Anti- Ehrlichia properties of the dichloromethane extract of Ageratum conyzoides

Ehrlichia canis is an intracellular bacterium that infects hematopoietic cells. It is the causative agent of canine monocytic ehrlichiosis (CME). The disease may be acute, subclinical, or chronic, and is treated with tetracyclines including doxycycline. However, this class of tetracyclines may cause several side effects due to prolonged treatment. Bacterial resistance to antimicrobials has been extensively reported. The present study aimed to assess the anti- Ehrlichia activity of the dichloromethane extract (DCM) of Ageratum conyzoides L. on Ehrlichia- infected DH82 cells. For this purpose, the DCM extract of A. conyzoides collected in the municipality of São Luís, State of Maranhão (MA), northeast Brazil, was obtained from the aerial parts of the plant by exhaustive percolation in H 2 O- CH 2 Cl 2 (2:8) and subsequent extraction of the chemical compound. The chemical composition of these samples was investigated. The anti-Ehrlichia properties of A. conyzoides were confirmed in Ehrlichia- infected DH82 cells at a concentration of 200 μg.mL -1 of its DCM extract. The results of the treatments were evaluated at 18h and 36h after the insertion of the treatments evaluated with A. conyzoides. Based on the results of the chemical analysis of the samples, we may attribute these antirickettsial properties to the compounds from the lignan family that are found in this medicinal plant .


Introduction
Ehrlichia spp. is an obligate intracellular bacterial pathogen that parasitizes hematopoietic cells (leukocytes). This rickettsial organism infect a wide range of mammalian hosts including dogs, cats, cattle, horses, and humans (Bogićević et al. 2017). Canine monocytic ehrlichiosis (CME) is caused by the bacterium Ehrlichia canis and has the ixodid tick Rhipicephalus sanguineus (common name: brown dog tick) as its main vector (Dagnone et al. 2001). The disease may be acute, subclinical, or chronic.
In the acute phase of the disease, clinical signs include depression, lethargy, anorexia, pyrexia, lymphadeno -and splenomegaly, and weight loss. Affected animals may exhibit bleeding especially petechiae and ecchymoses on the skin and mucous membranes and occasional epistaxis ( The drug of choice for the treatment of the disease in all its phases is doxycycline for 28 days (Tilley et al. 2003). However, the drugs of the tetracycline group may have some undesired effects when administered for prolonged periods of time. The most notable toxic effects of tetracyclines are observed in the bone and teeth. It causes a delay in bone formation and yellowish discoloration of teeth (Damaso 1990). Tetracycline bone toxicity results from neuromuscular blockade which is probably produced by the chelation of calcium ions (Goodman and Gilman 1990) and direct action on the mechanism of bone absorption and resorption by osteoclasts (Donahue et al. 1992) interfering with the deposition of calcium in bone tissue (Van Linthoudt et al. 1991).
In addition, photosensitivity manifested by hyperpigmentation, erythema, and ultimately skin ulceration is frequently associated with tetracycline therapy (Cunha 2001 Zimmerman 2000). This drug is able to cross the placental barrier and is therefore 4 contraindicated during pregnancy. Tetracycline should not be given to puppies (Gugliemo and Jacobs 2001).
According to González-Lamothe et al. (2009), the products of secondary metabolism accumulated by plants can act as "potentiators of antibacterial activity", favoring the activity of antibiotics whose action is limited by multidrug resistance mechanisms developed by microorganisms; or as "virulence attenuators", adapting the host's immune system response to infection.

Plant material
The plant species Ageratum conyzoides L. was cultivated and collected at the Berta

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 H 2 O-CH 2 Cl 2 (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.

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 6 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. 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 8 were run in 24-well culture plates at 37 °C and 5% CO 2 . 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 IC 50 (50% inhibition concentration) using the GraphPad Prism 7.0 software (Graph-Pad Inc., San Diego, CA, USA).

Results
This is the first study, the anti-Ehrlichia potential of A. conyzoides was calculated after 18h and 36h of treatment with the extract botanic (Fig 1). In the triplicate assays, the IC 50 of the proposed treatment was 200 μg.mL -1 .
Were assessed the viability of the DH82 cells against different concentrations of the tested treatment. Were noted that they were not toxic in the highest concentration, i.e. 500 μg.mL -1 (Fig 2) when compared with the control group which was formed by DH82 cells treated with ultrapure distilled water forlornly a 24-hour period.
In Fig. 3 shows the chromatogram of the extract dichloromethane of A. conyzoides with the UV absorption profiles of the 5 chromatographic peaks as evidenced by overlapping peaks high resolution in reverse phase (Table 1) 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.