Antimicrobial activity of Brazilian plants of the genera Leguminosae and Myrtaceae

Five Brazilian plant extracts from Pimenta pseudocaryophyllus, Erythrina speciosa, Tephrosia toxicaria, Inga marginata and Cassia leptophylla found in Paraná State and currently used in folkloric medicines, were assayed for antibacterial, antifungal and toxic activities. The antibacterial and anti-yeast activities were assessed by a diffusion assay on Gram (+) bacteria (Bacillus subtilis and Staphylococcus aureus), Gram (-) bacteria (Escherichia coli and Pseudomonas aeruginosa), and the yeasts (Candida albicans, Candida krusei, and Candida tropicalis). The extracts were also tested against the phytopathogenic fungi (Lasiodiplodia theobromae, Botryosphaeria ribis, Botryosphaeria rhodina, and Fusarium verticillioides) by growth inhibition using CaptanTM as control. The toxicity was evaluated in Artemia salina through LC50 (50% median lethal concentration) using Probit analysis. P. aeruginosa was inhibited by P. pseudocaryophyllus stems, E. speciosa and C. lephophylla leaves. E. coli by P. pseudocaryophyllus stems, B. subtilis by C. leptophylla leaves, and T. toxicaria roots. C. albicans by P. pseudocaryophyllus stems and E. speciosa leaves, while C. krusei by P. pseudocaryophyllus stems, and C. tropicalis by P. pseudocaryophyllus leaves, E. speciosa leaves and I. marginata leaves. E. speciosa and P. pseudocaryophyllus leaf extracts also inhibited all the phytopathogenic fungi examined. T. toxicaria roots showed stronger toxicity towards A. salina.


INTRODUCTION
The interest in plant natural products with various therapeutic applications has been increasing considerably because of the biological properties detected in different medicinal plant extracts.These plants are widely used as folkloric medicines in rural and urban areas, as infusion or cold macerated homogenates (Rates, 2001).
Pimenta pseudocaryophyllus (Gomes) L. R. Landrum belongs to the Myrtaceae family, and is a native species of the Brazilian flora with a clove-like taste.It has been used for culinary and medicinal purposes such as flavour enhancer in cooking, tea preparations, refreshing drinks, for treatment of fatigue, colds and menstrual problems, and as agents to treat diuretic and digestive problems (Landrum and Kawasaki, 1997;Lima et al., 2006;Paula et al., 2008Paula et al., , 2010)).Pentacyclic triterpenes, glycosylated flavonoids, gallic acid, and ellagic acid have been isolated from ethanolic leaf extracts (Paula et al., 2012).
The Tephrosia genus presents some bioactive metabolites such as rotenoids and flavonoids.The species Tephrosia calophylla, Tephrosia purpurea and Tephrosia maxima of this genus was described in the treatment of various ailments, including cancer, in the Indian subcontinent (Subhadra et al., 2011), piscicides and insecticides (Cronquist, 1981).
The I. marginata fruit has been used for the treatment of vaginal ulcers, and the cooked stem works as an astringent and a vasoconstrictor (Lopez et al., 1987).Activities such as antibacterial, antifungal (Alvarez et al., 1998), anti-inflammatory and anti-diarrheic (Silva et al., 2007) have been described in various species of this genus.The secondary metabolites include phenolic compounds, saponins and non-protein amino and imino acids, and an over expression of protein amino acids, and galloyl tyrosine (Alvarez et al., 1998;Lokvam et al., 2004Lokvam et al., , 2006Lokvam et al., , 2007;;Dias et al., 2010).
Cassia species have been described to present laxative, purgative, antimicrobial, antipyretic, antiviral and anti-inflammatory properties, and have been used as folk medicines in some regions of India, Asia and Africa (Agarkar and Jadge, 1999;Viegas-Junior et al., 2006).The pharmacological potential of the aforementioned plants may be due to the presence of a broad range of secondary metabolites such as phenolic compounds, anthraquinones, steroids and stilbenoids, and piperidine alkaloids.
Considering the resistance of microorganisms to antibiotics and the biological activities of some plants, the aim of the present work was to screen five Brazilian medicinal plant extracts collected in the region of Daniel et al. 959 Londrina-Paraná State, in order to identify the most active to be submitted for further studies related to the control of human or plant diseases.The phytochemical analysis of all the plants cited earlier were previously reported by Custódio (2009), Faria et al. (2007), Martinez et al. (2012), Rieger (2011), andBurgo (2010).All the results obtained herein are described for the first time with P. pseudocaryophyllus stems, E. speciosa leaves, T. toxicaria roots, and C. leptophylla leaf extracts, as well as the toxicity and antifungal assays using Lasiodiplodia theobromae, Botryosphaeria ribis, Botryosphaeria rhodina and Fusarium verticillioides with the P. pseudocaryophyllus and I. marginata leaf extracts are also described.Brazil (latitude 23° 19' 25'' S,and longitude 51° 12' 65''W), under an UEL botanist supervision.

