Antimicrobial Screening of Medicinal Plants Popularly used in Mato Grosso for Treating Infections : Advances on the Evaluation of Conyza bonariensis ( L . ) Cronquist in vitro and in vivo Antibacterial Activities

Human infections are a serious public health problem because many pathogens such as bacteria and yeast are becoming increasingly resistant to antibiotics.1 Despite the great advances achieved by science, among the ten main causes of death worldwide, which include ischaemic heart disease, stroke, diarrhea, HIV/ AIDS, malaria, tuberculosis, pre-term birth complications and birth asphyxia and birth trauma, the lower respiratory infections top the list.2 It is estimated that the so called ‘super-microorganisms’ alone will be responsible for over 10 million deaths by 2050, making it imperative the search for alternative treatments to microorganisms which are unresponsive to most modern antibiotics.3 Similarly, opportunistic fungal Objective The aim of this study was to screen a group of medicinal plants’ extracts used in the treatment of ailments related to infections in the Brazilian popular medicine. And to carry out in vivo toxicity and antibacterial studies on Conyza bonariensis (Asteraceae) leaves and roots methanolic extracts selected based on the screening. Methods: Eleven methanolic extracts obtained from nine plants, reportedly used in the treatments of infections from the state of Mato Grosso, Brazil, were initially screened for their in vitro antibacterial and antifungal activities employing disc diffusion and broth micro dilution assays. Preliminary phytochemical analysis was carried out. The most promising extract based on our results and previous literature reports was then evaluated in the in vivo antibacterial activities using mouse model of bacterial infection induced by Staphylococcus aureus and Escherichia coli. In addition, in vivo acute toxicity was conducted to evaluate the safety profile of the extracts. Results: All of the extracts tested were active against at least one of the bacterial and fungal strain tested with activities ranging from moderate to weak. Phytochemical analyses of MECbl and MECbr demonstrated the presence of free steroids and coumarins in MECbl and flavonoids, tanins, free steroids, reduced anthraquinones and coumarins in MECBr. Oral administration of MECbl and MECbr up to 5000 mg/kg did not provoked any toxicological events in the mice, thus suggesting that the LD50 is higher than 5000 mg/kg. In vivo antibacterial assay demonstrated superior prophylactic activity of MECbl compared to MECbr. Conclusion: MECbl and MECbr are safe when administered acute orally at doses up to 5000 mg/kg. Methanolic extracts of Conyza bonariensis possessed in vitro antibacterial and antifungal activities. Considerable in vivo antibacterial activities were observed in bacterial infection model for both MECbl and MECbr, effects comparable to that of meropenem, in some cases. Both extracts present in common free steroids and coumarins. The current in vivo antibacterial activity study further lend supports to the use of Conyza bonariensis in the treatment of infections in many traditional medicines. Cristiane Coimbra de Paula1*, Domingos Tabajara De Oliveira Martins1, Karuppusamy Arunachalam1, Sikiru Olaitan Balogun1, 2, Quessi Irias Borges1, Marcelo Garcia Picone1, Wander Miguel de Barros3, Regilane Matos da Silva Prado 1, 4


