C with a gradient of 3°C min−1 followed by a gradient of 10°C min−1 up to 280°C, keeping a 5 min isotherm, was used. The ionisation energy of the detector was kept at 70 eV, and the sample injection volume following dilution in hexane ( 10%) was 0.5  L. The analysis was carried out in the scanning mode, with a 40–400 m/z mass interval and 1 : 5 injection ratio. The quantitative analysis was obtained by integrating the total ion chromatogram (TIC). The identification of the components was performed by comparing the mass and retention indices (RI) calculated using values for the mass and retention indices available in the literature [33]. The retention indices were calculated by coinjection with a mixture of hydrocarbons, C8–C32 (Sigma, MO, USA), applying the Van Den Dool and Kratz equation [34].

The mass spectra of the flavonoids were collected using a coupled LC/EM/EM: Varian 1200L (Walnut Creek, CA, USA) system with a quadrupole ion analyser and ionisation through electron impact, 70 eV in positive mode m/z [M + H]+. The m/z scanning spectrum was 100–900, and the ionisation chamber was kept at room temperature.

The HPLC was held on Waters equipment (MA, USA) equipped with quaternary pump, e2695 separation module, 2998 diode array detector (PDA), and Empower 2.0 data processing system. The Varian C-18 (250 × 4.6 mm) column was used at room temperature. The detection system used was monitored at 360 nm for detection of flavonoids. The injection volume was of 30  L and the run in isocratic mode was used as mobile phase 75% of A [acetonitrile/water (0.1% acetic acid)—90 : 10] and 25% of B [MeOH (0.1% acetic acid)] at a constant flow of 1 mL min−1. The maximal running time was 20 min. Quercitrin (Sigma) was used as reference standard. The samples were previously filtered through Millex (Millipore, MA, USA) membrane and the mobile phase in 0.45  m PVDF membrane (Millipore).

For analytical and preparative thin-layer chromatography (ATLC, PTLC), chromatography plates prepared with G60 F254 Vetec silica gel, or F254 silica plates manufactured by Merck, RJ, Brazil, were used. Mixtures of organic solvents with the proper polarities for each analysed fraction were used as mobile phases. For detection of the components, the plates were observed under UV light at 254 nm and 365 nm and developed by the spraying of sulphuric vanillin followed by heating or exposure to a solution of 1% ethylamine diphenyl borate in methanol (NP) [35].

The column chromatography used silica gel G60 0.05–0.2 mm (Vetec, Brazil) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) as stationary phases. As mobile phases, mixtures of organic solvents in increasing order of polarity were used, according to the polarity profiles of the fractions undergoing the process.

2.2. Plant Material

The leaves of P. pseudocaryophyllus of the (E)-methyl isoeugenol and citral chemotypes were collected in São Gonçalo do Abaeté, MG, Brazil, in February 2008, at 18°20′58.4′′S, 45°55′23.4′′W, and 864 m altitude. These two chemotypes are the most common found in the region of São Gonçalo do Abaeté and therefore were selected for this study.

The plant material was identified by Professor Carolyn Elinore Barnes Proença, Ph.D., of the Universidade de Brasília, and a voucher specimen (UFG-27159) was deposited at the herbarium of the Universidade Federal de Goiás.

2.3. Preparation of Extracts and Fractions

Air-dried leaves were ground in a knife mill. The crude ethanol extract was obtained by maceration of the powdered material (1 : 5 w/v) of each chemotype in 95% EtOH (v/v) at room temperature, followed by filtration, and concentration on a rotary evaporator at a temperature below 40°C. The extracts were concentrated to a constant weight and named crude ethanol extract of (E)-methyl isoeugenol chemotype (EEM) and crude ethanol extract of citral chemotype (EEC).

Fifty grams of each extract (EEM and EEC) were dissolved in 250 mL MeOH/H2O (7 : 3) and subjected to liquid/liquid partitioning with solvents of increasing polarity (hexane, dichloromethane, and ethyl acetate). The final MeOH/H2O residual was concentrated in a rotary evaporator for elimination of the MeOH, and the resulting aqueous fraction was lyophilised. Therefore, four fractions were obtained from the crude extract of each chemotype: the hexane fraction (HFM and HFC), the dichloromethane fraction (DFM and DFC), the ethyl acetate fraction (EAFM and EAFC), and the aqueous fraction (AFM and AFC).

For obtaining of essential oils, leaves from each chemotype were subjected to hydrodistillation for 3 h in a Clevenger apparatus. The essential oils were dried with anhydrous Na2SO4, packaged in amber glass vials, and stored at −20°C until use. They were named essential oil of the citral chemotype (EOC) and essential oil of the (E)-methyl isoeugenol chemotype (EOM).

2.4. Isolation of the Chemical Constituents

HFC
The HFC (2.5 g) was fractionated on a silica gel column (1 : 40) eluted with a gradient of hexane-EtOAc (5–100%), EtOAc-MeOH (1 : 1), and MeOH (100%). Seventy-five 20 mL fractions were collected and evaluated by TLC [hexane-EtOAc mixtures (10–30%)], observed under 254/365 nm UV light, and detected with sulphuric vanillin reagent. Similar fractions were pooled, resulting in 14 new fractions (CH-1 to CH-14). Fractions CH-8 through CH-11 were rechromatographed on a silica gel column eluted isocratically with CH2Cl2-petroleum ether (7 : 3), resulting in Ppc-1 (133.5 mg).

DFC
The DFC (1.0 g) was fractionated on a silica gel column (1 : 40) and eluted with a gradient of CH2Cl2-petroleum ether (7 : 3 and 9 : 1), CH2Cl2-EtOAc (5–100%), EtOAc-MeOH (1 : 1), and MeOH (100%). One hundred and one 5 mL fractions were collected. Using TLC [mobile phases composed of mixtures of CH2Cl2-EtOAc (75 : 25, 80 : 20, and 95 : 5)] with 254/365 nm UV light, and sulphuric vanillin reagent for detection, the fractions were combined into 20 new fractions (CD-1 to CD-20). Fractions CD-9, CD-10, and CD-11 were fractionated on a silica gel column eluted isocratically with petroleum ether-EtOAc (90 : 10). The semipurified fractions were rechromatographed on a silica gel column eluted isocratically with petroleum ether-acetone (40 : 10), resulting in Ppc-2 (22.6 mg).

EAFC
In a column packed with Sephadex LH-20, 1.5 g of EAFC was subjected to chromatography, using MeOH (100%) as the mobile phase. Two hundred and sixty-one 10–20 mL fractions were collected. The fractions were evaluated by TLC [EtOAc-formic acid-acetic acid-H2O (100 : 11 : 11 : 26)], observed under 254/365 nm UV light and developed with sulphuric vanillin and NP reagents. Similar fractions were pooled, resulting in 12 new fractions (CEA-1 to CEA-12).
CEA-12 showed a single spot at close to 1, resulting in Ppc-3 (59.7 mg). Fractions CEA-7, CEA-8, and CEA-9 showed typical spots of flavonoids with similar values, so they were gathered and rechromatographed on a Sephadex LH-20 column eluted isocratically with MeOH-H2O (1 : 1). From this column, 175 fractions were collected and lyophilised after MeOH evaporation, evaluated by TLC, and grouped into 17 fractions (CEA-A to CEA-Q). The CEA-N fraction was subjected to preparative TLC [EtOAc-formic acid-acetic acid-H2O (100 : 11 : 11 : 26)], resulting in Ppc-4 (18.7 mg). CEA-K, CEA-L, and CEA-M were gathered and subjected to preparative TLC, and the semipurified fraction was rechromatographed on a Sephadex LH-20 column eluted with a gradient of acetone-H2O (1 : 1, 6 : 4, and 7 : 3). From this column, 93 1 mL fractions were collected and subjected to solvent evaporation, lyophilisation, and monitoring by TLC, which led to Ppc-5 (73.2 mg).