Preparation of the different plant extracts
Air-dried plant materials were ground in a knife mill, and the powder extracted exhaustively with different organic solvents [ethyl acetate (EA), and ethanol (ET)], and filtered through filter papers.The organic solvents were removed by vacuum distillation at 55 and 60°C using a rotary evaporator.The roots from T. toxicaria were directly and exhaustively extracted with EA to obtain the EA extract.Then it was fractionated by chromatography on a column of silica gel (50 cm × 10 cm diameter), and was eluted with hexane (HE) and methanol (ME).Parts of these fractions were used to develop the biological tests of this work.The results of all extracts and the yields are presented in Table 1.

Bacterial, yeast and filamentous fungal isolates
Two Gram (+) bacterial strains, Bacillus subtilis (ATCC 8272) and Staphyloccocus aureus (ATCC 25923), and two Gram (-) bacteria, Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922), were kindly donated by Dr. R. M. B. Quesada (Department of Microbiology, University Hospital, UEL, Londrina, PR-Brazil), and were also used to develop the screening tests.The bacterial strains were grown on Müeller-Hinton agar (MHA) medium (Oxoid), and the yeasts on Sabouraud Dextrose Agar (SDA) medium (Difco).After growth, bacteria were maintained in The dried leaves were pulverized and exhaustively extracted with ET at room temperature.

(LE)
Tephrosia toxicaria (IAC 17211) a "timbó de caiena" The roots were dried, ground in a knife mill, and then submitted to exhaustive extraction with EA.This extract was fractionated using HE and ME.
Eppendorf tubes on MHA, and yeast on SDA at 4°C.The strains were activated by subculture at 371°C over 18 to 24 h on an appropriate freshly poured agar plate prior to any of the antimicrobial tests.
Candida albicans, Candida krusei and Candida tropicalis used to develop the antimicrobial assays were isolated at UEL in the microbial laboratory of the University Hospital (HU), Londrina, PR-Brazil, where these strains were deposited.

Antimicrobial activities
Two different methods were employed to determine in vitro antimicrobial activities from the Brazilian plants extracts.

Antimicrobial activity: Assays by the well-plate diffusion method
Plant extracts were prepared at a concentration of 5 to 7 mg/ml in dimethyl sulfoxide (10%; DMSO, Merck) as solvent, and sterilized by filtration (0.20 μm Millipore filters).Extracts were screened for antimicrobial activity using the well-plate diffusion method (NCCLS, 1997(NCCLS, , 2001)).Cell suspensions of 2.0 × 10 8 CFU/ml (24 h cultures) were used as inocula.Aliquots of a 50 µl suspension containing the tested microorganism were overlaid on Petri dishes containing 14 ml of Müeller-Hinton agar for bacteria, or Sabouraud Dextrose agar for yeasts.Wells (6 mm diameter) were cut out using a cork borer, and were individually impregnated with 30 µl of extracts dissolved in DMSO (10%, v/v) and 0.5% Tween 80. Wells prepared under the same condition with the same volume of 10% DMSO solution and 0.5% Tween 80 were used as a negative control.Thirty (30 µg) of anfotericin B (Fungizon) and 50 µg of the tetracycline (tetracycline hydrochloride), penicillin (Penicillin G,crystalline) and Nystatin (Mycostatin) were prepared in 0.5% (v/v) Tween 80 in 1 ml of water.The antimicrobial references referred to earlier were used as positive controls to determine the sensitivity of one microbial strain/isolate in each of the species tested.Inoculated plates were incubated at 37°C for 24 h for bacterial strains, and 48 h for yeasts.After the incubation period the inhibition zones were measured (mm) with a ruler, against the test organisms.All the tests were performed in duplicate.