INTRODUCTION
Human infections are a serious public health problem because many pathogens such as bacteria and yeast are becoming increasingly resistant to antibiotics. 1 Despite the great advances achieved by science, among the ten main causes of death worldwide, which include ischaemic heart disease, stroke, diarrhea, HIV/ AIDS, malaria, tuberculosis, pre-term birth complications and birth asphyxia and birth trauma, the lower respiratory infections top the list. 2 It is estimated that the so called 'super-microorganisms' alone will be responsible for over 10 million deaths by 2050, making it imperative the search for alternative treatments to microorganisms which are unresponsive to most modern antibiotics. 3Similarly, opportunistic fungal infections and resistance to antifungal agents have increased significantly in immunocompromised patients and these infections are responsible for a high rate of morbidity/mortality in severe cases. 1,4he growing need for more effective and safe antimicrobial agents has led to the renewal of multidisciplinary investigation on natural products, where new approaches combined with traditional techniques, are accelerating tracking of substances, which present antimicrobial activities.In addition, it has also allowed identification of the molecular targets responsible for their effects, moreover, many of these substances present new mechanisms of action. 4edicinal plants constitute an arsenal of chemicals S153 that could be exploited by human to prevent microbial invasion and have been a major source for drug development.All over the world, plant extracts and their products are used in the treatment of bacterial, fungal and viral infections. 57] Many plants are used in Brazil in the form of crude extracts, infusion or poultice to treat common infections, without any scientific evidence.] Brazil possesses the largest floristic diversity on Earth, containing six continental biomes, the Amazon rainforest, the Cerrado the Caatinga, the Atlantic forest, the Pantanal and the Pampas, with Amazon rainforest the most noteworthy, since it is the largest tropical forest in the world.In addition, the diversity of plant species constitutes an endless source for the research on herbal remedies for the development of new molecules with biological activities. 2 The state of Mato Grosso (MT), the largest farming and livestock producer in Brazil, contains three important bio geographical regions (Amazon rain forest, Brazilian Savannah and Pantanal) and a rich ethnic-cultural diversity, represented by 42 indigenous groups and traditional Quilombola, Cabocla and Riverine Communities.In vitro antimicrobial tests allow selection of crude extracts of plants with potential properties through use of chemical and pharmacological studies.1][12] However, in vitro susceptibility testing is only one-step in the evaluation of the potential efficacy of antimicrobial agents against microbial organisms.Based on the aforementioned, this study aimed at screening selected medicinal plants from Mato Grosso, through in vitro antimicrobial activity methods with the view of selecting the most promising for in vivo antibacterial study.As part of our on-going research towards development of new antimicrobial for use in humans, the aim of the present study was to screen medicinal plants used popularly in the state of Mato Grosso for treating infections, with the sole purpose of selecting the most promising plant.Conyza bonariensis extracts were selected for further studies based on its in vitro antimicrobial activities and the availability of extensive reports of its use in ethnomedicine.The important uses include among others microbial infections; wound healing, constipation, diarrhea, and inflammation, just to mention but few.Numerous species of the genus Conyza have been extensively used in popular folk medicine.][15][16][17][18][19][20] There are also reports of several biological, phytochemical and pharmacological studies of different extracts or derivatives from this plant that have supported its popular use in many cases.Despite several in vitro studies, none has ventured to evaluate Conyza bonariensis in vivo antibacterial activity in experimental rodents.Thus in the present work, we present its in vitro activities using different methods, the acute toxicity and it's in vivo prophylactic effect in systemic infection model.

Experimental Animals
Albino mice Mus musculus, Swiss-Webster strain (25-30 g) were used for the in vivo anti-bacterial studies.Animals were maintained in polypropylene cages at 26°C in a 12 h light-dark cycle, with free access to standard laboratory chow and water.Groups of six animals were used for each experiment.The experimental protocol followed the International Principles for the Biomedical Research Involving Animal 13 and was approved by the Committee on the Use of Animal for experimentation (CEUA/UFMT) with protocol number 23108,047577/09-1.The number of animals and the intensity of the stimuli used were minimum required to demonstrate in a consistent manner the effect of treatments.

Extract preparations
The extracts of the plants were prepared at the Natural Products Laboratory of Pharmacology, Faculty of Medicine, UFMT.The parts of the plants were collected cleaned and dried in the shade at room temperature for a period of 7 days, were milled and sieved using electric miller, result-ing in 100 g of powdered plant material.

Preliminary phytochemical analysis
Preliminary phytochemical tests were performed to identify the following principal secondary metabolite groups: tannins, flavonoids, steroids and triterpenoids, saponins, alkaloids, coumarins and quinones, through a process of qualitative prospecting. 16The preliminary phytochemical analysis was carried out by using the following standard methods.
Test for tannins: 10 mL of bromine water was added to the 0.5 g crude extracts.Decoloration of bromine water showed the presence of tannins.
Tests for flavonoids shinoda test: Pieces of magnesium ribbon and HCl concentrated were mixed with crude plant extracts after few minutes and pink color showed the presence of flavonoid.
Test for steroids: steroids was sought by the reaction of Liebermann, 10 mL of crude extracts were evaporated.The residue was dissolved in 0.5 mL of hot acetic anhydride; we added 0.5 mL of the filtrate chloroforme.Treated with the reagent of Libermann Burchardt.The appearance, at the interphase, a ring of blue-green, showed a positive reaction.

