Antimicrobial activity and bioautographic study of antistaphylococcal components from Caesalpinia pyramidalis Tull

The antimicrobial activity of dry methanol and ethyl acetate extracts for the leaves, bark of the stem, peel of the root, flower, fruit and seed of Caesalpinia pyramidalis Tull. (catingueira) was performed against seventeen isolates of Staphylococcus aureus MRSA multiresistant strains, which included two isolates of S. aureus MSSA and two ATCC strains. The antimicrobial activity was tested by the agar diffusion method and the Minimum Inhibitory Concentration (MIC) was determined. The dry methanol extract of the root showed good antimicrobial activity with a MIC of less than 0.5 mg.mL-1. The dry ethyl acetate extracts exhibited lower antimicrobial activity, which might be explained by solubility problems and less diffusion in the agar medium. Results of the bioautographies also confirmed inhibition halos corresponding to the active substances present in the leaves, as well as in the flower of C. pyramidalis. The phytochemical study of the leaves, bark of the stem, peel of the root, flower and fruit of extracts from C. pyramidalis confirmed the presence of a number of known antimicrobial agents including ursolic acid, quercetin, catechin, ellagic acid, sitosterol, flavonoids, proanthocyanidins and gallic acid.


INTRODUCTION
The importance of nosocomial infection caused by Staphylococcus aureus, especially by methicillin resistant S. aureus (MRSA) is well known for its frequency, morbidity, mortality and principally for its difficulty to treat (Nascimento et al., 2000).The strains of MRSA are resistant to all β-lactamics, macrolides, tetracycline, aminoglycosides, while the two glycopeptides (vancomycin and teicoplanin) remain the only alternatives for clinical treatment against infections of MRSA multiresistant S. aureus (Shibata et al., 2005).However, S. aureus with intermediate susceptibility to vancomycin (VISA) or S. aureus glycopeptides (GISA) has recently been identified in different countries.Subsequently, strains of S. aureus resistant to vancomycin (VRSA) have also emerged, possessing a different mechanism to those of VISA strains (Nishi et al., 2004).
Studies conducted in Brazil have described a clone of methicillin resistant Staphylococcus aureus (Brazilian Epidemic Clone-BEC, ST247-SCCmecIIIA) that is disseminated and predominates within hospitals throughout the country.It was also observed that this clone has spread to other countries in South America (Argentine, Chile, Colombia, Peru, Ecuador, Uruguay), besides Europe (Portugal, Italy and the Czech Republic) and Asia (Miranda et al., 2007;Sola et al., 2002;Rodríguez-Noriega et al., 2010).In light of this evidence, the need to identify new antimicrobial agents is clear.
Caesalpinia pyramidalis Tull., Leguminous family (Fabaceae) is a tree found in the Northeastern region of Brazil, popularly known as "catingueira", "pau-de-porco" and "mussitaiba".In folk medicine, its flowers, seeds, leaves and bark of the stem are used for the treatment of catarrhal infections, diarrheas and dysentery, besides being endowed with antipyretic and diuretic properties (Mendes et al., 2000;Braga, 1960;Rêgo Júnior et al., 2011).

Collection and identification
The plant was collected in the town of Carnaubeira da Penha, in the hinterland of Pernambuco (State), at a latitude of 08°19'09", longitude of 38º44'41" and altitude of 446 meters (MME, 2005), between the months of March and June, 2004.Samples were identified by employees of the Herbarium of the Empresa Pernambucana de Pesquisa Agropecuária (IPA), Dr. Rita de Cássia Pereira, and deposited with voucher nº 70.008.

Preparation of extracts
For determination of the antibacterial activity of C. pyramidalis, different fresh material parts of the plant including leaves, bark of the stem, peel of the root, flower, seeds and fruit were triturated, weighed and submitted to three successive extractions by the process of infusion, with medium intervals of 72 hours for each solvent.The order of solvents used was n-hexane, followed by ethylic acetate and lastly methanol.The extracts thus obtained were filtered and solvents evaporated at 40 ºC under reduced pressure.These were subsequently weighed and their output calculated.
The dry extracts obtained from the methanol extraction were reintroduced into water/DMSO (1:1 v/v) (Sakagami et al., 2005) at a concentration of 100 mg.mL -1 .The dry extracts obtained from the ethyl acetate and n-hexane extractions were also reintroduced in tween 80/water (4.8:0.2 v/v) at a concentration of 100 mg mL -1 .