Test for antifungal activity
The in vitro biological activity of ethanolic extracts was assessed based on the radial hyphal growth rate of the fungi tested in the presence and absence of the plant extracts.Agar plugs of 7-mm diameter colonized with five filamentous phytopathogens were used as inocula.Plant extracts of concentrations of 1.48 to 1.54 mg/ml were dissolved in DMSO containing three drops of Tween 80. Aliquots (0.8 ml) of each extract were added to the assay in flasks containing 17 ml of sterile growth medium (PDA).After vortexing, 17.8 ml was poured into Petri dishes (60 × 15 mm).Plates containing only PDA with DMSO (0.8 ml) were used as negative controls.Positive controls contained the fungicide Captan™ [4cyclohexene-1, 2-dicarboximide, N-(trichloromethyl)thiol] (0.18%).The assay was performed by placing a 7-mm diameter agar plug containing the growing fungal mycelium at the center of a Petri dish, and left to growth.Three replicates were run simultaneously (Quiroga et al., 2001).In all the plates, the radial mycelia growth was measured after 7 to 10 days, which was the time required for the micro-organisms to grow in the culture medium containing only PDA at 28 ± 2°C.Each data point represented the mean of at least four measurements of a growing colony (MGC: mycelial growth control; MGPE: mycelial growth with the plant extract).Growth inhibition (GI) was calculated from the expression: GI (%) = MGC -MGPE/MGC.All experiments were conducted in replicates of three.

Toxicity to Artemia salina
A. salina (brine shrimp) assay was performed according to Meyer's method (Meyer et al., 1982) with some modifications.An amount of A. salina eggs (Maramar, Rio de Janeiro) was incubated in fresh artificial sea water.All plant extracts and the positive control using potassium dichromate solution (PDS, K 2 Cr 2 O 7 ) were dissolved in three drops of Tween 80, 2 ml of dimethyl sulfoxide (DMSO), nine to twelve nauplii larvae were deposited in each glass tube, and the volume was completed to a final 5 ml with artificial sea water.
After 24 h incubation, the number of survivors and dead-hatched, brine shrimp larvae, (nauplii) in each one of the tubes were noted and registered.LC 50 (50% median lethal concentration values) and 95% confidence limits were estimated using Finney's statistical method by Probit analysis (Finney, 1952).Two control samples, one of which contained fresh artificial sea water, and other with PDS, were run simultaneously under the aforementioned conditions.The experiments were performed in quadruplicate and developed according to Sam (1993).

Statistical analysis
The filamentous antifungal data were expressed as mean ± standard error of mean (SEM), and data evaluated by statistical analysis (ANOVA) to determine the significance level of the differences (p<0.05).The statistical significance for the differences between extracts was detected by ANOVA, followed by the Tukey test.The p values under 0.05 (p<0.05) were considered significant.Data analysis was performed by R software (R Core Team (2013), http:/www.R-project.org/).A pairwise comparison of each treatment was performed using Tukey's multiple-range comparison tests to identify the significant differences between the fungal bioassay results.Finney's statistical method by Probit analysis (Finney, 1952) was used to analyse the toxicity to A. salina.

RESULTS AND DISCUSSION
The phytochemical analysis of the ethanolic extracts was obtained from the leaves and stems of P. pseudocaryophyllus, and was previously described for having triterpenes by Custódio (2009).Paula et al. (2008), by contrast reported phenolic compounds, flavonoids and tannins to be present in ethanolic extracts of the leaf, collected in the state of Minas Gerais (Brazil).More recently, however, pentacyclic triterpenes, Daniel et al. 961 glycosylated flavonoids, gallic acid, and ellagic acid were also isolated (Paula et al., 2012).
The alkaloid nororientaline was isolated from E. speciosa leaves (Faria et al., 2007), and the extract was studied in this work.A further two alkaloids, erysotrine and erythartine, were described by Faria et al. (2007), and were isolated from the flowers of this plant.Moreover, other species showed the presence of alkaloids (Folkers and Koniuszy, 1940;Folkers et al., 1941;Soto-Hernandez and Jackson, 1994;Chacha et al., 2005;Cui et al., 2009).
The ethyl acetate extract of T. toxicaria roots used in this work was evaluated for antimicrobial and toxic activities, and was previously described by Martinez et al. (2012) to contain rotenoids, flavonoids, a biflavonoid, flavanols and coumarins, and also possessed antioxidant activity.In the same way, the extract of I. marginata leaves used herein, were reported by Rieger (2011) to contain triterpenes and antraquinones.On the other hand, Alvarez and collaborators (1998) showed the presence of saponins, tannins, phytosterols, and triterpenes in the stem extracts of this last plant.
The ethyl acetate extract of C. leptophylla leaves used in this work had the phytochemistry studied by Burgo (2010), who reported the presence of sitosterol and antraquinones.Phenolic compounds, anthraquinones, steroids, triterpenoids, saponins, stilbenoids, and piperidine alkaloids have been described in different Cassia species by Bolzani et al. (1995) and Yang et al. (2003).