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mL of concentrated sulphuric acid was added along the sides of the test tube.Formation of a pink colour indicates the presence of triterpenoids.Test for saponins: 5.0 mL of distilled water was mixed with crude plant extracts in a test tube and it was mixed vigorously.The frothing was mixed with few drops of olive oil and mixed vigorously and the foam appearance showed the presence of saponins.
Test for anthraquinones: 10 mL of benzene was added in 6 g of the crude plant extracts in a conical flask and soaked for 10 min and then filtered.Further 10 mL of 10% ammonia solution was added to the filtrate and shaken vigorously for 30 s and pink, violet, or red color indicated the presence of anthraquinones in the ammonia phase.
Test for alkaloids: Dragendorff 's test To 2 mg of the crude extracts 5 mL of distilled water was added, 2 M HCl was added until an acid reaction occurs.To this 1 mL of Dragendorff 's reagent was added.Formation of orange or orange red precipitate indicates the presence of alkaloids.
Test for coumarins: Evaporate 5 mL of ethanolic solution, dissolve the residue in 1-2 mL of hot distilled water and divide the volume into two parts.Take half the volume as a witness and to add another volume of 0.5 mL 10% NH 4 OH.Put two spots on filter paper and examined under UV light.Intense fluorescence indicates the presence of coumarins.

Disc diffusion assay
The disc diffusion method was used for the tests disc.Sterile Filter papers (7 mm in diameter, (Sensibiodisc-Cecon, São Paulo, Brazil) impregnated with extract solution (20 µL) were placed on Muller-Hinton agar (Oxoid, Thermo Fisher Scientific, São Paulo, Brazil) and Saubouraud agar (Oxoid, Thermo Fisher Scientific, São Paulo, Brazil), according to the method of Kirby et al. 17 against nine bacteria species, being 6 Gramnegative and 3 Gram-positive, and 5 leveduriforms (Candida spp.).The test plates were prepared with Müller-Hintonand Saubouraud agar and were inoculated on the surface with bacterial and fungal suspension respectively, prepared in sterile saline (0.9%).
The concentration of the bacterial suspension was adjusted to 0.5 Mac-Farland scale (1x10 5 CFU/mL) and the fungal suspension was adjusted to 1 MacFarland scale (1x10 5 UFC/mL).The extracts were tested at different concentrations (20 -0.009 µg/disc), using chloramphenicol (30 μg/disc, Sensibiodisc-Cecon, São Paulo, Brazil) and amphotericin B (100 μg/disc, Sensibiodisc-Cecon, São Paulo, Brazil) as the standard drugs.The negative controls for the extracts were as follows: distilled water for MEAhl, MEAHc, MECr, MEHh, DMSO (0.04%) and Tween 80 (2%) in distilled water for MEPm, MEGb, MECp, MECbl, MECbr and MELp.The plates were placed in a refrigerator for 4 h, so that the test drug will diffuse throughout the medium.After this period, the plates were incubated at 37°C for 24 h and we subsequently proceeded to measure the zones of inhibition of bacterial growth, considering the active zones of inhibition of bacterial growth ≥ 10 mm. 6 Tests were performed in duplicates.

