IN VITRO ANTIBACTERIAL EFFECT OF EUTERPE OLERACEA MART . AND THEOBROMA GRANDIFLORUM HYDROALCOHOLIC EXTRACTS ( Efeito antibacteriano In vitro de extratos hidroalcoólicos de Euterpe oleracea Mart . E Theobroma grandiflorum )

This study evaluated the in vitro antimicrobial activity of these species against strains of Gram-positive and Gram-negative bacteria. The hydroalcoholic extracts were prepared from dried leaves, pulp and seeds of E. oleracea Mart. and T. grandiflorum by continuous percolation with 70% ethyl alcohol. The antimicrobial activity was evaluated against four microorganisms by the agar disc diffusion method and the minimal inhibitory concentration (MIC) assay. The antimicrobial activity showed that the açai pulp and seeds possessed significant inhibition in Clostridium perfringens (320 and 640 MIC), Staphylococcus aureus (80 and 320 MIC) and Pseudomonas aeruginosa (640 and 2560 MIC). Cupuassu extracts showed no effect on any bacteria. The use of açai extract products can be a sustainable, viable and an accessible alternative for antimicrobial treatment. New studies should be conducted to determine better results for acai herbals and new formulations of cupuassu extracts.


INTRODUCTION
Due to existing cases of antimicrobial resistance against a diverse number of bacteria, new alternatives to combat these pathogens are being investigated.
In recent years, phytotherapy research has increased, opening a new possibility as a viable complementary treatment for bacterial infections (Arantes et al., 2014).Besides being a new solution against resistant pathogens, these agents are readily accepted, due to the fact that they are generally less toxic to the host organism and to the environment (Souza et al., 2013).
The Amazon Forrest has the largest plant diversity in the world with various plants with known antimicrobial properties (Suffredini et al., 2006;Ranilla et al. 2012).However, more than 80% of the plants still have to be studied and their medicinal properties elucidated (Silva & Franco 2010).Açai (Euterpe oleracea Mart.) and cupuassu (Theobroma grandiflorum) are among the most studied plants in Brazil (Carvalho et al., 2009;Santos et al., 2011).Studies have demonstrated the versatility of producing different pharmaceutical compounds using these plants using raw material, due to the diversity of the existing active compounds (Hanada et al., 2010;Gonçalves et al., 2012).
Clostridium perfringens is a group of obligatory anaerobic bacteria highly pathogenic to both animal and humans.
Due to the inadequate use of possesses a notable ability to develop resistance to medications shortly after it's used (Weese & van Duijkeren, 2010).
The objective of this study was to evaluate the effectiveness of different formulations of phytotherapeutic agents as antimicrobials, produced from leaves, fruits and seeds from açai and cupuassu.