Antimicrobial activities: Bacteria and yeast
According to the results shown in Table 2, the Gramnegative bacterium, P. aeruginosa, was sensitive to three extracts tested.The P. pseudocaryophyllus stem extract was active in two Gram-negative bacteria.Only T. toxicaria (fraction methanol) and C. leptophylla extracts, showed antibacterial activities against Gram-positive B. subtilis, and neither extract inhibited S. aureus.P. aeruginosa is responsible for a wide variety of acute and chronic human infections, including patients with severe burn wounds, urinary tract infections, AIDS, lung cancer, chronic obstructive pulmonary disease, and cystic fibrosis (Balasubramanian et al., 2013).This pathogen exhibited an inhibition zone of 12 mm using extracts of E. speciosa and C. leptophylla.
The Gram-negative bacterium, E. coli, was susceptible only to the stem extract of P. pseudocaryophyllus.This pathogen can cause serious intestinal or extra-intestinal diseases in humans and animals (Leimbach et al., 2013).This activity may be explained by the presence of triterpenes in this extract.Literature data indicated that betulinic, ursolic and oleanolic acids and derivatives showed antimicrobial activities (Silva et al., 2012).Also, amyrins, ursolic acid and their 3-O-fatty acid ester chains were active against a series of Gram-positive and Gram- negative bacteria (Mallavadhani et al., 2004).The antimicrobial activity of the ethanolic extract of P. pseudocaryophyllus leaves collected in different Brazilian states have been described be active against Gram-positive bacteria and the yeast C. albicans (Paula et al., 2008(Paula et al., , 2009(Paula et al., , 2012)).Antimicrobial results on Gram-positive bacteria showed the efficacy of C. leptophylla and T. toxicaria (fraction methanol) extracts against B. subtilis.In addition, C. fistula is considered a medicinal plant with pharmacological activity, which includes antibacterial and antifungal activities (Kumar et al., 2006).

Antimicrobial activities: Phytopathogenic filamentous fungi
As shown in Table 3, the Brazilian plant extracts tested were found to be active towards four phytopathogenic fungi at low concentrations.Among the tested extracts, only P. pseudocaryophyllus and E. speciosa leaves inhibited all of the phytopathogens examined.
The fungus L. theobromae, an anamorphic form of B. rhodina (Saldanha et al., 2007), has caused serious crop damages in Brazil, particularly to tropical fruits and mango trees.It has been associated with leaf spots, necrosis, gummosis and even death of many host plants (Costa et al., 2010).
The statistical analysis of the colony diameter using P. pseudocaryophyllus leaf and stem extracts (Table 3) showed no significant difference at the 5% level to inhibit L. theobromae (p=0.4411), and B. rhodina (p=0.5173)growth, considering that the inhibitions were 36.63%(leaves), 30.30% (stems), 29.90% (leaves), and 36.85%(stems), respectively.Then both plant extracts can be used to inhibit these fungi.Similar results were obtained for F. verticillioides (p=1.0000),considering that the percentage of inhibition was 6.20% (leaves) and 6.98% (stems), respectively.On the other hand, the P. pseudocaryophyllus stem extracts did not inhibit B. ribis.
negative control.
Additionally, E. speciosa extracts were effective for all of the filamentous fungal species tested (Table 3) in this work, particularly F. verticillioides.These results were in accordance with those presented by Soto-Hernández and Miguel-Chávez (2006), which showed that the interaction of the various alkaloids detected in the Erythrina coralloides extract could cause growth inhibition of fungi.In contrast, the phytopathogen B. ribis was susceptible only to the leaf extracts of P. pseudocaryophyllus and E. speciosa.
The analysis of C. leptophylla and I. marginata leaf extracts revealed that there was significant difference (p=0.0001) on L. theobromae inhibition (27.37 and 46.40%, respectively), and similar results were found for B. rhodina (43.81 and 26.35%, respectively).However, F. verticillioides inhibition did not show significant differences (p=1.000).These extracts did not inhibit B. ribis.
Results  ).Among all of the plant extracts evaluated within this work, I. marginata ethanolic extract showed the highest growth inhibition for L. theobromae (46.40%), while C. leptophylla ethyl acetate extract was the highest for B. rhodina (43.81%).Results suggest that the antifungal activity can be correlated with the presence of sitosterol in the C. leptophylla extract, and stigmasterol in I. marginata.These compounds according to Haraguchi et al. (1999) could change the membrane permeability arising from membrane lipid alteration, thus promoting the fungal inhibition.