Broth micro dilution
The antibacterial activities of the extracts were evaluated by determining the minimal inhibitory concentration (MIC) according to guidelines established by Clinical and Laboratory Standards Institute (CLSI).MICs were determined using micro plates of 96 wells according to CLSI guidelines. 18Stock solutions of the extracts in distilled water were diluted to give serial twofold dilutions that were added to each medium, resulting in concentrations ranging from 1000 -1.9 μg/mL of the extracts.Inoculum of 100 μL (final concentration 10 4 CFU/mL) were added to Mueller-Hinton broth.Chloramphenicol (50 -3.1 μg/mL) (Sigma, São Paulo, Brazil) was used as positive control.The culture medium 0.04% DMSO served as the negative control.Plates were incubated for 24 h at 35ºC.
The same procedure was used to evaluate the antifungal activity, using the Saubouraud medium (Acumedia, São Paulo, Brazil) incubated for 24 h.Amphotericin B (100 -3.25 μg/mL) (Sigma, São Paulo, Brazil) was used as standard drug.The reading of MIC was performed manually or visually, considering the presence of turbidity in each microplate. 19he reading was performed using the microplate reader method.The criteria used to classify the activity of the extracts were: MIC ≤ 100 μg/ mL good antimicrobial activity; when the MIC between 100 -500 μg/ mL, moderate activity; MIC above 500 -1000 μg/mL, weak activity and MIC ≥ 1000 μg/mL inactive. 20The MIC is the lowest concentration of the test drug that was able to inhibit completely the bacterial growth in the medium.All tests were conducted in duplicates.

Acute toxicity screening test
The effect of MECbl and MECbr on the general behavior of conscious animals was evaluated in mice, as previously described by Malone and Robichaud. 21

Systemic bacterial infection in mice
For the systemic infection experiments, 22 the MECbl and MECbr were used against two bacterial clinical isolates of S. aureus and E. coli.Swiss albino male and female mice, weighing between 25-35 g were allocated into 10 groups of 10 animals each.The negative control group received distilled water (vehicle) orally and the positive control group received meropenem (Biochimico, São Paulo, Brazil) 20 mg/kg subcutaneously as treatment.In the test groups, different doses (0.01; 0.1; 1; 10; 50; 100; 200; 300 and 500 mg/kg) of the tested extracts were given orally.The bacterial strains were plated on nutrient agar, 24 h before the experiment (Biobrás, São Paulo, Brazil).The bacterial inoculum of S. aureus was adjusted to MacFarland 6 scale (21x10 8 CFU/mL), for E. coli the scale was MacFarland 3 scale (9x10 8 CFU/mL).These bacterial concentrations are capable of inducing systemic infection in the animals and causing death in 100% of the animals in less than 14 days.Bacterial infection was induced by the intraperitoneal administration (0.2 mL) of the bacterial suspension in BHI broth (Biobrás®, São Paulo, Brazil).Treatments of the animals were done immediately and 4 h after inoculation of the animals, and they were observed for 14 days to record mortality.

Data analysis
The Bartlett's test was used to test for homogeneity of variance between groups.When no significant heterogeneity was detected, one-way analysis of variance (ANOVA) was applied, followed by Student-Newman-Keuls multiple comparison test.P < 0.05 level was considered as significant.Graph Pad Prism© version 5.01 for Windows (Graph Pad Software, USA) was used for statistical analysis.

Preliminary phytochemical analysis
Preliminary phytochemical analysis of the extracts revealed the presence of flavonoids, tanins, alkaloids, free steroids, saponins, reduced anthraquinones, triterpenes and coumarins (

Antimicrobial activity
Disc diffusion assay Antibacterial activities of the plants' methanolic extracts obtained against the Gram-positive and Gram-negative bacteria organisms in the disc diffusion method are shown in Table 3.All the plant extracts tested demonstrated antibacterial activity against one or more bacterial agents.However, they differ in their spectrum of activities against the microorganisms.On one hand, none of the extracts was active against K. pneumoneae, S. flexneri and P. mirabilis, whereas, E. feacalis was the most sensitive bacterial strain.Chloramphenicol, the standard antibiotic used in this assay was active against all the tested strains (

Disc diffusion assay
The antifungal activities of the extracts using disc diffusion method can be seen in

Broth microdilution assay
Similar to the results obtained in the antibacterial micro broth dilution assay, all plants' extracts demonstrated moderate to weak activity against all the fungal strains tested.However, Amphotericin B showed superior activity against all the leveduriform strains with MIC ranging between 0.25 and 1.0 μg/mL (Table 6).criteria.These criteria specifically were: the preponderance of reports that have demonstrated scientific evidence of the ethnomedicinal uses of the plant in question; the potential antibacterial and antifungal activities observed; literature evidence concerning its pharmacological and biological activities, reports of toxicity, if any, and if there is study with human subject.We therefore proceeded only with the methanolic extracts of Conyza bonariensis leaves and root for the in vivo toxicological and antimicrobial evaluations.

