Antibacterial evaluation of Styrax pohlii and isolated compounds

The antibacterial activity of the compounds egonol (1) and homoegonol (2), of the crude ethanolic extract of Styrax pohlii (Styracaceae) aerial parts (EE), and of its n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions was evaluated against the following microorganisms: Streptococcus pneumoniae (ATCC 6305), S. pyogenes (ATCC 19615), Haemophilus influenzae (ATCC 10211), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 10031). The broth microdilution method was used for determination of the minimum inhibitory concentration (MIC) during preliminary evaluation of antibacterial activity. The EE yielded MIC values of 400 μg/mL for S. pneumoniae and P. aeruginosa and 300 μg/mL for H. influenzae. The HF and EF fractions exhibited enhanced antibacterial activity, with MIC values of 200 μg/mL against S. pneumoniae, but only EF displayed activity against H. influenzae (MIC 200 μg/mL). The best MIC value with compounds 1 and 2 (400 μg/mL) was obtained for (1) against S. pneumoniae and P. aeruginosa. Therefore, 1 exhibited weak antibacterial activity against these standard strains.


INTRODUCTION
A major concern in the public health field is the ease of development of antibiotic resistance by bacteria, since infectious diseases caused by resistant microorganisms are responsible for increased health costs as well as high morbidity and mortality, particularly in developing countries (Nickerson et al., 2009a;2009b;Gangoué-Piéboji et al., 2009).The rapid emergence of bacterial resistance is attributable to such diverse factors as the complex genetics of the microorganism; the increasing transport of humans, animals, and goods between countries; the widespread use of antibiotics; and the lack of precise therapeutic choices for high-risk patients (Stefani, 2009).
Natural medicinal products have been used for millennia to treat various ailments.Although many have been superseded by conventional pharmaceutical approaches, there is a resurgence of interest in natural products by physicians (Boukraa, 2008;Shimizu et al., 2001).In addition, the existence of resistant bacterial strains reinforces the need for development of novel safe and effective antibacterials that can combat infectious diseases caused by bacteria (Gangoué-Piéboji et al., 2009;Coutinho et al., 2009).
Therefore, as part of our ongoing biological studies into the antibacterial activity of natural compounds (Pauletti et al., 2000;Scalon Cunha et al., 2007;Cunha et al., 2010;Silva et al., 2009), we now provide a preliminary report on the antibacterial activities of compounds 1-2, the crude ethanolic extract (EE), and the n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions of Styrax pohlii against the microorganisms Streptococcus pneumoniae, S. pyogenes, Haemophilus influenzae, Pseudomonas aeruginosa, and Klebsiella pneumoniae, which have not yet been described.

Collection and identification
S. pohlii A. DC. was collected in the municipality of Luis Antonio, state of São Paulo, Brazil, in October 2008, and identified by Prof. V. M. M. Gimenez and Prof. M. Groppo.A voucher specimen (SPFR12168) was deposited in the Herbarium of the Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo.

Extraction and isolation
The air-dried, powdered stems and leaves (2.43 kg) of S. pohlii were extracted with EtOH by maceration at room temperature.After filtration, the solvents were removed under reduced pressure to yield 87.2 g of extract.The EE (20.3 g) was then dissolved in MeOH/H 2 O (1:4, v/v) and successively partitioned with n-hexane, EtOAc, and n-BuOH.After solvent removal using a rotary evaporator, each partition phase yielded 1.39, 2.97, and 3.80 g of extract respectively.Additionally, the residual hydromethanolic phase furnished 4.12 g.In a previous study, our research group had fractionated the n-hexane fraction and obtained compounds 1 (27.3 mg) and 2 (22.0 mg) (Bertanha et al, 2012).

Antibacterial assay
The following microorganisms were used for evaluation of antibacterial activity: Streptococcus pneumoniae (ATCC 6305), S. pyogenes (ATCC 19615), Haemophilus influenzae (ATCC 10211), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 10031).Minimum inhibitory concentration (MIC) values for each sample were determined in triplicate by using the broth microdilution method (CLSI, 2009).The samples were dissolved in DMSO (Merck) at 0.5 mg/mL and then diluted in Brain Heart Infusion broth or, for H. influenzae only, BD Haemophilus Test Medium Agar (Difco Labs), to concentrations ranging from 400 to 20 μg/mL.The final DMSO concentration in the culture medium was 5% (v/v), which was used as a negative control.The inoculum was adjusted to each organism to yield a cell concentration of 5×10 5 colony forming units (CFU/mL).One inoculated well was included to control the adequacy of the broth for organism growth.One non-inoculated well, free of antibacterial agent, was also included to ensure medium sterility.Chloramphenicol and vancomycin were used as controls, dissolved in DMSO (Merck) at 0.1 and 10 mg/mL respectively, and then diluted in Brain Heart Infusion broth or BD Haemophilus Test Medium Agar to 0.02 mg/mL.The controls were evaluated at final concentrations ranging from 0.01 to 5.9 μg/mL.The microplates (96-well) were incubated at 37 °C for 24 h.Then, 30 μL of resazurin 0.01% aqueous solution (Sigma-Aldrich) was added to determine microorganism viability (Sarker et al., 2007).The MIC was determined as the lowest concentration of the sample capable of inhibiting microorganism growth.