MATERIAL AND METHODS
The açai (Euterpe oleracea Mart.) leaves, fruits and seeds were collected The hydroalcoholic extracts were obtained utilizing an amber flask and sealing all its lateral extensions with aluminum foil.Seventy-two grams of each plant part were placed in separate flasks with 720 ml of 70% ethyl alcohol (v/v).Subsequently, the content was stored for 30 days in the refrigerator at 4 0 C and stirred periodically.to 100 (susceptible).
The açai pulp showed the lowest DPPH scavenging potential with IC50 values of 52.7μg/ml (Table 1) followed by the açai at the Biological Science Institute of the Federal University of Para, city of Belem, Brazil in July 2014.The cupuassu (Theobroma grandiflorum)leaves, fruits, and seeds were collected at Ver-o-Peso Market, in the same city and time period.The material was identified by its standard botanical description(Henderson, 1995).After collection, the material was dried at room temperature and subsequently grounded in a mill.This was followed by inspection and removal of foreign bodies and the product was subsequently stored in a dark recipient, protected from light and hermetically sealed, up to the time to be used in the production of the extracts.
corresponding to 0.5 McFarland turbidity standard equivalent to 1.5x10 6 UFC ml -1 .The agar diffusion test was done according to the method described by Karaman et al. (2003).With the aid of a sterile swab, the microbial inoculum was streaked onto the surface of the Muller-Hinton agar.The agar was poured at a depth of 4 mm within a Petri dish.Filter paper discs of 6 mm in diameter were impregnated with 10 μl of extract.Using the serial dilution method, a total of eight different concentrations (10-2.560μg/ml) of the plant extracts were obtained.The disks were deposited at a distance of 30mm from each other and the periphery of the petri dish to reduce overlaying of the inhibition zone.The negative controls were prepared utilizing the same solvents used to dissolve the extracts.For the positive control Ciprofloxacin (40 μg/disk) and Gentamicin (30 μg/disk) were utilized.The plates were incubated at 37 o C for 24h.Each test in this experiment was done in triplicates.The antimicrobial activity was evaluated by measuring the inhibition zone formed around the disks.The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the extract able to cause the death of the inoculum.The microbial susceptibility index (MSI) was adapted from Eloff (2004) method, to compare the relative susceptibility among the microbial strains.MSI values ranged from 0 (resistant to all extracts) above 5.0.The antimicrobial effect of tannins has been confirmed in several studies.Its activity is due to enzyme inhibition in a direct action to the membrane, competing for metallic ions, essential for microbial metabolism (Shohayeb et al., 2013), whereas the antimicrobial activity of sterols occurs due to the alteration in the pH of the media and the alkylation of important proteins of the microorganisms (Schinor et al., 2007).Anthocyanins are known for their medicinal properties, such as its antimicrobial properties due to their antioxidant action (Rocha et al., 2011).Saponins, catechins and triterpenes possess relative bactericidal activity, especially against Gram-positive bacteria (Avato flavonoids were found to be the major polyphenols in these plants.Many studies have demonstrated that flavonoids have strong anti-oxidant activities and others biological properties (Kang et al., 2010).In these plants, this compound is found primarily within the pulp of the fruit/plant.Other polyphenols that are also responsible for antioxidant activity are tannins and lignins (Gülçin, 2012).To establish the toxicity of A. annua, the lethal-dose test against A. salina was used as a highly reliable assay to determine the toxicity of phytotherapeutics agents (Fernández-Calienes Valdés et al., 2009).The brine shrimp bioassay is a simple, rapid and low cost test that can be performed in vitro (Lieberman, 1999).It is important to analyze the toxicity of new therapeutic agents to verify the possible side effects when the product is tested in vivo.The results of the toxicity test are shown in Table 1.None of the extracts were toxic with values exceeding 1000 μg/ml.The antimicrobial activity of the plant extracts is shown in Table 2.The extracts produced from the açai fruits and seeds, demonstrated antimicrobial activity against C. perfringens, S. aureus and P. aeruginosa.However, the extract produced from the açai leaves presented antimicrobial activity only against S. aureus.The extracts produced with the different parts of the cupuassu were not effective against any bacteria.None of the products had any effect against E. coli.MSI values were useful in evaluating the susceptibility of the different strains of microbes to the tested material.S. aureus (ATCC 29213) strain was found to be the most susceptible organism (MSI of 50) (Table 3).On the other hand, E. coli (ATCC 25922) showed to be resistant to all products.Araújo et al., (2013) demonstrated that extracts from the seed of the açai inhibit the growth of S. aureus.Melhorança-Filho & Pereira, (2012) studied the antimicrobial activity of the oils produced from the açai fruit and observed antimicrobial activity against S. aureus.This bacteria is highly sensitive to antibiotics and substances with a similar mode of action, and as such, it can be used as a biological indicator for substances with potential antimicrobial activity (Oliveira et al., 2008).However, there are a few reports of the germicidal action of the extracts produced from the açai plant against gram-negative organisms.It is believed that this occurs due to the more complex structure and a greater lipid content of the cell wall of the plant, which could make the bacteria more resistant to phytochemicals (Efstratioi et al., 2012).