In vivo acute toxicity study
The in vivo oral acute toxicity study of the two extracts MECb extracts (root and leaves) demonstrated that both extracts are safe at doses up to 5000 mg/kg, as no behavioural or deaths were recorded after 14 days of observations (Table 7).

Systemic bacterial infection in mice
Table 8, shows the protective effects of MECbl, MECbr and meropenem, on a murine systemic infection model induced by a variety of pathogens.The protective effect of MECbl was comparable to that of imipenem and stronger than that of MECbr for infections induced by S. aureus.For the other gram-negative bacterial infection i.e.E. coli, its protective effect was inferior to that meropenem, but superior to MECbr, which lack effect on the E. coli.In of general, by observing the in vitro and in vivo results, it is evident that S. aureus was more susceptible to the extracts than the E. coli.Intriguingly, at the maximum dose of 500 mg/kg there seems to be reductions in the prophylactic activities of the two extracts.

DISCUSSION
As part of our research goals, in identifying medicinal plants with potential for phytotherapeutic ends, we screened selected medicinal plants from the Cerrado of the state of Mato Grosso for potential antimicrobial use.Generally, the antimicrobial activities of natural products are screened using in vitro biological assays susceptibility testing. 37Several plants were selected based on initial ethnobotanical survey using for screening of their antibacterial activity the disc diffusion, agar diffusion and micro dilution methods, that are the most commonly used for screening plant extracts with potential antimicrobial activities. 38nitial screening of the 11 extracts showed that all the extracts displayed antibacterial and antifungal activities to more than one pathogen tested, although at varying degrees.4][25][26][27][28][29][30][31][32][33][34][35][36] Although, there are some reports concerning antimicrobial activities of some of the plants tested (Table 9), the main difficulties in comparing previous studies, lies in the fact that the criteria, method and end-points used for reporting the activity are very diverse.As can be seen in the table in the case of Gossypium barbadense, the minimum concentration used in the study by Ikobi et al. 32 and regarded to represent antibacterial activity, is considered in our study to be too high (10 folds increase compared to the maximum dose we utilized), and regarded as not having activity.The genus Conyza(Asteraceae) is comprised of approximately 400 species and several species are known for their use in traditional medicine. 39][47][48][49][50][51] C. bonariensis (leaves (MECbl) and root (MECbr) extracts) were selected based on its modest in vitro antibacterial activity results and the vast amount of studies done on different parts of the plants from different parts of the world.In the popular medicines, different parts of C. bonariensis, in the form of infusion or decoction of its parts, are used as antiseptic, anti-ulcer-