RESULTS AND DISCUSSION
A previous study on the CH 2 Cl 2 -CH 3 OH (2:1, v/v) crude extract from the leaves of S. ferrugineus demonstrated antimicrobial activity, as detected by inhibition of Cladosporium sphaerospermum, Candida albicans, and Staphylococcus aureus (Pauletti et al., 2000).This finding has encouraged us to conduct biological and chemical investigations of metabolites isolated from other species of this genus.
Regarding the antibacterial assay, the crude EtOH extract (EE), as well as the n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions of S. pohlii, were screened for their antibacterial activity against five different bacterial strains, three of which were Gram-positive and two were Gram-negative.The preliminary antibacterial activities, expressed as the MIC, are shown in Table I.
The EtOH extract (EE) yielded MIC values of 300 μg/mL against H. influenzae and 400 μg/mL against S. pneumoniae and P. aeruginosa.EE did not inhibit the growth of the two other tested strains.Therefore, EE exhibits moderate activity against H. influenzae, S. pneumoniae and P. aeruginosa, as described by Holetz et al. (2002).
Compared with EE, the n-hexane fraction (HF) provided superior antibacterial activity against the Grampositive S. pneumoniae, with a MIC value of 200 μg/mL.However, unlike EE, HF did not significantly reduce the growth of H. influenzae.In addition, HF displayed an MIC of 400 μg/mL against P. aeruginosa, but was considered ineffective against S. pyogenes, H. influenzae, and K. pneumoniae.According to these results, the HF fraction exhibits promising activity against S. pneumoniae.
The best MIC value obtained for the EtOAc fraction (EF) was 200 μg/mL, against the Gram-negative H. influenzae and the Gram-positive S. pneumoniae.This result is superior to those obtained with EE against the same strains (MIC = 300 and 400 μg/mL, respectively).In addition, EF displayed an MIC of 400 μg/mL against P. aeruginosa and S. pyogenes.Thus, EF was the only assayed sample that exhibited moderate antibacterial activity against S. pyogenes, as compared with the data obtained by Holetz et al. (2002).
The n-BuOH fraction (BF) exhibited moderate antibacterial activity against P. aeruginosa, S. pneumoniae, and H. influenzae (MIC = 400 μg/mL), and did not significantly inhibit the growth of the two other tested strains, just as found for EE.The hydromethanolic fraction (HMF) displayed moderate antibacterial activity against P. aeruginosa.Thus, the BF and HMF fractions are not promising antibacterials against the strains tested in this work when compared with the HF and EF fractions.
In conclusion, differences in antibacterial effect were more evident for the HF fraction when assayed against the tested Gram-positive bacteria.HF was not effective against Gram-negative bacteria, perhaps due to the presence of the outer membrane in the latter microorganisms (Horiuchi et al., 2007).The HF fraction was thus selected for a further purification process as described previously (Bertanha et al., 2012), which yielded egonol (1) and homoegonol (2) (Figure 1).
Regarding the antibacterial activity of the isolated compounds (Table I), egonol (1) displayed weak activity against P. aeruginosa and S. pneumoniae (MIC = 400 μg/mL), according to Ríos and Recio (2005).As HF displayed the same activity as 1 against P. aeruginosa, it might be suggested that 1 is the compound responsible for the activity initially observed for the HF fraction, at least against P. aeruginosa.However, data for S. pneumoniae showed that 1 displayed half of the activity found for HF against this same strain.Similar findings are frequently observed in the area of natural product research after fractionation processes (Harvey, 2009;Yeh et al., 2012).Homoegonol (2) was considered inactive, at least in the present assay conditions.
Compounds 1 and 2 are quite similar, differing mainly in the presence of a methylenedioxyl group or two methoxyl groups at the phenyl ring connected to C-2 of the 5-propylbenzofuran core, respectively.Therefore, considering their antibacterial activity, it can be suggested that the presence of a methylenedioxyl group in compound 1 may improve its activity, since it was more active than compound 2. The mechanism whereby benzofuran derivatives exert their in vitro antibacterial effect is unclear.However, 1, 2, and some semi-synthetic egonol derivatives have been shown to possess antifungal and antibacterial activities against several species, including Staphylococcus aureus, Candida albicans, Cladosporium sphaerospermum, Bacillus subtilis, and Escherichia coli (Pauletti et al., 2000;Öztürk et al., 2008).Nevertheless, further studies are still required to improve the antibacterial activity of these natural products and obtain a better understanding of the mode of action of benzofuran derivatives.

CONCLUSION
The EtOH extract (EE) of S. pohlii showed moderate activity against the Gram-negative bacterial species H. influenzae.Chemical analysis of the EE yielded four fractions.The n-hexane fraction exhibits promising activity 1 -Chemical structures of the compounds isolated.
against the Gram-positive S. pneumoniae bacterium and was thus selected for further purification, which yielded egonol (1) and homoegonol (2).Furthermore, biological assay results suggested that compound 1 inhibits the growth of P. aeruginosa and S. pneumoniae.Further studies are ongoing to assess other potentially important biological effects of this plant species.

TABLE I -
Minimum inhibitory concentration values (MIC) obtained for the crude ethanolic extract of S. pohlii, fractions and isolated compounds a Positive Controls: (c)-Chloranphenicol (v)-Vancomycin