Abstract
Antimicrobial activity of medicinal plants has been searched in several species worldwide. The aim of this study was to evaluate antibacterial activity in vitro of the ethanol extract from the peel fruit of Syzygium cumini (L.) - Skeels. To this end, were used four bacterial strains standard American Type Culture Collection (ATCC), three Gram-negative bacteria: Salmonella enteritidis (ATCC 13076), Escherichia coli (ATCC 25922) and Enterobacter aerogenes (ATCC 13048) and a Gram-positive Staphylococcus aureus (ATCC 25923). The antibacterial action was evaluated through the inoculation of Mueller Hinton agar plates. Cultures were suspended in sterile saline 0.85% (w/v) read in spectrophotometer at 600.0 nm, by adjusting of 0.5 McFarland standard turbidity standard equivalents (1.5 x 108 CFU mL-1 - colony-forming units per milliliter). The antimicrobial susceptibility testing (Antibiogram) was performed according to the method of diffusion disk or Kirby-Bauer. Under sterile conditions, were used 8 paper disks, being a disk with sterile water, 4 commercial antibacterial disks and 3 disks embedded with 50.0 µL of the jambolan ethanol extracts in concentrations 350.0 mg mL-1; 750.0 mg mL-1 and 1000.0 mg mL-1. The plates were inverted and incubated at 35.0 ºC for 24 hours, and their reading of inhibition hales diameter was done. The concentration of extract used corresponded to the minimum inhibitory concentration (MIC) of this extract, previously determined in another experiment. The experiment was conducted with 3 replicates per treatment and each treatment consisted of a series in triplicate (3 plates). Statistical analysis was performed using one-way analysis of variance (ANOVA) and the means were compared by Tukey test at 5% significance level. As verified, the ethanol extracts tested showed no antibacterial activity against ATCCs used in this study, only Staphylococcus aureus showed inhibition, but when compared to other antibiotics, was difference by Tukey test with significance level of 5%.
Keywords
Jambolan, Secondary metabolites, Antibiogram, Antimicrobial Activity, McFarland, Kirby-Bauer, minimum inhibitory concentration (MIC).
Introduction
The jambolan (Syzygium cumini (L.) Skeels), it is a plant originating from eastern India (MORTON, 1987). Its synonym names Eugenia jambolana (Lam.), Myrtus cumini L., Syzygium jambolanum (Lam.) DC andEugenia cumini Druce (MARCHIORI; SOBRAL, 1997).
The fruit has an average size of 1.0 cm diameter by 2.0 cm long and ovoid shape. The mature peel is purple, thin, glossy and sticky and the seed is surrounded by fleshy pulp and edible, sweet and astringent, and pleasant to taste (LAGO; GOMES; SILVA, 2006).
The medicinal plants therapy has been widely used in several countries as an alternative treatment for diarrhea and gastrointestinal disorders, by the bark decoction (Bragança, 1996), sedative action and anticonvulsant (LIMA et al., 1998).
Several studies have evaluated the hypoglycemic action of S. cumini using the sheet (SOARES; COSTA; CECIM, 2000) and seed (PRINCE; MENON; PARI, 1998; GROVER; VATS; RATHI, 2000) and antioxidant effect (PRINCE; MENON; PARI, 1998).
In Brazil, the jambolan fruit is usually eaten fresh, but this fruit can be processed in various forms i) jam, ii) liquor, iii) wine, iv) vinegar, v) jelly, vi) pie, vii) sweet (VIZZOTTO; FETTER, 2009).
Isoflavones, indoles, phytosterols, alkaloids, tannins, vitamins, are produced by plants against environmental aggression and thus adapting to it. These substances have antimicrobial and immunomodulatory properties (WILLIAMS, 2001).