Extract
6][47][48][49][50][51] In fact, promising results were obtained with the methanolic extract of C. bonariensis from Pakistan, as it demonstrated to be active in DMBA-induced skin carcinogenesis in vivo studies. 52easonable comparisons with previous studies could not be made for many reasons.We have summarized these studies in Table 9, with short comments added for clarifications.These include among others, the use of different antimicrobial assay methods from those we employed in this work and/or sometimes the experimental conditions were poorly described.For example, Avancini and Wiest 53 (only reported that 1g of the extract was macerated in 10 mL of hydroethanolic solution of C. bonariensis without stating the concentration of ethanol used, nor the yield of the extract so as to ascertain the active concentration.Sometimes different parts of the plants are used and or its essential oils 54 or different solvents in most cases.Moreover, in some occasions, the concentrations used are ten or more folds higher than the maximum concentration we employed (Table 9).We encountered similar impediments, as in the case of the in vitro antibacterial studies of C. bonariensis, while trying to compare our results with previously reported in vitro antifungal studies.Most reports with previous studies.See Table 9 for more details on these issues.. 55 In vivo acute toxicity is usually performed on drug candidate for the purposes of: classification and labeling, to provide basic information on the mode of toxic action of a substance if any, to help in the choice of dose of a new compound, as well as to help in dose determination in animal studies. 56We therefore conducted the Hippocratic screening, to evaluate the potential toxic properties of the extracts.The acute toxicity test of the extracts administered orally demonstrated the high safety margin of MECbr and MECbl, suggesting lack of toxicity at the level of dose to be used in the in vivo studies.The no adverse effect level (NOAEL) in the oral acute toxicity study of MECbl and MECbr was calculated to be above 5000 mg/kg b.w.The human equivalent dose (HED) of 5000 mg/ kg in the rats using body surface area was 405.4 mg/kg b.w. 57Although, there are no reports of the toxicity studies of Conyza bonariensis in the literature, toxicity of some other species of Conyza have been studied.][47][48][49][50][51] Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 S161 With these promising results of both the in vitro antimicrobial activities and the lack of in vivo acute toxicity, we proceeded to evaluate the in vivo antibacterial effects of the plant using S. aureus and E. coli systemic infection models.However robust might be the results of in vitro studies, in vivo testing is without doubt one of the recognized, if not the most important, essential links between in vitro sensitivity testing and clinical studies in humans.

MEAhl
Based on this fact, several regulatory agencies in many countries have made it as explicit requirements of experimental evaluation of new compounds in animals, destined for human, as part of guidelines for the clinical evaluation of efficacy and toxicity of anti-infective drugs, being prerequisites to clinical trials. 58taphylococcus aureus is an important human pathogen responsible for many infectious diseases, sometimes life-threatening, including skin and soft tissue infections (SSTIs), foreign-body infections, bloodstream infections, just to mention but few, in both hospital and community settings.On the other hand, E.coli, is a known pathogen and one of the most frequent and lethal causes of bloodstream infections. 59We therefore selected these two bacterial strains for the in vivo antibacterial activity, based on their clinical relevance.Our results from in vivo murine systemic infection model revealed that treatments with MECbl and MECbr demonstrated potential antibacterial activities, particularly, their prophylactic activity in the systemic infections caused by Gram-positive and Gram-negative microorganisms (S. aureus and E. coli).
The effect of MECbl was similar to that of the standard antibiotic, meropenem, in the case of S. aureus systemic infection, but milder in the case of E. coli.Thus, demonstrating the potential of this plant as an anti-bacterial agent.Considering the maximum dose (500 mg/kg) used in these studies, the HED is estimated at 40.5 mg/kg.Simple comparison of this value with HED of the NOAEL shows that it is 10 folds less, further testifying to its high safety margin.In general, the in vivo antibacterial effect of MECbl and MECbr seem to be more effective on the Gram-positive bacterium (S. aureus) than the Gram-negative (E.coli) bacterium.The Gram-negative bacteria are implicated in the pathogenesis of severe sepsis and septic shock, although the exact mechanism is uncertain.A number of studies have been conducted to decipher the pathophysiological differences in bacteraemia with different causative bacterial species.In the study of patients admitted to the general intensive care unit (ICU) of a university teaching hospital by Abe et al. 60 the authors observed that the incidence of Gram-negative bacteraemia was significantly higher in bacteraemia ICU patients with septic shock than in those with sepsis or severe sepsis.They concluded that the Gram-negative bacteraemia induces greater magnitude of inflammatory response than Gram-positive bacteraemia.In fact, these authors showed that the C-reactive protein and IL-6 levels were significantly higher in Gram-negative bacteraemia than in Gram-positive bacteraemia.These may actually explain the difference in the response to the extracts by these two bacterial strains, representing the Gram-negative and Gram-positive strains. 61e also observed that maximal positive response occurred at up to certain dose level, beyond which it declines (Table 8).The exact mechanism responsible for this effect is not known, but is sometimes seen in the effects of plants extracts and phytochemical compounds. 62However, the disc diffusion method is restrict to evaluate antimicrobial activities of plant extracts because the activity of the substances present in the extracts depends on the solubility of metabolites in the medium to act in the micro-organism.In the case of MESr the preliminary phytochemical analysis indicated the presence of various classes of secondary metabolites (free steroids, coumarins, reduced anthraquinones, saponins, tan-nins and flavonoids) many of non-polar categories.So it is probable that these substances have difficulty to diffuse across the agar, but if these are in direct contact with the bacterium like in the broth micro dilution method the solubility of the substances is not an impairment factor. 63nother possibility is that Enterobacter aerogenes is a Gram-negative bacterium and consequently is more resistant to antibiotic, because it has outer membrane that is not present in Gram-positive bacterium like Streptococcus pyogenes.The presence of saponins in the MESr may facilitate the penetration of the compounds across the outer membrane of bacterium.On the other hand, MECb1 presented in preliminary photochemical analysis only two classes of secondary metabolites (free steroids and coumarins).It is possible that the substances presents in MECbl have sufficient capacity to diffuse on the agar and exert their action against-Streptococcus pyogenes, a Gram-positive bacterium.It is noteworthy that we are talking about two different species of bacteria, one Gram-positive that is usually more sensitive to antibiotics and another Gram-positive in general, more resistant to antibiotics. 64Moreover, various hypotheses have been postulated to explain this phenomenon.These include the fact that many phytochemical compounds are pleitropic molecules that may act by binding to certain receptor.Desensitization of such receptor(s) may occur at higher drug dose, thereby resulting in little or no effect as compared to the lower dose.Increases in the dose may also triggered an untoward effect on other body systems, thereby provoking a negative response toward the antibacterial activity of observed.The induction or enzymatic systems (phase II), responsible for detoxification of xenobiotic.Taken together, it is probable that a higher dose predisposes the physiological system to excrete more of MECbl and MECbr, thus lowering their effective physiological concentration, and hence, diminished protective effects.Preliminary phytochemical analysis of MECbl revealed the presence of flavonoids, coumarins and free steroids.There are considerable in formation in the literature detailing the antibacterial activities of the identified phytochemical constituents.The antibacterial effects of MECbl and MECbr may therefore be due in part to the presence of the aforementioned metabolites, and possibly through a synergistic and or combined effects and may be responsible for its antibacterial activity established in this study.To the best of our knowledge, this is the first study dealing with the in vivo antibacterial activity of MECbl and MECbr.