AFC
The AFC (1.2 g) was chromatographed on packed column with Sephadex LH-20 eluted with MeOH (100%). Approximately 180 fractions of 1–10 mL were collected. The fractions were evaluated by TLC [isobutanol-acetic acid-water (14 : 1 : 5)] and exposed with UV light and 1% FeCl3 in 0.5 M hydrochloric acid. Fractions with the same chromatographic profiles were pooled, resulting in 11 new fractions (AC-1 to AC-11). AC-9 was reactive to FeCl3, resulting in Ppc-6 (83.1 mg).

HFM
The HFM (2.5 g) was fractionated on a silica gel column (1 : 40) and eluted with 100% hexane-EtOAc (5–100%), EtOAc-MeOH (1 : 1), and MeOH (100%). One hundred 10 mL fractions were collected and evaluated by TLC [hexane- EtOAc (10–30%)], observed under UV light, and detected with sulphuric vanillin reagent, resulting in 14 new fractions (MH-1 to MH-14). Fractions MH-4 and MH-5 were rechromatographed on a silica gel column eluted isocratically with CH2Cl2-petroleum ether (7 : 3), resulting in Ppm-1 (106.2 mg) and Ppm-2 (75.8 mg). Fractions MH-9 and MH-10 were gathered and subjected to isocratic silica gel column chromatography [hexane-CH2Cl2-MeOH (10 : 10 : 1)], which resulted in Ppm-3 (36.9 mg).

DFM
The low output from DFM hindered the use of chromatography columns in isolating possible compounds. Thus, TLC of the DFM was carried out with some of the semipurified fractions for comparison to verify whether some of the previously isolated compounds could also be found in this fraction. Runs were performed with DFM, Ppc-1, Ppm-1, and Ppm-2 in CH2Cl2-petroleum ether (7 : 3), DFM and Ppc-2 in petroleum ether-acetone (40 : 10), and DFM and Ppm-3 in hexane-CH2Cl2-MeOH (10 : 10 : 1).

EAFM
The EAFM (1.2 g) was subjected to chromatography on a packed column with Sephadex LH-20 [MeOH (100%)]. Approximately 159 1-mL fractions were collected and analysed by TLC [EtOAc-formic acid-acetic acid-H2O (100 : 11 : 11 : 26)]. Based on observation in UV light and detection with NP reagent, the fractions were pooled into four new fractions (MEA-1 through MEA-4). Fractions MEA-1 and MEA-2 showed typical spots of flavonoids with yellow-orange and greenish-yellow fluorescence after spraying with NP and observation under 365 nm UV light, with similar values; thus, they were pooled and rechromatographed on a Sephadex LH-20 column eluted with a sequence of acetone-H2O mixtures (1 : 1, 6 : 4, and 7 : 3). From this column, 99 fractions were collected and, after evaluation by TLC, resulted in Ppm-4 (273.4 mg).

AFM
The AFM (1.2 g) was subjected to chromatography in a packed column with Sephadex LH-20 [MeOH (100%)]. Approximately 117 1-mL fractions were collected. The fractions were monitored by TLC [isobutanol-acid acetic-water (14 : 1 : 5)] with use of the UV light and 1% FeCl3 in 0.5 M hydrochloric acid. Fractions with the same chromatographic profiles were pooled, resulting in nine new fractions, and one of the most reactive to the 1% FeCl3 was named Ppm-5 (203.6 mg).

2.5. Antimicrobial Activity

The extracts, fractions, essential oils, citral (Sigma), and semipurified substances were subjected to the microdilution test in broth for determining the minimum inhibitory concentration (MIC) in sterile 96-well microplates with “U-”shaped wells, as recommended by the Clinical and Laboratory Standards Institute (CLSI) [36, 37]. The experiments were performed in duplicate and repeated twice, independently, except for the semipurified substances.

American Type Culture Collection (ATCC) standard strains and clinical isolates were used (Table 1); these are kept in the Laboratories for Bacteriology and Mycology at the Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, GO, Brazil.

tab1
Table 1: Microorganisms used in determining the minimum inhibitory concentration (MIC).

Prior to testing, to reactivate the microbial cultures, the bacteria were transferred to Casoy broth and incubated at 37°C for 24 h, then transferred to inclined Casoy agar, and incubated at 37°C for an additional 24 h. The fungi were transferred to Sabouraud dextrose agar and incubated at room temperature for 24 to 48 h (Candida spp.) or 48 to 72 h (Cryptococcus spp.).

The culture medium used in the antibacterial activity test was 2x Müeller Hinton broth (MH) and that used in the antifungal activity test was RPMI 1640. The samples were solubilised in 10% dimethyl sulfoxide (DMSO) and diluted in MH broth (antibacterial activity) to obtain a concentration of 2000  g mL−1 or in RPMI (antifungal activity) to obtain a concentration of 1000  g mL−1. The semipurified substances were used at concentrations according to the available amount of the substance. For the preparation of samples with essential oils, 0.02% Tween 80 was added. Vancomycin (Sigma; 32  g mL−1) and gentamicin (Sigma; 128  g mL−1) were used as controls for Gram-positive and Gram-negative bacteria, respectively, and itraconazole (Sigma) at an initial concentration of 16  g mL−1 was used as a control for fungi.

The microplates inoculated with bacteria were incubated at 35°C 2°C for 16–20 h and for 24 hours at this temperature for Staphylococcus. One hour before the end of the incubation period, each well received 20  L of 0.5% triphenyl tetrazolium chloride (TTC), and the microplates were reincubated for approximately 30 min. The appearance of reddish colour was considered as proof of bacterial growth. The microplates inoculated with fungi were incubated at room temperature for 48 h (Candida spp.) and 72 h (Cryptococcus spp.). Fungal growth was checked visually, and the MIC was defined as the lowest concentration ( g mL−1) of the sample fully capable of inhibiting bacterial and fungal growth.

2.6. Antinociceptive and Anti-Inflammatory Activity

These experiments were approved by the Animal Ethics Committee of the Federal University of Goiás, Protocol number 146/2008. Male, young adult mice, weighing between 25 and 30 g, were transferred to the experimental room two days before the tests, kept in a light/dark cycle of 12 h at 22 2°C in a noise-free facility, with water and food ad libitum. The food was taken away 12 h before the test, keeping the water available ad libitum at all times.