Bacteria strains
A total of twenty-one Staphylococcus aureus strains, comprising nineteen clinical isolates and two standard strains, were used in the study (Table I).These consisted of eight multiresistant S. aureus MRSA of the Brazilian epidemic clone (BEC), five isolates of the pediatric epidemic clone, four isolates of the sporadic clones and two MSSA strains which were only resistant to the antibiotics penicillin, erythromycin and gentamycin.The multiresistant S. aureus MRSA and S. aureus MSSA were strains from the Microbiological Analysis Laboratory collection, derived from a study on the susceptibility/resistance profile of S. aureus in Recife-Pernambuco state (Cordeiro, 2004).

Preparation of inoculates
The inoculates were prepared starting with the 24 h

Agar well diffusion method
The inoculum were applied to the surface of the Mueller-Hinton agar and after perforation of the wells (perforator 6 mm in diameter), 100 µL of the extracts (concentrations of 100 mg .mL - and 50 mg mL -1 ), standard antibiotic (300 µg mL -1 ) and of the control solution (DMSO at 50% (v/v) or tween 80 at 4% (v/v)) were added to each well.After incubation at 37 ºC ± 1 for 24 hours, the diameter of the inhibition halos were measured and results evaluated according to the following scale: inhibition halos of 9 mm -inactive; 9-12 mm -somewhat active; 13-18 mm -active; 18mm -very active (Alves et al., 2000).

Agar dilution method -Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration for the extracts (basic solution of 20 mg.mL -1 ), dilutions in water with concentrations of 312.5 µg.mL -1 to 20.10 3 µg.mL - , had been prepared and incorporated to Mueller-Hinton agar (1:9), thus final concentrations of 31.5 µg.mL -1 to 2000 µg.mL -1 of the extracts remained.The standard antibiotics used were tetracycline and oxacillin (Sigma), with concentrations as indicated by the CLSI ( 2003) norms.
The twenty-one inoculates were distributed aseptically in the Stears multi-inoculator, and then deposited on the surface of the culture medium, and incubated at 36 ºC ± for 24 hours.
Duplicate controls were performed at the beginning and end of the inoculation control process.Control of the diluents was also done (tween 80 at 4% and DMSO at 50%).
The results were interpreted comparatively to the strain controls and determined by the first plate whose concentration inhibited growth.
The visualization of TLC was carried out under UV light (254 and 366 nm), as shown in Table II.

Bioautographic technique
The developed TLC according to system (1) was subjected to aseptic current air for 5 minutes, and another developed by system (2) for 6h, respectively.On the TLC surface, melted Mueller-Hinton agar (MH) was added, inoculated with a saline suspension of S. aureus ATCC 6538 (bacterial suspension at 10 8 UFC/mL) and homogeneously distributed over the TLC plate.After solidification of inoculated MH, it was left for 30 minutes at the surrounding ambient temperature of 25 ºC for predifusion of the active components.Subsequently, it was incubated for 24 to 36 hours ±1 ºC.After this period the bioautography was revealed with a solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 2.5 mg mL -1 and incubated for a further 4 hours.
The presence of an inhibition zone indicated the existence of active components (Pessini et al., 2003).

Antimicrobial activity
The results of methanol and ethyl acetate extracts from C. pyramidalis against four clinical isolates and one standard strain (AM103) of S. aureus are given in Table III.The hexane extracts showed no activity.The diameters of the inhibition halos expressed in millimeters are expressed in the form of a table for each extract and each bacterium.
The MIC values of the assayed extracts are shown separately, according to type of S. aureus MRSA multiresistant clone.

Bioautography
The bioautographic results for the leaves and flower extracted in methanol and ethyl acetate, and peel of the root extracted in ethyl acetate, from C. pyramidalis are listed in Table V.