CONCLUSION
In conclusion, systemic infection studies demonstrated that C. bonariensis had in vivo antimicrobial activity comparable to that of meropenem.This in vivo antimicrobial activity study confirmed that methanol extracts of C. bonariensis has high activity and deserves further investigation.The present results confirm previous in vitro studies by many researchers on different extracts of C. bonariensis further lending support to its use as anti-infective in traditional medicine.There is need for further studies to identify probable metabolites responsible for the in vivo antibacterial activity and possible mechanism of action of the extracts.

Table 1 : Plants collected, place of collection in Mato Grosso State, Brazil, and voucher number. Scientific name/ Family Main vernacular name Part collected/Medicinal use Place of collection Voucher number Reference
S154Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Briefly, male and female mice (n=3/group) received by gavage (p.o.) MECbl and MECbr at doses of 500, 1,000, 2,000 and 5,000 mg/kg body weight (b.w.).One control animal per group, received the vehicle (distilled water, 10 mL/kg).Animals were observed individually in open field at 5, 10, 15, 30, 60, 120 and 240 min and once a day, for a period of 14 days, noting any clinical signs or mortality.

Table 4 : Antibacterial activity in broth microdilution assay of selected medicinal plant methanolic extracts from the state of Mato Grosso, Brazil.
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Table 8 : In vivo antibacterial activity methanolic extracts of Conyza bonariensis leaves and root in the systemic infection models in mice by Staphylococcus aureus and Escherichia coli.
Sa: Staphylococcus aureus; Ec: Escherichia coli; MECbl and MECbr: methanolic extract of Conyza bonariensis leaves and root, respectively.