Action of Essential Oils Obtained from Baccharis coridifolia D . C . ( Asteraceae-Astereae ) on the Activity of Antibiotics *

The aim of this study was to investigate the effects of Baccharis coridifolia essential oil on the Activity of Antibiotics. Assays were performed with ampicillin (10 μg), cephalothin (30 μg), chloramphenicol (30 μg), gentamicin (10 μg) and tetracycline (30 μg) alone and in combination with the essential oil (4% v/v) through the disk diffusion susceptibility test. The results showed the effects of essential oil on the activity of the antibiotics tested. Zones of inhibition of bacterial growth with different diameters were observed surrounding the antibiotic disks, whether or not they were impregnated with the essential oil. The occurrence of the synergistic or antagonistic effect was observed in both bacterial strains assessed—Staphylococcus aureus (ATCC-25923) and Escherichia coli (ATCC-25922). These results show that the use of products derived from plants can, in some cases, interfere with the effectiveness of antibiotics during clinical therapy.


Introduction
The use of natural products as a therapeutic resource is as old as human civilization.Even with the growing economic power of the large pharmaceutical companies, natural products have not lost their place in therapy, being mistakenly regarded by the population as safe medicines and being increasingly used [1].
Plants with therapeutic properties used in traditional health care are an important source of new biologically active compounds.They have appeared as part of the traditional health care in many parts of the world for decades and have attracted the interest of several researchers [2][3][4][5][6].
In folk medicine, plants are used concomitantly with conventional medicaments [7].In this combined use, medicinal plants and/or their byproducts may act by inhibiting or enhancing the therapeutic effect of conventional drugs, as well as not interacting in the expected way [8].In dermatology, such associative use often puts the patient at risk, as it can trigger phytodermatitis due to irritation or photosensitivity mechanisms.This practice may also hinder the clinical diagnosis, as often the patients are not aware of the importance of informing health care professionals about the use of medicinal plants during a medical appointment.
Oils and plant extracts have long been used in numerous applications in folk medicine, including the production of topical antiseptics.This was the basis for several scientific investigations aimed at confirming the antimicrobial activity of essential oils [9][10][11][12][13].
Celiktas et al. [14] related the increase of antimicrobial activity of Rosmarinus officinalis essential oil in samples collected during the spring.Moon et al. [15] found significant variation in the size of inhibition zones when testing the antimicrobial activity of essential oils from different samples of Lavandula angustifolia against the same microorganisms.
The active ingredients present in plants have been extensively studied in many countries, because new drugs can be developed with this knowledge.Understanding the interactions between drugs and plants can also be useful in therapy (beneficial interaction) and for disease prevention.
Plants of the family Asteraceae are extensively studied for their chemical composition and biological activity, with some supporting the development of new drugs, insecticides and other products [16][17][18].
Assessing the biological activity of essential oils produced by B. dracunculifolia, B. uncinella and B. coridifolia Ferronato et al. [13] observed the antioxidant and anti-inflammatory effect of these oils on Escherichia coli ATCC, Staphylococcus aureus ATCC, Pseudomonas aeruginosa ATCC and Streptococcus mutans isolated from saliva, thus showing the importance of these plants.Besides the antimicrobial activity observed in several plant species, natural compounds can also exhibit synergism when used in conjunction with other drugs [21].Thus, this study evaluated the effect of essential oil and extracts produced by B. coridifolia D. C. (Asteraceae) (Figure 1) on the activity of some antibiotics used in clinical therapy.

Botanical Material
The plant studied was B. coridifolia D. C. (Asteraceae) collected in the Southwest region of Paraná, Brazil, from November 2006 to November 2007.The essential oils were obtained followed the method described by Onofre et al. [13,17,18,22].This species was chosen because it is widely used in folk medicine and it contains antimicrobial substances described in previous studies [13,22].

Microorganisms
S. aureus (ATCC-25923) and E. coli (ATCC-25922) were used as test microorganisms.Suspensions were prepared in a 0.85% (w/v) NaCl solution, with turbidity level compared to the 0.5 McFarland standard, which approximately corresponds to a concentration of 10 8 CFU/mL.

Effect of Essential Oil on Antibiotic Activity
The disk diffusion susceptibility test was carried out to analyze the effect of the essential oil on antibiotic activity.The essential oil was studied at a 4% concentration, which corresponds to the MIC determined in previous studies for the microorganisms tested here [2][3][4]13,[24][25][26].The disks containing the antibiotics in their respective concentrations were impregnated with 20 μL of 4% essential oil (MIC), and then placed on sterile Petri plates containing Mueller-Hinton agar inoculated with 1 mL of bacterial suspensions.After incubating the plates at 37˚C for 48 h, the effect of the essential oil MIC on the activity of antibiotics was observed.Synergistic effect was considered when inhibition zones ≥2 mm diameter were observed surrounding disks containing the essential oil (EO) and the antibiotic (AB).Antagonistic effect was considered when an inhibition zone of smaller diameter was observed around the disks with AB and EO, compared to that developed by the AB disks.Indifferent effect was considered when there was a zone of inhibition surrounding the disks with AB and EO, with a diameter equal to that resulting from the application of the AB alone [27].All assays were performed in duplicate and the results were obtained by averaging the results of the parallel tests.