Phytochemical study
The secondary metabolites found in the extracts of the parts studied from C. pyramidalis with well-known antimicrobial activity are shown in Table VI.
Among the two techniques used for the determina- tion of antimicrobial activity, the agar well diffusion technique, despite using larger volumes (100 µL) (Caetano et al., 2002) compared to disks (10 µL) (Voravuthikunchai and Kitppipit, 2005), has the advantage of allowing the use of adjuvant to improve the solubility of the extract constituents and to permit radial as well as superficial diffusions, conditions resulting in better inhibition halos.
With regard to the five Staphylococcus aureus strains studied, four represent the three types of MRSA multiresistant clones and one the standard strain.
In Table III, the extract of the flower methanol (FLM) showed the largest inhibition halos for the five tested strains, with values in the order of 22 mm, at the highest concentration (10 mg/well), corresponding to the very active classification (Alves et al., 2000), and halos in the order of 16 mm for concentrations of 5 mg per well , with extracts of leaves, peel of the root, fruit and seed also having good activity.
In general, extracts from retrieval in ethyl acetate showed smaller inhibition halos compared with those extracted from methanol.This fact can be due to the polarity characteristics of its constituents which resulted in lower diffusion in aqueous environs and therefore produced smaller inhibition halos despite having substances with antimicrobial activity (Lenette et al., 1987).
Observing Table III of the corresponding extracts (leaves, bark of the stem, peel of the root, fruit and seed) in ethyl acetate, the extract of peel of the root produced inhibition halos in the order of 20 mm, while the extract of the leaves showed no activity except against the standard strain (AM103).Experimentally, this difference may be explained by the fact that the dry extract of peel of the root is in the form of powder and dissolved very well in tween 80/water 4% whereas extract from the leaves had a pasty consistency and was not well dissolved, hindering its solubility.
The inhibition zone produced by the standard antibiotic, tetracycline, confirmed the resistance phenotype of the clone strains, i.e.Staphylococcus aureus MRSA AM594, S. aureus AM723 and S. aureus AM942, resistant to tetracycline had inhibition halos in the order of 13 mm or less while the strains S. aureus MRSA AM922 and S. aureus AM103 sensitive to tetracycline had inhibition halos in the order of 26 mm.
The presented data are mean values of two determinations.
On the other hand, the presence of active substances in the leaves as well as in the flower for both types of extracts was evidenced in the results of the bioautographies (Table V).
The bioautography technique permits the detection of inhibition halos that reveal the presence of active substances in the TLC (Pessini et al., 2003) against S. aureus ATCC 6538 in the methanolic extracts, one in Rƒ 0.48 for the flower extract and Rƒ 0.55 for the leaves extract.Similarly, for the extracts in ethyl acetate, one showed the presence of inhibition halos in Rƒ 0.62 for the flower extract, the Rƒ 0.51, Rƒ 0.62, Rƒ 0.80 and Rƒ 0.94 for the leaves extract and the Rƒ 0.26, Rƒ 0.39, Rƒ 0.58, Rƒ 0.68, , Rƒ 0.77 and Rƒ 0.92 for the stem bark extract (Table V).
The phytochemical investigation evidenced the presence of substances such as flavonoids (flavonoid aglycones), sitosterol and cinnamic derivatives for all the extracts of C. pyramidalis tested.The presence of gallic acid and ellagic acid was also evidenced in the flower extract in methanol .Other components identified were polyphenols (phenolic acid) in the fruit, and ursolic acid and quercetin from the bark of the stem and peel of the root (root and bark of the stem) thus confirming data from the literature (Bahia et al., 2010;Bahia et al., 2005).
With reference to MIC (Table IV), analysis of the results reveals that, in the case of S. aureus MRSA Brazilian epidemic clone, that some of the values are less than 0.5 mg.mL -1 for the methanol extracts as well as ethyl acetate extracts of the peel of the root.
Similar results were found for the methanol extract of the flower.
The MIC values for the five pediatric clones were again low for the extracts of the peel of the root, being in the order of 0.25 mg.mL -1 for both methanol and ethyl acetate extracts.
Also, in Table IV featuring MIC for the four sporadic clones together with the susceptible S. aureus MSSA strains and standard strains, the extracts of the peel of the root in ethyl acetate had some values as low as 0.125 mg mL -1 , even less than those of the MSSA strains (0.5 mg mL -1 ) and standard strains (0.25 mg mL -1 ).
The MIC values of tetracycline and oxacillin confirmed the phenotype resistance and MRSA or MSSA character of isolates of S. aureus strains, respectively.
In relation to the DMSO diluents at 50% and tween 80 at 4%, these showed no inhibition against any of the twenty-one strains, confirming earlier data for DMSO by Sakagami et al. (2005).

CONCLUSION
The extracts from C. pyramidalis showed good antimicrobial activity against the S. aureus multiresistant strains.Also, the bioautography technique allowed visualizing six inhibition halos for the extracts in ethyl acetate from stem bark and three from the leaves, which allows us to conclude that there are at least six plus three active compounds, respectively.
Further experiments aimed at the isolation and structural identification of the active compounds are needed, as well as more pharmacological data to validate the popular use of the extracts from the C. pyramidalis.such as data determining its safety and toxicity.

TABLE I -
List of Staphylococcus aureus strains assayed Nº: Register Number; AM: Microbiological analysis laboratory collection -Pharmaceutical Science Department -UFPE; ATCC: American type culture collection; Table IV summarizes the results of eight Brazilian epidemic clones, five pediatric clones, four sporadic multiresistant clones, two strains of S. aureus MSSA, and finally two standard strains.

TABLE II -
Chromatographic systems used for phytochemical screening of Caesalpinia pyramidalis Tull.

TABLE VI -
Secondary metabolites from Caesalpinia pyramidalis with well-known antimicrobial activity