2.6.1. Evaluation of Antinociceptive Activity by Testing Abdominal Contortions Induced by Acetic Acid

The evaluation of abdominal contortions induced by acetic acid was carried out according to Koster et al. [38] and Lapa et al. [39]. Test groups consisting of 10 mice per dose of extract, fraction, standard drug or vehicle were used. The mice from the different experimental groups received intraperitoneal injections of 1.2% acetic acid solution (v/v, 10 mL kg−1) 1 h after oral intake (gavage) of the extracts or fractions (in different doses) or indomethacin (standard drug) or vehicle (control). They were then placed under glass funnels and the contortions, contractions, and rotation of the abdomen followed by the extension of one or both back paws were counted over the subsequent 30 min.

The extracts, fractions, and essential oils were tested in the following doses: EEM and EEC—2000, 1000, and 500 mg kg−1; FHC—400 mg kg−1; DFC—240 mg kg−1; EAFC—1160 mg kg−1; AFC—480 mg kg−1; EOC—60, 200, 600 mg kg−1. Indomethacin was used at a dose of 10 mg kg−1. The control groups received the vehicle used in the solubilisation of each extract, fraction, or essential oil (10 mL kg−1), so there were three control groups that received either 10% propylene glycol in CMC gel, 10% DMSO in water, or only water.

2.6.2. Anti-Inflammatory Activity by Testing Ear Oedema Induced by Croton Oil

Tests of ear oedema induced by croton oil were carried out according to Zanini et al. [40]. Groups of mice ( ) were treated (v.o.) with dexamethasone (2.0 mg kg−1), vehicle (10% propylene glycol in CMC gel, 10 mL kg−1), or EEC (2000, 1000, and 500 mg kg−1). One hour after treatment, each animal received 20  L of a freshly prepared solution of croton oil (2.5% v/v) in acetone on the surface of the right ear. The left ear received the same volume of acetone. After 4 h, the animals were sacrificed and identical segments were taken from both ears. The formations and intensities of the oedema were expressed as the mean of differences in weight between the segments of the animals’ ears: the smaller the weight difference, the greater the potential for inhibition.

2.7. Statistical Analysis

The antinociceptive effects of the different extracts, fractions, and essential oils were expressed as the means ( SEM) and are shown as percentages relative to the control group (vehicle). The significant differences between treated and control groups (vehicle) were assessed by an ANOVA and a multiple comparison by Tukey-Kramer. values 0.05 were considered statistically significant. The data analysis used the application GraphPad Prism 3.0.

3. Results and Discussion

3.1. Phytochemical Analysis

The phytochemical analysis of the fractions obtained from the crude ethanol extracts of P. pseudocaryophyllus leaves of the citral and (E)-methyl isoeugenol chemotypes enabled the identification of pentacyclic triterpenes (lupeol, -amyrin, -amyrin, oleanolic acid, betulinic acid, and ursolic acid), flavonoids (quercetin 3-O- -L-rhamnopyranoside, quercetin 3-O- -glucopyranoside, kaempferol 3-O- -L-rhamnopyranoside, quercetin 3-O- -arabinofuranoside, quercetin 3-O- -arabinopyranoside, quercetin 3-O- -arabinopyranoside, and catechin), gallic acid, and ellagic acid (Table 2). These constituents, except ursolic acid, were found for the first time in this species. They were identified based on spectra of 1D and 2D 1H and 13C NMR (HSQC and HMBC) and with comparison with data in the literature (copies of the original spectra can be obtained from the corresponding author). The flavonoid quercitrin was also identified based on the mass spectrum and analysis by HPLC in comparison with an authentic sample.

tab2
Table 2: Substances identified in Pimenta pseudocaryophyllus citral and (E)-methyl isoeugenol chemotypes.

The results from the qualitative and quantitative analysis of volatile oils of P. pseudocaryophyllus, with the volatile constituents listed in order of elution, are found in Table 3. A total of 31 compounds were identified, accounting for 94–100% of the volatile components. There was a predominance of phenylpropanoid derivatives (97.5%) among the volatile components of the (E)-methyl isoeugenol chemotype, and almost all of the oil consisted of (E)-methyl isoeugenol (93.9%). The essential oil of the citral chemotype consisted mainly of oxygenated mono- and sesquiterpenes (69.65% and 13.7%, resp.), and the monoterpene aldehydes neral and geranial, which are referred to as citral when their isomers are mixed, were the major components (27.59% and 36.49%, resp.).

tab3
Table 3: Percentage chemical composition of essential oils from leaves of P. pseudocaryophyllus ( )-methyl isoeugenol and citral chemotypes.
3.2. Antimicrobial Activity

The MIC values of extracts, fractions, and essential oils, as well as citral (Sigma), are described in Table 4. Table 5 shows the MIC of the semipurified substances and of vancomycin, gentamicin, and itraconazole, which were used as controls. The results were discussed taking into account the classification by Holetz et al. [41], which has also been adopted by other authors [4244], where a MIC lower than 100  g mL−1 represents good antimicrobial activity; a MIC from 100 to 500  g mL−1 represents moderate antimicrobial activity; a MIC from 500 to 1000  g mL−1 represents weak activity; a MIC above 1000  g mL−1 suggests that the substance is inactive. EEC showed the lower MIC compared to EEM for Gram-positive bacteria, and both were inactive for Gram-negative bacteria (Table 4). EEC was the most active for fungal strains, with a MIC between 7.8 and 62.5  g mL−1. The lowest MIC of this extract was found for the strains of Cryptococcus (MIC = 7.8–15.6  g mL−1), while EEM showed good-to-moderate activity (MIC = 15.6–125  g mL−1) (Table 4) for the yeasts studied.

tab4
Table 4: Minimum inhibitory concentration ( g mL−1) of extracts, fractions, and essential oils from leaves of P. pseudocaryophyllus ( )-methyl isoeugenol and citral chemotypes, and of the citral substance.
tab5
Table 5: Minimum inhibitory concentration ( g mL−1) of semipurified substances of P. pseudocaryophyllus.

Regarding the fractions and semipurified substances, the dichloromethanic fractions of both chemotypes (DFM and DFC) were the most active for Gram-positive bacteria (MIC = 250–500  g mL−1) and were less active for the fungal strains (MIC 500  g mL−1) (Table 4). The antibacterial activity of DFC is due to substance Ppc-2, which had the greatest inhibitory effect on Gram-positive bacteria of the compounds studied (MIC = 31.2  g mL−1 for S. aureus ATCC 6538, S. epidermidis ATCC 12229, and B. cereus ATCC 14579; MIC = 62.5  g mL−1 for S. aureus ATCC 25923, M. roseus ATCC 1740, and B. atrophaeus ATCC 6633; MIC = 125  g mL−1 for M. luteus ATCC 9341) (Table 5). The phytochemical analysis showed that Ppc-2 is oleanolic acid, a pentacyclic triterpene with antibacterial activity [4547], which was especially effective against Gram-positive bacteria. The TLC analysis of the DFM showed the presence of oleanolic, betulinic, and ursolic acids, which probably contributed to the observed antibacterial activity of this fraction [4547]. Ppm-4, isolated from EAFM and composed of quercitrin, afzelin, isoquercitrin, avicularin, guaijaverin, catechin, and gallic acid, showed activity against C. neoformans L2 and L1 (CIM = 62.5  g mL−1) but was not active against Candida (Table 5). Studies have shown that catechin and quercitrin, the main components of Ppm-4, inhibit the growth of some pathogenic fungi [4850]. Ppm-5, obtained from the AFM, showed an activity profile similar to this fraction, except for C. parapsilosis 11A (Table 5). The phytochemical analysis carried out on Ppm-5 verified the presence of gallic acid and its derivatives, which are known for their antimicrobial activities [5153], as well as the presence of sugars. The Ppc-4 isolated from EAFC, which was composed of quercitrin, showed moderate inhibition of Candida strains (MIC = 250  g mL−1) and good-to-moderate inhibition of the strains of Cryptococcus (MIC = 62.5 to 250  g mL−1) (Table 5), partly contributing to the antifungal activity of this fraction. Ppc-5, which was also isolated from fraction EAFC, consisted of a mixture of flavonoids of which the major component is quercitrin and did not show activity under the conditions tested. Ppc-6 contributed in part to the antifungal activity of AFC, with MICs ranging from 62.5 to 125  g mL−1 for Candida strains (Table 5). The phytochemical analysis of Ppc-6 showed ellagic acid to be the major component. Similar results for Candida spp., due to ellagic acid, were reported by Silva et al. [54]. These findings are promising in the search for new options against infections caused by Candida spp. and Cryptococcus spp., particularly with the continuous increase of opportunistic fungal resistance to available treatments, the emergence of rare species of Candida [55], and the increasing number of infections by Cryptococcus neoformans and C. gattii [56].

