Anti-Ehrlichia properties of the essential oil of Ageratum conyzoides L. and its interaction with doxycycline

Essential oil extraction

In this study, we used 200 g of fresh aerial parts of A. conyzoides collected in the early morning of July of 2017 at the Horto Berta Langes de Morretes, Federal University of Maranhão (UFMA), located in the municipality of São Luís, State of Maranhão (MA), northeast Brazil, Lat. 2°33′13.5″S 44°18′20.8″W. Samples were taxonomically identified and desiccated (voucher) specimens were deposited in the Herbarium of Maranhão—UFMA under the ID number Nº. 9.099. The essential oil of A. conyzoides was extracted from by hydrodistillation in a clevenger apparatus coupled to a Quimis ultrathermostatic bath with a temperature less than 12 °C. The aerial parts of the plant were crushed and placed in a conical flask added with ultrapure distilled water. After 2 h of distillation, the oil was removed from the water surface, centrifuged, and carefully separated from the water added with anhydrous sodium sulfate (JT Baker Chemical Co.), stored in an amber glass ampoule, hermetically sealed, and then stored in a cooled 4 °C for further analysis (Coutinho et al. 2007).

The access was registered under the ID number ADBBA07 in the National System of Management of Genetic Heritage and Associated Traditional Knowledge according to art. 41 of Decree No. 8.772/2016 of the Ministry of the Environment in Brazil.

Characterization of the essential oil by GC/MS

The chemical composition of the essential oil was analyzed by gas phase chromatography/mass spectrometry (GC/MS) with the injection of 1 μL (Auto Injector AOC-20i) in a GCMS-QP 2010 Ultra (Shimadzu) equipped with a Rtx-5MS silica capillary column (Restek, USA) 30 m long × 0.25 mm inner diameter coated with 5%—diphenyl/95%—dimethyl-polysiloxane (0.25 μm film thickness).

The temperature of the GC oven was programmed from 60 to 240 °C at 3 °C/min, injector (1:20 split). Transfer line and ionization chamber temperatures were 250 °C, 250 °C, and 200 °C, respectively. Helium was used as the entrainment gas at a rate of 1 μL/min.

The mass spectra were obtained by electronic impact at 70 eV with automatic scans in the mass range between 35 and 400 m/z at 0.30 scans/s.

The identification of the components was based on the time and linear retention index (series of C8–C28 n-alkanes) and on the interpretation and comparison of the mass spectra obtained from the libraries (Adams 2012; NIST 2011).

Microorganism

DH82 cells (Canine Histiocyte: ATCC No. CRL-10389) infected with 35th passage of the Cuiabá #1 strain of E. canis were cultured in Dulbecco’s Modified Eagle’s (DMEM) medium (Sigma Chemical Co., St. Louis), MO, USA) supplemented with 5% fetal calf serum (HyClone Laboratories, Logan, Utah, USA) and maintained in a 25 cm² culture bottle at 37 °C with 5% CO₂ as recommended by Aguiar et al. (2007). E. canis infection rate was determined by examining (screening) smears from a monolayer cell stained by the Diff-Quik Kit (Laborclin, Pinhais, PR, Brazil) under the light microscope.

When an rickettsial infection rate of 70% was detected using this method, the cells were resuspended with the same effect and the cell suspension was centrifuged at 4000g for 5 min. The experiments were run on 24-well culture plates at 37 °C with 5% CO₂. The bacterial rate was standardized as 3000 cells per well and 70% of the cells infected with the rickettsia.

The access was registered under number A9463BB in the National System of Management of Genetic Heritage and Associated Traditional Knowledge according to art. 41 of Decree No. 8.772/2016 of the Brazilian Ministry of the Environment.

Biological assay

The assays were performed in triplicate at concentrations of 25, 50, 100, 200, 300, 400, and 500 µg/mL of the essential oil of A. conyzoides L. plus 1% Dimethylsulfoxide-DMSO (Merck Chemical Co.) in order to solubilize the sample and at concentrations of 0.25, 0.50, 0.75, 1.0, 1.5 µg/mL of the doxycycline plus 1% Dimethylsulfoxide-DMSO (Merck Chemical Co.). Analyses were performed at 18 h and 36 h after addition of the treatments to the medium. The analyses consisted of counting the percentage of infected cells on Diff-Quik (Laborclin, Pinhais, PR, Brazil) stained cell monolayer smear preparations examined under the light microscope (Aguiar et al. 2007).