Results and Discussion
Table 1 shows the antimicrobial susceptibility of the tested strains.The Gram-positive S. aureus was susceptible to all antibiotics tested, showing growth inhibition zones with diameters ranging from 12 to 24 mm.The Gram-negative E. coli was susceptible to cephalothin, gentamicin and tetracycline.These results are in agreement with the CSLI [28].
Table 2 shows the essential oil effects on the activity of antibiotics used in clinical therapy.When comparing the zones of inhibition diameters observed in the assays with antibiotics alone and in combination with the essential oil, the effects of the essential oil on the antibiotic activity can be observed in some cases.This effect was observed with both strains tested, at different susceptibility levels.The greatest synergistic effect was observed with cephalothin, for the two strains tested.Ampicillin and chloramphenicol showed antagonistic effects for both bacteria.Gentamicin activity was inhibited (antagonistic effect) when evaluated for S. aureus, but an indifferent effect was observed when tested for E. coli.Tetracycline had indifferent effect on S. aureus and synergistic on E. coli.Generally, the effect of the essential oil on antibiotic activities varied according to antibiotic type, essential oil combination and bacterial strain tested.
The present results corroborate Canton and Onofre [29], who used the same methodology to assessed the interference of ethanol extracts obtained from B. dracunculifolia on 15 antibiotics, reporting activity changes that caused synergistic or antagonistic behaviors, and no alteration in some cases.
In a study on the extracts of Alternanthera brasiliana (L.) O. Kunt.(Amaranthaceae) and their effect on the activity of antibiotics used in clinical therapy, Araújo and  Onofre [30] found that when such extracts are associated to 18 inhibitors of protein synthesis, altered behavior and, consequently, synergism or antagonism, are observed.Indifferent behavior, though, was also observed.
It should be noted that the sale of medicinal plant products currently happens in pharmacies and health food stores, where they are marketed according to an industrialized labeling system.In general, these plant preparations have no certificate of quality and are produced from cultivated plants, which mischaracterize traditional medicine that uses almost always plants of the native flora.
The use of medicinal plants from the traditional Chinese and Hindu medicines, completely unknown to westerner civilizations, is increasingly common.These plants are marketed through advertisements that promise safe benefits, because they come from a natural source.However, often the supposed pharmacological properties advertised have no scientific validity, because they have not been investigated, or have not had their pharmacological actions proven through scientific tests in preclinical or clinical trials.
Media calls for the consumption of products made from natural sources increase every day in both developing and developed countries.Herbalists promise health and long life, based on the argument that plants used for millennia are safe for the population.
In Brazil, medicinal plants from the native flora are consumed with little or no evidence of their pharmacological properties, and their consumption is propagated by users or dealers.Frequently, these plants are even used for medicinal purposes different from those originally used by the native people.Compared to the drugs prescribed in conventional treatments, the toxicity of medicinal plants and herbal medicines may seem trivial, but this is not true.The toxicity of medicinal plants is a serious public health problem.
Research to evaluate the safe use of medicinal plants and herbal medicines is still incipient in Brazil, as well as the control of these products' sales by official bodies in public markets or health food stores.
In this sense, this work has contributed to the elucidation of the effects caused by the use of B. coridifolia, in relation to the metabolites present in two fractions, showing that such metabolites can interfere positively, causing synergism, or negatively, causing antagonism in the activity of antibiotics commonly employed in clinical therapy.
Multidisciplinary studies involving ethnobotanists, chemists, pharmacologists and agronomists are needed in order to expand the knowledge on medicinal plants.This would bring a better understanding on how their compounds act, what are the toxic and adverse effects, how they interact with new allopathic medicines and what are the most appropriate strategies for production and quality control of herbal medicines, observing the regulatory agencies standards and resolutions.
Raising awareness in the professional team involved in the prescribing, dispensing and administering medications is important.Professionals from different healthcare areas must always question and warn their patients about the non-prescribed use of medicinal herbs.

Conclusion
According to the obtained results, it is observed that the essential oil analyzed can have a greater or lesser interfering effect on the activity of the antibiotics tested, either through a synergistic or antagonistic effect.This interference was observed mainly in the assays involving both strains evaluated.Therefore, concomitant use of plant products and conventional medicines deserves careful attention, because of the possibility of interfering with the treatment of diseases of bacterial etiology.