Flavonoids are present in jambolan fruit (anthocyanins, quercetin, rutin, mirecetin) and hydrolyzable tannins that are naturally produced to protect the attack of pests and diseases and, in addition, help to withstand adverse environmental conditions. Studies have revealed that these phytochemicals prevent cancer and improve circulation and heart function (VIZZOTTO; FETTER, 2009).
Bragança (1996) showed that the stem bark of S. cumini provides antimicrobial action.
The aim of this study was to evaluate antibacterial activity in vitro of the ethanol extract from the peel fruit of Syzygium cumini (L.) - Skeels.
Methods
The study was conducted at the Federal Institute of Triângulo Mineiro - Uberlândia Campus, located at Farm Sobradinho, Rural Zone of Uberlândia-MG. Analyses were performed in physical chemistry and Microbiology Laboratories, from August 2011 to July 2012.
Jambolan fruits (Illustration 1) were collected in Uberlândia city, (18 ° 55 '8 "S, 48 ° 16' 37" W), and stored at -12.0 °C (Consul® CVU30), being transported in cool boxes with mineral ice, to the physical chemistry and microbiology laboratory.
Botanical identification was held at the Biology Institute of the Federal University of Uberlândia, compared to the voucher specimen deposited under number 62245 HUFU (Herbarium Uberlandense) reference and classified by Master Priscila Oliveira Rosa.
Jambolan fruits were sanitized in chlorinated water (Start® L. 231197), 200.0 mg mL-1 for 15 minutes of contact. After this period, the triple rinsing was done in sterile distilled water, followed by manual pulping.
Fruit peels were dried at 45.0 °C (± 0.5 °C) (Quimis® Q. 317.D222) to constant mass (Illustration 2).
The dehydrated fruit peels, protected white light, were fractionated and subjected to three stages of extraction in the presence of ethanol (CH3CH2OH) AP (ACS Vetec®, L. 0,901,291), for thirty six days at room temperature 28.0 °C (± 2.0 °C). Ethanol was added to the fractionated peel in a proportion of 4.0 g mL-1. The infusion was subjected to reduced pressure (-650.0 mm Hg) on rotaevaporator (Biothec®, M. BT351) at 45.0 °C (± 2.0 °C) until the elimination of the extractor.
The yield was based on the topic "Determination of Loss on Drying" the Brazilian Pharmacopoeia V (ANVISA, 2010), with some modifications and was calculated according to the formula Re = (Pext Ppeel-1) x 100, where Re is the yield (%); Pext corresponds to the weight of dry extract (g) and Ppell is the weight of the fresh or dried peels (g).
Standard bacterial strains from American Type Culture Collection (ATCC) of second generation were used in this study, Salmonella enteritidis (ATCC 13076), Escherichia coli (ATCC 25922) and Enterobacter aerogenes(ATCC 13048) (Gram negative) and Staphylococcus aureus (ATCC 25923) (Gram-positive) from the bacterial collection of the Microbiology Laboratory of the Federal University of Uberlândia where it remains frozen at -12.0 °C (Consul® I Facilitate).
Suspensions for each strain were prepared by transferring five or six colonies on BHI (Brain Heart Infusion) agar at 37.0 °C for a period of 24 to 48 hours, depending on the bacterial genus.
Cultures were suspended in sterile saline 0.85% (w/v) read in spectrophotometer (DR Hexis® 2800) at 600.0 nm, by adjusting of 0.5 McFarland standard turbidity standard equivalents (1.5 x 108 CFU mL-1 - colony-forming units per milliliter).
The antimicrobial activity of S. cumini extract was determined by the antimicrobial susceptibility testing (Antibiogram) according to the Disk Diffusion Method or Kirby-Bauer (BAUER, 1966).
Disks were spread over the surface of the Mueller-Hinton agar, previously inoculated with 50.0 µL of suspensions of the test organisms prepared in sterile saline 0.85% (w/v).
With four treatments; treatment 1 consisted disk containing sterile distilled water, 4 disks commercial antibiotic that show inhibition in the sensitivity test for Gram Negative and 3 disks containing ethanolic extracts at concentrations 350.0 mg mL-1, 750.0 mg mL-1 and 1000.0 mg mL-1.