The essential oil of the citral chemotype (EOC) showed good activity (MIC = 62.5 μg mL−1) against B. cereus ATCC 14579 and moderate activity (MIC = 125–250 mL g  g mL−1) against the remaining Gram-positive bacteria; in some cases, this essential oil showed better inhibition than citral (MIC = 125–250  g mL−1) (Table 4). Citral, the major component of the EOC, is a monoterpene aldehyde mixture of the isomers neral and geranial. Aldehydes, such as formaldehyde and glutaraldehyde, are known to have strong antimicrobial activity, and several researchers have demonstrated the antimicrobial effect of citral [5762]. It is suggested that the aldehyde group conjugated to the carbon-carbon double bond, which is present in neral and geranial, provides a highly electronegative chemical structure that may explain its activity [57]. The EOM was inactive against bacteria under the conditions tested. Its major component, the phenylpropanoid (E)-methyl isoeugenol, comprises almost all of the oil (93.9%). Structurally, (E)-methyl isoeugenol differs from eugenol because it has a methoxyl group at the C1 position of the ring instead of a hydroxyl group, and the double bond in its propenyl group is in a different position. The absence of the hydroxyl group in the phenolic structure of (E)-methyl isoeugenol may have contributed to the lack of activity of this compound. Griffin et al. [63] observed that the methylation of the hydroxyl group in eugenol, producing methyleugenol, resulted in a loss of activity for Gram-negative bacteria.

The EOC showed good activity against Cryptococcus (MIC = 15.6  g mL−1 to 62.5  g mL−1), but the EOM showed moderate activity against strains of Cryptococcus (MIC = 125  g mL−1 to 250  g mL−1) and showed no activity for species of Candida obtained from clinical isolates (MIC 500  g mL−1) (Table 4). The structure-activity relationship of the components of certain essential oils has been researched in phytopathogenic fungi, and there is little information on fungal pathogens in humans. For example, carbonyl , -unsaturated compounds, such as the monoterpenic aldehydes neral and geranial, have strong antifungal activity [27, 64, 65], similar to their antibacterial activity. Little information regarding the antifungal mechanism of action of these aldehydes is available. The evaluation of the effects of citral on the membranes, organelles, and intracellular macromolecules of Aspergillus flavus spores has shown that citral damages the cell walls and membranes of spores, reducing their elasticity [66].

3.3. Antinociceptive and Anti-Inflammatory Screening

The EEM (v.o. 2000, 1000, and 500 mg kg−1) significantly reduced the number of abdominal contortions caused by the intraperitoneal injection of acetic acid in mice compared to animals that received only vehicle (control group) (Figure 1). There was no significant difference observed between the analgesic effects of the three doses used (data not shown).

420715.fig.001
Figure 1: EEM antinociceptive effect in the abdominal contortion model induced by acetic acid (1.2% v/v, i.p.) in mice previously treated with EEM (2000, 1000, and 500 mg kg−1, v.o). Vehicle (CMC + propylene glycol) 10 mL kg−1, v.o. Indomethacin was used as positive control (10 mg kg−1, v.o). Each column shows the mean ( SEM) of number of contortions in percentages relative to control (vehicle). *Indicates the significance level compared to the control group (vehicle). mice. * , *** .

The EEC (v.o. 2000 and 1000 mg kg−1) was able to inhibit, significantly and in a dose-dependent manner, the abdominal contortions in mice induced by the intraperitoneal acetic acid compared with the control group and showed no significant difference compared to the group of animals treated with indomethacin (10 mg kg−1) (Figure 2).

420715.fig.002
Figure 2: EEC antinociceptive effect in the abdominal contortion model induced by acetic acid (1.2% v/v, i.p.) in mice previously treated with EEC (2000, 1000, and 500 mg kg−1, v.o). Vehicle (CMC + propylene glycol) 10 mL kg−1, v.o. Indomethacin was used as positive control (10 mg kg−1, v.o). Each column shows the mean ( SEM) of number of contortions in percentages relative to control (vehicle). *Indicates the significance level compared to the control group (vehicle). mice. * , ** , *** .

Given the dose-dependent antinociceptive effect shown by EEC (Figure 2), this extract was selected to perform the screening of the antinociceptive activity of its fractions and essential oils, as well as to evaluate its anti-inflammatory activity by testing ear oedema induced by croton oil. HFC (400 mg kg−1), DFC (240 mg kg−1), and EAFC (1160 mg kg−1) significantly decreased the number of abdominal contortions induced by the intraperitoneal injection acetic acid compared with the control group. However, there was no significant difference in the antinociceptive effect shown by the three fractions. The group of animals that received AFC (480 mg kg−1) showed a number of contortions similar to that of the group that received vehicle (Figure 3).

420715.fig.003
Figure 3: Antinociceptive effect of fractions in the abdominal contortion model induced by acetic acid (1.2% v/v, i.p.) in mice previously treated with HFC (400 mg kg−1, v.o), DFC (240 mg kg−1, v.o), EAFC (1160 mg kg−1, v.o), and AFC (480 mg kg−1, v.o). Vehicle (water + DMSO 10%) 10 mL kg−1, v.o. Each column shows the mean ( SEM) of number of contortions in percentages relative to control (vehicle). *Indicates the significance level compared to the control group (vehicle). mice. * , ** , *** .

The EOC at doses of 60, 200, and 600 mg kg−1 (v.o.) showed significant dose-dependent inhibitory effects on the abdominal contortions induced by intraperitoneal acetic acid in mice compared to the control group (Figure 4).

420715.fig.004
Figure 4: EOC antinociceptive effect of abdominal contortion model induced by acetic acid (1.2% v/v, i.p.) in mice previously treated with EOC (600, 200, and 60 mg kg−1, v.o). Vehicle (water + DMSO 10%) 10 mL kg−1, v.o. Each column shows the mean ( SEM) of number of contortions in percentages relative to control (vehicle). *Indicates the significance level compared to the control group (vehicle). mice. * , *** .