The experiments were performed according to the method published by Chou (2006). The treatments were tested at constant ratios of equipotent concentrations, ranging from 0.0625-, 0.125-, 0.25-, 0.5-, 1-fold of their respective IC₅₀ value that was determined for each experiment. The synergism, antagonism, or additive effect of each of the combinations was assessed by calculating the combination index value (CI). According to Chou (2006) and Chou (1976):1\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \frac{{d_{1} }}{{d_{1} x}} + \frac{{d_{2} }}{{d_{2} x}} $$\end{document}d1d1x+d2d2xin which D₁ and D₂ are the doses of doxycycline (1) and EO (2), respectively, that are responsible for an effect x in combination whereas D₁x and D₂x are the doses of 1 and 2, respectively, that are responsible for the same effect individually. If CI < 1, the treatments have a synergistic effect; if CI > 1, they are antagonistic; if CI = 1, an additive effect is observed. A normalised isobologram was created by plotting the normalised concentrations \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \frac{{d_{1} }}{{d_{1} x}} $$\end{document}d1d1xof 1 and \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \frac{{d_{2} }}{{d_{2} x}} $$\end{document}d2d2xof 2 on the y- and x-axis, respectively, in which the denominators represent the respective doses of c 1 and 2 alone reducing antibacterial load by x%, and the numerators represent the respective doses of 1 and 2 reducing bacterial load by x% in combination. The normalised concentrations were calculated considering that2\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \frac{{D_{1} }}{{D_{1} x}} = \frac{{D_{1} }}{{Dm_{1} \cdot \left( {\frac{fa}{1 - fa}} \right)^{{\frac{1}{{m_{1} }}^{{}} }} }} $$\end{document}D1D1x=D1Dm1·fa1-fa1m1in which \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ D_{{m_{1} }} $$\end{document}Dm1is the IC₅₀ of 1 in vitro, f_a is the fraction affected (or (% effect) ÷ 100), and m₁ is the slope of linear regressions from median effect plots using the function3\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ log\left( {\frac{fa}{1 - fa}} \right) = f\left( {log\left( {D_{1} x} \right)} \right) $$\end{document}logfa1-fa=flogD1x

The CI-effect graph representing the CI as a function of the associated antibacterial effect was also plotted as well as the log (DRI)-effect plot representing the log of the dose reduction index (DRI) as a function of the associated antibacterial effect. The DRI is the ratio of the concentration of a treatment resulting in an effect x alone (D₁x) to the concentration of the same treatment resulting in an effect x in combination (D₁):4\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ DRI = \frac{{D_{1} x}}{{D_{1} }} $$\end{document}DRI=D1xD1

Ehrlichia canis suspensions were standardized at 800 cells/well with a 70% infection rate using 96-well culture plates. Solutions of the tested products were used in concentrations determined from their respective IC₅₀ The protocol used to determine the antimicrobial effect of the essential oil and doxycycline were an adaptation from Rolain et al. (1998) and Rolain et al. (2002) regarding serial dilutions 0.0625 0.25, 0.5 and 1 times the respective IC₅₀ value that was determined for each treatment. Initially, we added 200 μL of medium into each well of a sterile microplate. Subsequently, 50 μL of each product tested in serial dilutions were arranged in an orderly fashion so that we were able to evaluate activity according to the decrease of the essential oil and the synthetic drug. From top to bottom, there is decrease of the essential oil IC₅₀, and horizontally from the right to the left there is a decrease of the synthetic drug (Nightingale et al. 2007).

Our results show that in each well there is a unique combination of concentrations between the two substances (i.e., essential oil of A. conyzoides and doxycycline).

Statistical analysis

The analyses were performed using the software GraphPad Prism 5.0 (GraphPad Software, La Jolla, California, USA). The Student’s test was for toxicity analysis and the analysis of variance (ANOVA) was used to obtain data on the rate of inhibition of the microorganisms and the treatment time of the groups. Statistically, significant differences were found with values of p < 0.05. The IC₅₀ value was also acquired by linear regression using the software GraphPad Prism 5.0 (GraphPad Software, La Jolla California USA). The effects of the interaction between the treatments and doxycycline were also evaluated by the analysis of the combination of multiple drugs using the software CompuSyn^® (Chou and Talalay 1984).

Biological assay

To the authors’ knowledge, this is the first alternative treatment (alternative therapy) based on bioactive compounds with antibacterial activity against E. canis. Figure 1 shows the percentage of inhibition of E. canis infection in cells after 18 h and 36 h of treatment with the essential oil of A. conyzoides. It is noted that at the concentration of 200 µg/mL at 36 h this hydrophobic liquid inhibited a percentage greater than 50% of E. canis morulae formation. This result demonstrates the in vitro efficacy of this oil.

Fig. 1

Percent of inhibition of Ehrlichia canis in DH82 cells, after 18 and 36 h of treatment as essential oil of A. conyzoides, in different concentrations. Each line represents medium and deviant pad of three independent trials

Cell viability

The cytotoxicity of the treatments tested was demonstrated on DH82 cells after 24 h of incubation, and the viability was determined by the trypan blue dye exclusion test. None of the treatments showed cytotoxicity up to a maximum concentration of 500 µg/mL (Fig. 2).