Series of treatments 2 and 3 showed the distribution of treatment 1, with the replacement of bacterial inoculum. Series of treatments 4 received the inoculum Gram Positive and disks with commercial antibiotics showing inhibition for Gram Positive.
After apposition the disks, plates were inverted and incubated in BOD - Biological Oxygen Demand (Solab®, M. SL200 / 250) at 35.0 °C for 24 hours.
Plates were analyzed by measuring the halos of inhibition of the microorganism, according to NCCLS (2003). The concentration of extract used corresponded to the minimum inhibitory concentration (MIC) of this extract, previously determined in another experiment. Three replicates were performed per treatment.
Statistical analysis was performed using one-way analysis of variance (ANOVA) and the means were compared by Tukey test at 5% significance level.
Results and Discussion
Yield, calculated on the dry basis, was 60.85%.
To measure the inhibition halos was used ruler 30.0 cm, with 18 hours for the Gram negative bacteria and 21 hours for the Gram positive. The measurement was performed taking as its starting point the circumference of the disk, measuring to the edge where there was growth of microorganisms.
The results of the inhibition halos of the four treatments and respective commercial antibiotics, diluted extracts and control (sterile distilled water) are shown in Illustration 3.
According to illustration 4, treatments T1, T2 and T3 no growth inhibition zone, however, no statistical analysis to these microorganisms.
Staphylococcus aureus showed inhibition by extracts 1, 2 and 3, but compared to commercial antibiotics, no difference by Tukey test at 5% significance level.
Regarding the size of inhibition zones, variations may occur by the differences in antimicrobial concentrations, where the plant is located, season in which the material was collected and even by genetic differences in plant species. The difference can also occur due to the use of microorganisms of different strains (SILVA et al., 2010).
The hydro-alcoholic extract of the bark Abarema cochliocarpos inhibited Gram positive strains showing no inhibitory effect on Gram-negative bacteria, it may be related to the cell wall (SANTOS et al., 2007). In this study, extracts only inhibited Gram positive and this correlates with the chemical composition of the cell wall.
The results of this study are different from those obtained by Loguercio et al. (2005), which showed antimicrobial activity on Gram positive and negative using hydro-alcoholic extract 10% (w/v) leaves of Syzygium cumini.
Flavonoids determine the color of flowers, fruit and leaves and tannins influence definition of the flavor, and these chemical classes show inhibiting ability of some microorganisms (FERNANDES, SANTOS, PIMENTA, 2005). Inhibition of Staphylococcus aureus in this study may be related to the flavonoids in peel jambolan fruit.
Michelin et al. (2005) showed antimicrobial activity of ethanol extract jambolan leaves on standards strains of Escherichia coli (ATCC 25922). In this study, there was no inhibition of this strain.
Studies show differences in the type of extractor used for each plant species and this is due mainly to the polarity of the extractor (CARDOSO et al., 2010).
For future studies, higher concentrations may be used, or alternatively use another extractor to evaluate the antimicrobial effect of Syzygium cumini extract.
Conclusion
Jambolan extracts tested showed no antibacterial activity against Salmonella enteritidis (ATCC 13076), Escherichia coli (ATCC 25922) and Enterobacter aerogenes (ATCC 13048) and, although there was dose dependent inhibition at concentrations of 350.0 mg mL-1, 750.0 mg mL-1 and 1000.0 mg mL-1 for Staphylococcus aureus (ATCC 25923), this was not statistically significant difference by Tukey test at 5% significance level.
Acknowledgements
This work was supported by grants from The National Council for Scientific and Technological Development-CNPq.
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Source(s) of Funding
Grant from the federal government (The National Council for Scientific and Technological Development-CNPq) to encourage the scientific research to Ludmila, during his graduation.
Competing Interests
Not Aplicable
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