The ear oedema induction test showed that the EEC has antiedematogenic activity that is significantly greater than that of the control at all doses tested (Figure 5). Pretreatment with EEC (2000, 1000, and 500 mg kg−1) reduced the oedema from 14.3 0.4 mg (control) to 10.9 0.5, 11.3 0.4, and 10.5 0.5 mg, respectively.

420715.fig.005
Figure 5: EEC anti-inflammatory effect under ear oedema induced by croton oil (2.5% v/v) in mice previously treated with EEC (2000, 1000, and 500 kg−1, v.o). Control (CMC + propylene glycol) 10 mL kg−1, v.o. Dexamethasone was used as positive control (2 mg kg−1, v.o). Each column expresses the mean ( SEM) of the difference of the weights of ear segments in percentages relative to control (vehicle). *Indicates the significance level compared to the control group (vehicle). mice. ** , *** .

The results obtained from this study showed that both EEM and EEC have antinociceptive activity. It was not possible to infer whether this action involves peripheral and/or central mechanisms, as the model of abdominal contortions induced by the intraperitoneal injection of acetic acid in mice is sensitive to analgesic substances that act centrally and/or peripherally and that show a wide range of mechanisms of action [39]. Similar results were observed in other species of the genus Pimenta [29, 30, 32].

The mixture of triterpenes lupeol and - and -amyrin isolated from HFC could be accountable for the observed activity. Several in vivo experiments have shown that, among the pharmacological activities attributed to these compounds, analgesic and anti-inflammatory effects are predominant [31, 6772].

Oleanolic acid, which is widely distributed in the vegetable kingdom, has strong anti-inflammatory effects [73] that may have contributed to reducing the number of abdominal contortions induced by chemical stimulation in mice, as observed for the DFC.

Quercetin, quercitrin, and kaempferol have anti-inflammatory activities [74, 75], which may also have contributed for the analgesic activity of EAFC observed in this study.

Data from the literature report the antinociceptive activity of citral [76, 77], a major component of the EOC. Moreover, sedative, anxiolytic, and anticonvulsant effects were observed from the essential oils of Cymbopogon citratus in mice through the use of motor activity tests (“rota-rod” and “open-field”), a hypnosedative activity test (sleep induced by barbiturate), an anxiolytic action test (“plus-maze” and “light-dark box”), and an anticonvulsant action test (seizures chemically induced by pentylenetetrazole) [78]. Therefore, the inhibition by the EOC of the contortions induced by chemical stimulation in mice in this study may be due to both peripheral and central mechanisms. However, the sedative and relaxing effects of citral, as well as the increase in sleep time induced by barbital, especially at high doses (200 mg kg−1) [79], may have exerted a greater influence on reducing the number of contortions than the analgesic effects themselves.

4. Conclusion

Until now, no systematic phytochemical and biological study on citral and the (E)-methyl isoeugenol chemotypes of P. pseudocaryophyllus had been reported. Among the isolated substances, oleanolic acid obtained from the dichloromethane fraction of the citral chemotype showed the best profile of antibacterial activity against the Gram-positive microorganisms used in this research (MIC = 31.2 to 125  g mL−1), followed by the essential oil of the citral chemotype, which showed good activity (MIC = 62.5 g  g mL−1) against B. cereus ATCC 14579 and moderate activity (MIC = 125–250  g mL−1) against the other Gram-positive microbes.

The extracts, fractions, and essential oils from P. pseudocaryophyllus leaves showed several levels of antifungal activity against Candida spp. and Cryptococcus spp. The antifungal activity shown by the ethyl acetate and aqueous fractions from both chemotypes, especially against Candida albicans (MIC = 31.2 to 62.5  g mL−1) and C. parapsilosis (MIC = 15.6 to 62.5  g mL−1), showed the potential of this species as a source of new antifungal alternatives.

In the models of abdominal contortions induced by acetic acid and ear oedema induced by croton oil in mice, the crude extract of the citral chemotype showed antinociceptive and anti-inflammatory effects, respectively. These effects may be related to the presence of the pentacyclic triterpenes lupeol, -amyrin, and -amyrin and the flavonoids quercetin, quercitrin, and afzelin.

Conflict of Interests

The authors have no conflict of interests to declare.

Acknowledgments

The authors acknowledge the financial support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG). Thanks are due to MCT/FINEP/CT-INFRA for financial support for Laboratório de Ressonância Nuclear Magnética of the Instituto de Química-UFG.