Fig. 2

Each row represents mean and standard deviation of three independent assays. Where *p < 0.05 demonstrated that there was no statistically significant difference in relation to the control group according to the unpaired Student’s t test with a 95% confidence index

Association of treatments

The possible interactions between doxycycline and the essential oil of A. conyzoides L. were assessed in vitro using the isobologram of non-fixed proportions modified using E. canis-infected DH82 cells after 24 h of incubation with the respective treatments tested.

Since doxycycline is the treatment of choice for treating canine ehrlichiosis, we performed the tests of association with the essential oil obtained from A. conyzoides to verify if at lower concentrations both treatments could be used to inhibit the percentage of infection in vitro by E. canis. We used the Chou and Talalay method to design the hypotheses and evaluate the various combinations (Chou and Talalay 1984; Chou 2010).

The treatments were tested in constant proportions of equipotent concentrations, ranging from 0.0625 to 1 times the respective IC₅₀ value that was determined for each treatment (Fig. 3a). The slope m was also determined from the linear regression of the median effect of the plots (Eq. 3), as they reflected the sigmoidicity of the dose–response curves and were used for the calculation of normalized concentrations and treatment reduction indices (Fig. 3b).

Fig. 3

Evaluation of the synergism of doxycycline in combination with essential oil (EO) of A. conyzoides. Data sets in blue, red and green correspond to doxycycline, EO, combinations, respectively. a Dose–response curves of each individual treatment. Doxycycline was evaluated twice, for each of the combinations with EO. The antibacterial effect was determined by measurement of foci size. b Median-effect representation of the dose–response curves for each individual compound, using Eq. (3). fa is the “fraction affected”, or (% effect) ÷ 100. c Normalised isobologram that represents, for each combination, the normalised dose of each treatment individually required to reach the observed effect in combination (Eq. 2). d CI-effect plot representing the combination index CI, calculated using Eq. (1), of each combination as a function of their associated antibacterial effect. e Log (DRI)-effect plot representing the drug reduction index (DRI) of compounds as a function of their antibacterial effect in combination. The DRI is calculated for each drug in each combination according to Eq. (4) and represents the dilution factor required for a drug to reach the same level of inhibition individually compared with it when in combination. The results are representative of 3 independent experiments

Standard isobologram is a graphical way of visualizing synergistic combinations with respect to concentrations. Since D1 is the concentration of treatment 1 responsible for an x effect in combination, and D₁x the concentration of treatment 1 responsible for an x effect alone, a normalized concentration \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{upgreek}\setlength{\oddsidemargin}{-69pt}\begin{document}$$ \frac{D1}{D1x} $$\end{document}D1D1x, calculated using Eq. (2), tends to zero as smaller concentrations of the treatments in combination are required to achieve an x effect.

As shown in Fig. 3c, virtually all data points are located in the region where combinations have a synergistic effect, suggesting that the essential oil obtained from A. conyzoides acts in synergy with doxycyclines in the in vitro treatment of infected DH82 cells E. canis. The IC effect of the graph also allows the visualization of combination effects, based on the combined CI index calculated using Eq. (1). The CI value is represented by a function of the antibacterial effect associated with each combination (Fig. 3d).

Similarly to Fig. 3c, the combination sites that were less than 1 indicate a synergistic effect between the essential oil obtained from A. conyzoides and doxycycline. The fact that the compounds show synergism means that their concentration in combination produces an effect that is stronger than when the treatments are used individually in a similar or greater concentration. For this synergistic property the inhibitors can be evaluated by calculating the drug dose reduction index (DRI, Eq. 4) for each treatment of each combination, and is plotted with the log (DRI) (Fig. 3e).

In our case the essential oil obtained from A. conyzoides and doxycycline make up combinations that inhibit E. canis infection in vitro, the effect becomes stronger, the greater the DRI. Although this result is expected, it does not translate into synergy. DRI is calculated for individual drugs in a given combination effect.

Based on the DRI effect plot, 100% inhibition of infection is achieved when the concentration of a compound in combination can be reduced tenfold compared to the concentration required to achieve the same effect individually.

EO analysis

Analysis of the essential oil was performed by gas chromatography/mass spectrometry (GCMS) using a Shimadzu^® equipment from GCMS-QP2010s; 8 compounds were detected in this oil, and we were able to identify 99.63% of these compounds. The composition of the oil is presented in Table 1.

The essential oil of A. conyzoides has precocene I as its main compound (92.75%) (Table 1) in terms of chemical composition.