Brine shrimp toxicity test
The toxicity of the different plant extracts is shown in Table 4. Furthermore, ethanolic extracts obtained from P. pseudocaryophyllus leaves were found to be toxic (LC 50 =141.41µg/ml) to A. salina.These extracts can be explored for future cytotoxic studies based on the literature data that have shown important activities of this plant or genus species.The presence of tannins in extracts from Pimenta dioica leaves and cytotoxic assessment showed activity against solid tumors and cancer cells (Marzouk et al., 2007).In addition, the extract of E. speciosa leaves (LC 50 =476.41µg/ml) was toxic to A. salina, while the dichloromethane extracts of Erythrina crista bark used in Argentina was also described as being cytotoxic, and presented DNA interaction (Mongelli et al., 2000).
The lethality of the ME fraction from T. toxicaria roots was notable on brine shrimp (LC 50 =1.71µg/ml), followed by the HE fraction (LC 50 =241.74µg/ml).These results are in agreement with data from others species of this genus used as folkloric medicines in the Indian subcontinent: T. calophylla, T. maxima and T. purpurea.Root chloroform extracts from these plants were active to hatchability and lethality of brine shrimp (LC 50 :70.7 to 100.0 µg/ml) assays, and these extracts were cytotoxic to animal cell-lines (Subhadra et al., 2011).According to these authors, the most active extracts bore high contents of phenolic compounds and flavonoids.It is well known that the various biological properties provided by these metabolites are due to antioxidant activity.Similarly, the powerful and very interesting toxic activity of the ME fraction from T. toxicaria roots can be explained by the presence of phenolic compounds, flavonoids along with rotenoids extracted with methanol.However, the activity of the HE fraction was probably due to the presence of the rotenoides (as tephrosin, rotenolone, deguelin) present in the EA extract.The presence of these compounds extracted with polar solvents had previously been described by Andrei et al. (1997).The anticancer (Kim et al., 2008) and insecticide (Khater, 2012) activities of degelin isolated from natural sources have been reported.Furthermore, the cancer chemopreventive properties of T. toxicaria were attributed to bioactive metabolites (Jang et al., 2003;Vasconcelos et al., 2009).
I. marginata leaves (LC 50 =285.84µg/ml) was toxic to A. salina, and the bark extract of this same plant was ichthyotoxic to the guppy fish, Poecilia reticulata, and also inhibited tumor growth (Alvarez et al., 1998).

Conclusion
Antibacterial activity was detected in the plant extracts from P. pseudocaryophyllus stems (P.aeruginosa and E. coli) and E. speciosa (P.aeruginosa) and antifungal activity (inhibited Candida species).Three other plant extracts from I.marginata, C. leptophylla and E. speciosa inhibited L. theobromae, B. rhodina and F. verticillioides.The methanol fraction from T. toxicaria showed higher toxicity towards A. salina in comparison to the five plants screened.The results suggest future investigations on human cells to search for new drugs, and on the studied fungi to control them in plants diseases.

Table 1 .
Method of extraction and yield (%) from Brazilian plant extracts.

Table 2 .
Antimicrobial activity from different extracts by the well-plate diffusion method.

Table 3 .
Growth inhibition of plant extracts on phytopathogens filamentous fungi.

Table 4 .
Toxic effects of Brazilian plant extracts using the A. salina lethality test.