References

  1. L. R. Landrum, “Monography 45: Campomanesia, Pimenta, Blepharocalyx, Legrandia, Acca, Myrrhinium, and Luma (Myrtaceae),” Flora Neotropica, vol. 4, pp. 72–115, 1986. View at Google Scholar
  2. M. Nakaoka-Sakita, O. T. Aguiar, M. Yatagai, and T. Igarashi, “Óleo essencial de Pimenta pseudocaryophyllus var. pseudocaryophyllus (Gomes) Landrum (Myrtaceae) I: cromatografia a gás/espectrometria de massa (CC/EM),” A Revista do Instituto Florestal, vol. 6, pp. 53–61, 1994. View at Google Scholar
  3. L. R. Landrum and M. L. Kawasaki, “The genera of Myrtaceae in Brazil: an illustrated synoptic treatment and identification keys,” Brittonia, vol. 49, no. 4, pp. 508–536, 1997. View at Google Scholar · View at Scopus
  4. M. E. L. Lima, I. Cordeiro, M. C. M. Young, M. E. G. Sobra, and P. R. H. Moreno, “Antimicrobial activity of the essential oil from two specimens of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) native from São Paulo State—Brazil,” Pharmacologyonline, vol. 3, pp. 589–593, 2006. View at Google Scholar
  5. J. A. M. Paula, J. R. Paula, M. T. F. Bara, M. H. Rezende, and H. D. Ferreira, “Pharmacognostic study about Pimenta pseudocaryophyllus (Gomes) L. R. Landrum leaves—Myrtaceae,” Brazilian Journal of Pharmacognosy, vol. 18, no. 2, pp. 265–278, 2008. View at Google Scholar · View at Scopus
  6. B. C. B. dos Santos, J. C. T. da Silva, P. G. Guerrero, G. G. Leitão, and L. E. S. Barata, “Isolation of chavibetol from essential oil of Pimenta pseudocaryophyllus leaf by high-speed counter-current chromatography,” Journal of Chromatography A, vol. 1216, no. 19, pp. 4303–4306, 2009. View at Publisher · View at Google Scholar · View at Scopus
  7. J. A. M. Paula, P. H. Ferri, M. T. F. Bara, L. M. F. Tresvenzol, F. A. S. Sá, and J. R. Paula, “Infraspecific chemical variability in the essential oils of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae),” Biochemical Systematics and Ecology, vol. 39, pp. 643–650, 2011. View at Publisher · View at Google Scholar · View at Scopus
  8. M. A. Pfaller and D. J. Diekema, “Epidemiology of invasive candidiasis: a persistent public health problem,” Clinical Microbiology Reviews, vol. 20, no. 1, pp. 133–163, 2007. View at Publisher · View at Google Scholar · View at Scopus
  9. S. J. Lee, H. K. Choi, J. Son, K. H. Kim, and S. H. Lee, “Cryptococcal meningitis in patients with or without human immunodeficiency virus: experience in a tertiary hospital,” Yonsei Medical Journal, vol. 52, no. 3, pp. 482–487, 2011. View at Publisher · View at Google Scholar · View at Scopus
  10. N. H. J. Leenders, J. J. Oosterheert, M. B. Ekkelenkamp, D. W. De Lange, A. I. M. Hoepelman, and E. J. G. Peters, “Candidemic complications in patients with intravascular catheters colonized with Candida species: an indication for preemptive antifungal therapy?” International Journal of Infectious Diseases, vol. 15, no. 7, pp. e453–e458, 2011. View at Publisher · View at Google Scholar · View at Scopus
  11. X. Liu, Z. Ling, L. Li, and B. Ruan, “Invasive fungal infections in liver transplantation,” International Journal of Infectious Diseases, vol. 15, no. 5, pp. e298–e304, 2011. View at Publisher · View at Google Scholar · View at Scopus
  12. M. F. Vicente, A. Basilio, A. Cabello, and F. Peláez, “Microbial natural products as a source of antifungals,” Clinical Microbiology and Infection, vol. 9, no. 1, pp. 15–32, 2003. View at Publisher · View at Google Scholar · View at Scopus
  13. M. A. Pfaller, S. A. Messer, G. J. Moet, R. N. Jones, and M. Castanheira, “Candida bloodstream infections: comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009),” International Journal of Antimicrobial Agents, vol. 38, no. 1, pp. 65–69, 2011. View at Publisher · View at Google Scholar · View at Scopus
  14. S. B. Mahato and A. P. Kundu, “13C NMR spectra of pentacyclic triterpenoids—a compilation and some salient features,” Phytochemistry, vol. 37, no. 6, pp. 1517–1575, 1994. View at Publisher · View at Google Scholar · View at Scopus
  15. J. Rodrigues, Uso Sustentável Da biodiversiDade Brasileira: Prospecção Químico-Farmacológica Em Plantas Superiores: Miconia Spp [Dissertation], Universidade Estadual Paulista, Araraquara, Brazil, 2007.
  16. G. Ye and C. Huang, “Flavonoids of Limonium aureum,” Chemistry of Natural Compounds, vol. 42, no. 2, pp. 232–234, 2006. View at Publisher · View at Google Scholar · View at Scopus
  17. M. Olszewska and M. Wolbiś, “Further flavonoids from the flowers of Prunus spinosa L.,” Acta Poloniae Pharmaceutica, vol. 59, no. 2, pp. 133–137, 2002. View at Google Scholar · View at Scopus
  18. M. Olszewska and M. Wolbiś, “Flavonoids from the flowers of Prunus spinosa L.,” Acta Poloniae Pharmaceutica, vol. 58, no. 5, pp. 367–372, 2001. View at Google Scholar · View at Scopus
  19. M. J. Jung, S. I. Heo, and M. H. Wang, “Free radical scavenging and total phenolic contents from methanolic extracts of Ulmus davidiana,” Food Chemistry, vol. 108, no. 2, pp. 482–487, 2008. View at Publisher · View at Google Scholar · View at Scopus
  20. X. C. Li, H. N. Elsohly, C. D. Hufford, and A. M. Clark, “NMR assignments of ellagic acid derivatives,” Magnetic Resonance in Chemistry, vol. 37, no. 11, pp. 856–859, 1999. View at Google Scholar · View at Scopus
  21. M. Miyazawa and M. Hisama, “Suppression of chemical mutagen-induced SOS response by alkylphenols from clove (Syzygium aromaticum) in the Salmonella typhimurium TA1535/pSK1002 umu test,” Journal of Agricultural and Food Chemistry, vol. 49, no. 8, pp. 4019–4025, 2001. View at Publisher · View at Google Scholar · View at Scopus
  22. D. Q. Falcão, S. O. B. Fernandes, and F. S. Menezes, “Triterpenos de Hyptis fasciculata Benth,” The Revista Brasileira de Farmacognosia, vol. 13, pp. 81–83, 2003. View at Google Scholar
  23. D. Fraisse, A. Heitz, A. Carnat, A. P. Carnat, and J. L. Lamaison, “Quercetin 3-arabinopyranoside, a major flavonoid compound from Alchemilla xanthochlora,” Fitoterapia, vol. 71, no. 4, pp. 463–464, 2000. View at Publisher · View at Google Scholar · View at Scopus
  24. V. Cattoir and C. Daurel, “Update on antimicrobial chemotherapy,” Medecine et Maladies Infectieuses, vol. 40, no. 3, pp. 135–154, 2010. View at Publisher · View at Google Scholar · View at Scopus
  25. R. P. Samy and P. Gopalakrishnakone, “Therapeutic potential of plants as anti-microbials for drug discovery,” Evidence-based Complementary and Alternative Medicine, vol. 7, no. 3, pp. 283–294, 2010. View at Publisher · View at Google Scholar · View at Scopus
  26. M. Oussalah, S. Caillet, L. Saucier, and M. Lacroix, “Antimicrobial effects of selected plant essential oils on the growth of a Pseudomonas putida strain isolated from meat,” Meat Science, vol. 73, no. 2, pp. 236–244, 2006. View at Publisher · View at Google Scholar · View at Scopus
  27. J. Kim, Y. S. Lee, S. G. Lee, S. C. Shin, and I. K. Park, “Fumigant antifungal activity of plant essential oils and components from West Indian bay (Pimenta racemosa) and thyme (Thymus vulgaris) oils against two phytopathogenic fungi,” Flavour and Fragrance Journal, vol. 23, no. 4, pp. 272–277, 2008. View at Publisher · View at Google Scholar · View at Scopus
