Plant material
The plant species Ageratum conyzoides L. was cultivated and collected at the Berta Langes de Morretes Medicinal Herb Garden of the Federal University of Maranhão (UFMA), municipality of São Luís, State of Maranhão (MA), northeast Brazil (2°33'13.5"S and 44°18'20.8"W) in July 2017 (rainy season), according to the method published in the Brazilian Pharmacopeia (ANVISA 2010). The plant was herborized and identified, and a sample (voucher specimen - MAR 9099) is deposited at the Herbarium of Maranhão (MAR), located in Federal University of Maranhão, Brazil.
The access was registered under the ID number ADBBA07 in the National System of Management of Genetic Heritage and Associated Traditional Knowlege. The collect did not involve endangered or protected species.
Method to obtain the extract and the extract of the plant
A total of 1.7 kg of the aerial parts of Ageratum conyzoides L. was collected in the early hours of the morning. Samples were dried at room temperature (25 °C) for a 7-day period. An infrared moisture analyzer (GEHAKA IV 2500) was used to measure the plant moisture and calculate the yield. Shortly after that, the dry aerial parts of A. conyzoides were crushed in a mechanical turbolizer. The exhaustive percolation in H2O- CH2Cl2 (2:8) was the extraction method used in this study. Percolation was carried out at room temperature (25 °C) and protected from light. The extractor liquid was completed every 24h Corresponding to a total of 72h until the plant material exhaustion was finished. These compounds were evaporated to dryness under vacuum at approximately 40 °C. Shortly after that, the specimen was ultra-refrigerated at -80 °C to be lyophilized and to obtain the dry extract.
Analysis of the Extract of Ageratum conyzoides L. by High Performance Liquid Chromatography (HPLC) with ultraviolet detection
After cleaning up the extract, the sample was analyzed by HPLC using a Shimadzu® chromatograph (Shimadzu Corp., Kyoto, Japan) consisting of a solvent injection module with a binary pump and UV detector -Vis (SPA-10A). The column used was a Luna 5 μm C18 100 A (150 μm x 4.6 μm). The elution solvents used were elution solvent A (water + 0.01% formic acid) and elution solvent B (methanol + 0.01% formic acid). Samples were eluted according to the following exploratory gradients: 5% to 100% B in 60 min and 100% to 100% in 60 to 70 min. The flow rate was 1 mL/min, and the column temperature was 20 °C. The injection volume of the sample was 20 μL. Data were collected and processed using the LC Solution software (Shimadzu). In this study, baseline separation for the major components of the sample was obtained in a 70-min chromatographic run and evaluated at a 254 nm wavelength.
Characterization of the components of Ageratum conyzoidesL. by liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry (FIA-ESI-IT/MS)
Characterization of the compounds extracted from Ageratum conyzoides L. was carried out at the Institute of Biosciences, São Paulo State University (Unesp), São Vicente, SP, southeast Brazil. By infusing the samples directly into a mass spectrometer with an ion-trap linear analyzer (Thermo Scientific LTQ XL) equipped with an electrospray (ESI) in negative mode (Thermo, San Jose, CA, USA). A stainless-steel capillary tube at 280 °C, a spray voltage of 5.00 kV, a capillary voltage of -90 V, and a -100 V tube lenses at a flow of 5 μL/min were used in this procedure. Samples were infused into the mass spectrometer from the HPLC system in which samples were analyzed online by ESI-MS in negative mode and with an associated UV detector. The mass spectra data were obtained in the same Fleet LCQ mass spectrometer from Thermo Scientific® with direct insertion of the sample device via continuous flow injection analysis (FIA). Samples were ionized with an ESI source. Fragmentations were obtained in multiple stages (MSn) in an ion trap (IT)-type interface. The negative mode was used for the generation and analysis of all spectra. The experimental conditions were the following: capillary voltage of −35 V, spray voltage −5000 V, capillary temperature 350 °C, carrier gas N2 and flow 60 (arbitrary units). The track acquisition was in a mass range of m/z 100–2000 with two or more sweep events performed simultaneously in the spectrum. Compounds were identified by comparing between data from literature and fragmentation patterns.
Culture of Ehrlichia canis-DH82 cells
The Ehrlichia canis strain was obtained from the 35th passage of E. canis of the Cuiabá #1 isolate which belongs to the collection (library) of Rickettsiae and Ehrlichia from the Laboratory of Virology and Rickettsioses of the School of Veterinary Medicine of the Federal University of Mato Grosso (FMVZ/UFMT), Cuiabá, MT, central-west Brazil. This rickettsial strain multiplied in DH82 cell monolayers (ATCC number: CRL-10389) and was maintained at 37 °C and 5% CO2.
The access was registered under the number A9463BB in the National System of Management of Genetic Heritage and Associated Traditional Knowledge according to art. 41 of Decree Nº. 8772/2016 of the Ministry of the Environment in Brazil.
DH82 cells (Canine Histiocyte: ATCC in CRL-10389) grew in Dulbecco's Modified Eagle's (DMEM) medium (Sigma Chemical Co., St. Louis, MO, USA), plus 5% fetal serum of calf (HyClone Laboratories, Logan, Utah, USA) and culture bottle of 25 cm2 at 37 °C and 5% CO2 as recommended by Aguiar et al. (2007). The rate of E. canis infection was determined by screening Diff-Quik stained cell monolayer smears (Laborclin, Pinhais, PR, Brazil) under the light microscope.
When an infection rate of 70% was detected, the cells were resuspended in the same medium and the cell suspension was centrifuged at 4,000 g for 5 min. The experiments were run in 24-well culture plates at 37 °C and 5% CO2. The infection rate was standardized at 3,000 cells per well and 70% of infected cells (Aguiar et al. 2007).
Anti-Ehrlichiaassay
The assays were performed in the IC50 determination of the treatments studied against E. canis was determined from the test concentrations of 25 μg.mL-1, 50 μg.mL-1, 100 μg.mL-1, 200 μg.mL-1, 300 μg.mL-1, 400 μg.mL-1 and 500 μg.mL-1 in cell monolayers DH82 infected with E. canis at a 70% infection rate, cell quantities were standardized at 3,000 cells / well, in 24-well plates, assays were performed in triplicate, where treatment control used spun doxicycline1μg.mL-1, according to the package insert, and as a control of bacterial culture, wells treated only with distilled water. The protocol used to determine the antimicrobial effect of the test treatments was an adaptation by Rolain et al. (1998) and Rolain et al. (2002).
Cell viability analyses were performed using the trypan blue assay (Trypan blue exclusion test of cell viability) (Sigma-Aldrich, St. Louis, MO) according to the protocol and guidelines provided by Barile (Barile 1994).
Statistical analysis
The experimental design used in all biological assays of this study was completely randomized. The mean of each treatment was compared to its respective control. Data were initially transformed to Log(X), normalized, and then nonlinear regression was calculated to obtain IC50 (50% inhibition concentration) using the GraphPad Prism 7.0 software (Graph-Pad Inc., San Diego, CA, USA).