  28. J. A. M. De Paula, J. R. De Paula, F. C. Pimenta, M. H. Rezende, and M. T. Freitas Bara, “Antimicrobial activity of the crude ethanol extract from Pimenta pseudocaryophyllus,” Pharmaceutical Biology, vol. 47, no. 10, pp. 987–993, 2009. View at Publisher · View at Google Scholar · View at Scopus
  29. A. Suárez, G. Ulate, and J. F. Ciccio, “Efectos de la administración aguda y subaguda de extractos dePimenta dioica(Myrtaceae) en ratas albinas normotensas e hipertensas,” Revista de Biologia Tropical, vol. 44, pp. 39–45, 1996. View at Google Scholar
  30. A. Benítez, J. Tillán, and Y. Cabrera, “Actividad analgésica y antipirética de un extracto fluido de Pimenta dioica L. y evaluación de su toxicidad aguda oral,” Revista Cubana de Farmacia, vol. 32, pp. 198–203, 1998. View at Google Scholar
  31. A. Fernández, A. Álvarez, M. D. García, and M. T. Sáenz, “Anti-inflammatory effect of Pimenta racemosa var. ozua and isolation of the triterpene lupeol,” Farmaco, vol. 56, no. 4, pp. 335–338, 2001. View at Publisher · View at Google Scholar · View at Scopus
  32. M. D. García, M. A. Fernández, A. Alvarez, and M. T. Saenz, “Antinociceptive and anti-inflammatory effect of the aqueous extract from leaves of Pimenta racemosa var. ozua (Mirtaceae),” Journal of Ethnopharmacology, vol. 91, no. 1, pp. 69–73, 2004. View at Publisher · View at Google Scholar · View at Scopus
  33. R. P. Adams, Identification of Essential Oil Components By Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, Carol Stream, Ill, USA, 4th edition, 2007.
  34. H. van Den Dool and K. P. Dec, “A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography,” Journal of Chromatography A, vol. 11, no. C, pp. 463–471, 1963. View at Google Scholar · View at Scopus
  35. H. Wagner and S. Bladt, Plant Drug Analysis: A Thin Layer Chromatography Atlas, Springer, Heidelberg, NY, USA, 2nd edition, 1996.
  36. Clinical and Laboratory Standards Institute (CLSI), Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard M27-A3, Clinical and Laboratory Standards Institute, Wayne, Pa, USA, 3rd edition, 2008.
  37. Clinical and Laboratory Standards Institute (CLSI), Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard M07-A8, Clinical and Laboratory Standards Institute, Wayne, Pa, USA, 8th edition, 2009.
  38. R. Koster, M. Anderson, and E. J. De Beer, “Acetic acid for analgesic screening,” Federation proceedings, vol. 18, pp. 412–421, 1959. View at Google Scholar
  39. A. J. Lapa, C. Souccar, T. R. Lima-Landman, M. A. S. Castro, and T. C. M. Lima, M.Plantas Medicinais: Métodos De Avaliação Da ativiDaDe Farmacológica, SBPC, Campinas, Brazil, 2008.
  40. J. C. Zanini, Y. S. Medeiros, A. B. Cruz, R. R. A. Yunes, and J. B. Calixto, “Action of compounds from Mandevilla velutina on croton oil-induced ear oedema in mice. A comparative study with steroidal and nonsteroidal antiinflammatory drugs,” Phytotherapy Research, vol. 6, no. 1, pp. 1–5, 1992. View at Google Scholar · View at Scopus
  41. F. B. Holetz, G. L. Pessini, N. R. Sanches, D. A. G. Cortez, C. V. Nakamura, and B. P. Dias Filho, “Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases,” Memorias do Instituto Oswaldo Cruz, vol. 97, no. 7, pp. 1027–1031, 2002. View at Google Scholar · View at Scopus
  42. R. Dall'Agnol, A. Ferraz, A. P. Bernardi et al., “Antimicrobial activity of some Hypericum species,” Phytomedicine, vol. 10, no. 6-7, pp. 511–516, 2003. View at Publisher · View at Google Scholar · View at Scopus
  43. J. C. Akio Tanaka, C. C. Da Silva, B. P. Dias Filho, C. V. Nakamura, J. E. De Carvalho, and M. A. Foglio, “Chemical constituents of Luehea divaricata Mart. (Tiliaceae),” Quimica Nova, vol. 28, no. 5, pp. 834–837, 2005. View at Google Scholar · View at Scopus
  44. M. C. C. Ayres, M. S. Brandão, G. M. Vieira-Jr et al., “Atividade antibacteriana de plantas úteis e constituintes químicos da raiz de Copernicia prunifera,” The Revista Brasileira de Farmacognosia, vol. 18, pp. 90–97, 2008. View at Google Scholar
  45. S. J. Shin, C. E. Park, N. I. Baek, I. S. Chung, and C. H. Park, “Betulinic and oleanolic acids isolated from Forsythia suspensa Vahl inhibit urease activity of Helicobacter pylori,” Biotechnology and Bioprocess Engineering, vol. 14, no. 2, pp. 140–145, 2009. View at Publisher · View at Google Scholar · View at Scopus
  46. A. Kurek, A. M. Grudniak, M. Szwed et al., “Oleanolic acid and ursolic acid affect peptidoglycan metabolism in Listeria monocytogenes,” Antonie van Leeuwenhoek, vol. 97, no. 1, pp. 61–68, 2010. View at Publisher · View at Google Scholar · View at Scopus
  47. K. I. Wolska, A. M. Grudniak, B. Fiecek, A. Kraczkiewicz-Dowjat, and A. Kurek, “Antibacterial activity of oleanolic and ursolic acids and their derivatives,” Central European Journal of Biology, vol. 5, no. 5, pp. 543–553, 2010. View at Publisher · View at Google Scholar · View at Scopus
  48. Y. H. Lu, Z. Zhang, G. X. Shi, J. C. Meng, and R. X. Tan, “A new antifungal flavonol glycoside from Hypericum perforatum,” Acta Botanica Sinica, vol. 44, no. 6, pp. 743–745, 2002. View at Google Scholar · View at Scopus
  49. X. Ma, W. Tian, L. Wu, X. Cao, and Y. Ito, “Isolation of quercetin-3-O-L-rhamnoside from Acer truncatum Bunge by high-speed counter-current chromatography,” Journal of Chromatography A, vol. 1070, no. 1-2, pp. 211–214, 2005. View at Publisher · View at Google Scholar · View at Scopus
  50. A. G. Báidez, P. Gómez, J. A. Del Río, and A. Ortuño, “Antifungal capacity of major phenolic compounds of Olea europaea L. against Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten,” Physiological and Molecular Plant Pathology, vol. 69, no. 4-6, pp. 224–229, 2006. View at Publisher · View at Google Scholar · View at Scopus
  51. G. Bisignano, R. Sanogo, A. Marino et al., “Antimicrobial activity of Mitracarpus scaber extract and isolated constituents,” Letters in Applied Microbiology, vol. 30, no. 2, pp. 105–108, 2000. View at Google Scholar · View at Scopus
  52. I. Kubo, P. Xiao, and K. Fujita, “Antifungal activity of octyl gallate: structural criteria and mode of action,” Bioorganic and Medicinal Chemistry Letters, vol. 11, no. 3, pp. 347–350, 2001. View at Publisher · View at Google Scholar · View at Scopus
  53. K. I. Fujita and I. Kubo, “Antifungal activity of octyl gallate,” International Journal of Food Microbiology, vol. 79, no. 3, pp. 193–201, 2002. View at Publisher · View at Google Scholar · View at Scopus
  54. I. F. Silva, M. Raimondi, S. Zacchino et al., “Evaluation of the antifungal activity and mode of action of Lafoensia pacari A. St.-Hil., Lythraceae, stem-bark extracts, fractions and ellagic acid,” Brazilian Journal of Pharmacognosy, vol. 20, no. 3, pp. 422–428, 2010. View at Google Scholar · View at Scopus
  55. M. A. Pfaller, D. J. Diekema, M. G. Rinaldi et al., “Results from the ARTEMIS DISK global antifungal surveillance study: A 6.5-year analysis of susceptibilities of Candida and other yeast species to fluconazole and voriconazole by standardized disk diffusion testing,” Journal of Clinical Microbiology, vol. 43, no. 12, pp. 5848–5859, 2005. View at Publisher · View at Google Scholar · View at Scopus
  56. K. Tintelnot, G. Schär, A. Polak et al., “Epidemiological data of cryptococcosis in Austria, Germany and Switzerland: part of the ECMM survey in Europe,” Mycoses, vol. 44, no. 9-10, pp. 345–350, 2001. View at Publisher · View at Google Scholar · View at Scopus
  57. H. J. D. Dorman and S. G. Deans, “Antimicrobial agents from plants: antibacterial activity of plant volatile oils,” Journal of Applied Microbiology, vol. 88, no. 2, pp. 308–316, 2000. View at Publisher · View at Google Scholar · View at Scopus
  58. S. Inouye, T. Takizawa, and H. Yamaguchi, “Antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact,” Journal of Antimicrobial Chemotherapy, vol. 47, no. 5, pp. 565–573, 2001. View at Google Scholar · View at Scopus
  59. K. Cimanga, K. Kambu, L. Tona et al., “Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo,” Journal of Ethnopharmacology, vol. 79, no. 2, pp. 213–220, 2002. View at Publisher · View at Google Scholar · View at Scopus
  60. D. R. Oliveira, G. G. Leitão, S. S. Santos et al., “Ethnopharmacological study of two Lippia species from Oriximiná, Brazil,” Journal of Ethnopharmacology, vol. 108, no. 1, pp. 103–108, 2006. View at Publisher · View at Google Scholar · View at Scopus
  61. M. Oussalah, S. Caillet, L. Saucier, and M. Lacroix, “Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes,” Food Control, vol. 18, no. 5, pp. 414–420, 2007. View at Publisher · View at Google Scholar · View at Scopus
  62. I. G. Sandri, J. Zacaria, F. Fracaro, A. P. L. Delamare, and S. Echeverrigaray, “Antimicrobial activity of the essential oils of Brazilian species of the genus Cunila against foodborne pathogens and spoiling bacteria,” Food Chemistry, vol. 103, no. 3, pp. 823–828, 2007. View at Publisher · View at Google Scholar · View at Scopus
  63. S. G. Griffin, G. Wyllie, J. L. Markham, and D. N. Leach, “The role of structure and molecular properties of terpenoids in determining their antimicrobial activity,” Flavour and Fragrance Journal, vol. 14, pp. 322–332, 1999. View at Google Scholar
  64. V. Moleyar and P. Narasimham, “Antifungal activity of some essential oil components,” Food Microbiology, vol. 3, no. 4, pp. 331–336, 1986. View at Google Scholar · View at Scopus
  65. Y. S. Lee, J. Kim, S. C. Shin, S. G. Lee, and I. K. Park, “Antifungal activity of Myrtaceae essential oils and their components against three phytopathogenic fungi,” Flavour and Fragrance Journal, vol. 23, no. 1, pp. 23–28, 2008. View at Publisher · View at Google Scholar · View at Scopus
  66. M. Luo, L. K. Jiang, Y. X. Huang, M. Xiao, B. Li, and G. L. Zou, “Effects of citral on Aspergillus flavus spores by quasi-elastic light scattering and multiplex microanalysis techniques,” Acta Biochimica et Biophysica Sinica, vol. 36, no. 4, pp. 277–283, 2004. View at Google Scholar · View at Scopus
  67. R. B. Agarwal and V. D. Rangari, “Antiinflammatory and antiarthritic activities of lupeol and 19α-H lupeol isolated from Strobilanthus callosus and Strobilanthus ixiocephala roots,” Indian Journal of Pharmacology, vol. 35, no. 6, pp. 384–387, 2003. View at Google Scholar · View at Scopus
  68. F. A. Oliveira, C. L. S. Costa, M. H. Chaves et al., “Attenuation of capsaicin-induced acute and visceral nociceptive pain by α- and β-amyrin, a triterpene mixture isolated from Protium heptaphyllum resin in mice,” Life Sciences, vol. 77, no. 23, pp. 2942–2952, 2005. View at Publisher · View at Google Scholar · View at Scopus
  69. M. F. Otuki, F. Vieira-Lima, A. Malheiros, R. A. Yunes, and J. B. Calixto, “Topical antiinflammatory effects of the ether extract from Protium kleinii and α-amyrin pentacyclic triterpene,” European Journal of Pharmacology, vol. 507, no. 1–3, pp. 253–259, 2005. View at Publisher · View at Google Scholar · View at Scopus
  70. R. Medeiros, M. F. Otuki, M. C. W. Avellar, and J. B. Calixto, “Mechanisms underlying the inhibitory actions of the pentacyclic triterpene α-amyrin in the mouse skin inflammation induced by phorbol ester 12-O-tetradecanoylphorbol-13-acetate,” European Journal of Pharmacology, vol. 559, no. 2-3, pp. 227–235, 2007. View at Publisher · View at Google Scholar · View at Scopus
  71. S. A. Holanda Pinto, L. M. S. Pinto, M. A. Guedes et al., “Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin,” Phytomedicine, vol. 15, no. 8, pp. 630–634, 2008. View at Publisher · View at Google Scholar · View at Scopus
  72. C. Soldi, M. G. Pizzolatti, A. P. Luiz et al., “Synthetic derivatives of the α- and β-amyrin triterpenes and their antinociceptive properties,” Bioorganic and Medicinal Chemistry, vol. 16, no. 6, pp. 3377–3386, 2008. View at Publisher · View at Google Scholar · View at Scopus
  73. M. J. Wu, L. Wang, H. Y. Ding, C. Y. Weng, and J. H. Yen, “Glossogyne tenuifolia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating LPS-induced NF-IκB,” Journal of Biomedical Science, vol. 11, no. 2, pp. 186–199, 2004. View at Publisher · View at Google Scholar · View at Scopus
  74. O. E. Ekpo and E. Pretorius, “Asthma, Euphorbia hirta and its anti-inflammatory properties,” South African Journal of Science, vol. 103, no. 5-6, pp. 201–203, 2007. View at Google Scholar · View at Scopus
  75. V. García-Mediavilla, I. Crespo, P. S. Collado et al., “The anti-inflammatory flavones quercetin and kaempferol cause inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive C-protein, and down-regulation of the nuclear factor kappaB pathway in Chang Liver cells,” European Journal of Pharmacology, vol. 557, no. 2-3, pp. 221–229, 2007. View at Publisher · View at Google Scholar · View at Scopus
  76. G. S. B. Viana, T. G. Do Vale, V. S. N. Rao, and F. J. A. Matos, “Analgesic and antiinflammatory effects of two chemotypes of Lippia alba: a comparative study,” Pharmaceutical Biology, vol. 36, no. 5, pp. 347–351, 1998. View at Publisher · View at Google Scholar · View at Scopus
  77. G. S. B. Viana, T. G. Vale, R. S. N. Pinho, and F. J. A. Matos, “Antinociceptive effect of the essential oil from Cymbopogon citratus in mice,” Journal of Ethnopharmacology, vol. 70, no. 3, pp. 323–327, 2000. View at Publisher · View at Google Scholar · View at Scopus
  78. M. M. Blanco, C. A. R. A. Costa, A. O. Freire, J. G. Santos, and M. Costa, “Neurobehavioral effect of essential oil of Cymbopogon citratus in mice,” Phytomedicine, vol. 16, no. 2-3, pp. 265–270, 2009. View at Publisher · View at Google Scholar · View at Scopus
  79. T. G. Do Vale, E. C. Furtado, J. G. Santos, and G. S. B. Viana, “Central effects of citral, myrcene and limonene, constituents of essential oil chemotypes from Lippia alba (mill.) N.E. Brown,” Phytomedicine, vol. 9, no. 8, pp. 709–714, 2002. View at Publisher · View at Google Scholar · View at Scopus