An ent-Kaurane-Type Diterpene in Croton antisyphiliticus Mart.

Croton antisyphiliticus is a medicinal plant widely used in the treatment of microbial infections, especially those affecting the genital tract. Crude extract, fractions and pure compound isolated from roots of this species were investigated to validate their antimicrobial activity against Escherichia coli and Staphylococcus aureus. The compound ent-kaur-16-en-18-oic acid was isolated as a major component (0.7% of crude extract), and its MIC value determined against S. aureus (ATCC 6538) was 250 μg/mL. This is the first phytochemical work on the species monitored with antimicrobial assay.

reported the antimicrobial activity of extracts of some endemic plants from the Cerrado against Staphylococcus aureus isolated from bovine mastitic milk. So far there is no study correlating the chemical structure of C. antisyphiliticus compounds and antimicrobial activity. The goal of this study was to perform a phytochemical screening by evaluating the antimicrobial activity of extracts, fractions and compounds isolated from Croton antisyphiliticus.

Results and Discussion
Fractionation of the C. antisyphiliticus chloroform root extract resulted in the isolation of compound 1, a white solid (0.670 mg) identified as ent-kaur-16-en-18-oic acid ( Figure 1).    Table 1).
The carbon of methyl group C-19 displayed long-range correlations in HMBC bidimensional spectrum with a hydrogen signal at  1.64 attributed to H-5. Also, the signal at  1.06 (s) attributed to H-19 showed correlations with carbon signals at  37.3 (C-3) and  50.6 (C-5). The signal at  1.71 (br s, H-3) indicated correlation with the quaternary carbon at  185.4, which is attributed to C-18. The 13 C-NMR spectrum presented 20 signals, among them five quaternary (C), three tertiary (CH), ten secondary (CH 2 ), and two primary (CH 3 ). The chemical shift values of C-5 at  50.6 and C-19 at  18.2 made it possible to verify that carboxyl group displayed cis-equatorial position in relation to H-5 and to establish that the structure of 1 is an epimer of ent-kaur-16-en-19-oic acid [12]. NOESY experiments corroborate structure identification, since irradiation at  Table 1.
Additionally, the heteronuclear correlations between HMQC and HMBC were similar to those reported for ent-kaur-16-en-18-oic acid [12]. Antimicrobial activity of C. antisyphiliticus CHCl 3 crude extract and fractions was insignificant compared to the antibiotic gentamicin sulphate used as positive control. Also, the pure compound ent-kaur-16-en-18-oic acid isolated from C. antisyphiliticus roots (MIC 0.250 mg/mL) was less active against S. aureus (ATCC 6538) than kaurenoic acid (MIC 0.125 mg/mL) ( Table 2). Although kaurenoic acid has not been isolated from Croton antisyphiliticus, studies concerning the evaluation of pharmacological activity of chiral compounds are always relevant and recommended.
Furthermore, the protective effect of plants against attacks by microorganisms and herbivores may be correlated with the presence of natural chiral compounds [13]. Like kaurenoic acid, ent-Kaur-16-en-18-oic acid is an epimer of kaurenoic acid and deviated the plane of polarized light to the left. Studies of kaurenoic acid epimers isolated from Mikania laevigata also showed antimicrobial activity at a level of 250 μg/mL [14]. The diterpenes ent-beyer-15-en-19-ol and its 4-epimer ent-beyer-15-en-18-ol isolated from Helichrysum tenax var tenax showed similar results in antimicrobial assays against the microorganisms tested here [15].
According to the literature, ent-kaurene compounds are inactive against Gram-negative [16] but they are active against Gram-positive bacteria [17]. Wilkens et al. [18] reported that this class of compounds is responsible for the lyses of protoplasts of these microorganisms.
Gil et al. [19] demonstrated the antimicrobial action of ent kaurenoic acid isolated from resin of Pseudognaphalium vira vira medicinal plant. According to Schmelz et al. [20], derivatives of ent-kaurene diterpenoids act as corn phytoalexins when accumulated at concentrations higher than 0.01%. Results showed that ent-kaur-16-en-18-oic acid represents 0.7% of the crude root extract of C. antisyphiliticus, meaning that this compound plays an important role in the defense against Gram-positive microorganisms present in this plant's natural habitat, indicating its ecological importance in the protection against attacks from insects and pathogens.

General
The IR spectra were recorded on a Nicolet Protégé-460 spectrophotometer operating in a 4,000-600 cm −1 range in the form of KBr pellets. 1 H-(300 and 400 MHz) and 13

Plant Material
Croton antisyphiliticus roots were collected in February of 2010 in Cerrado regions of Araxá, state of Minas Gerais, Brazil. A voucher specimen was deposited at the herbarium of the University of Ribeirão Preto (HPM-482). Roots were dried at 50 °C and powdered.

Extraction and Isolation
Powdered material (745 g) was extracted in CHCl 3 (2 L) at room temperature for 7 days. The CHCl 3 extract was concentrated to a small volume at reduced pressure to yield 8.93 g of oily residue. This residue was fractionated on a silica gel column (Silicagel Acrós®, 0.060-0.200mm, 60 A; Geel, Belgium), eluting with hexane, hexane/ethyl acetate, ethyl acetate and methanol gradient elution to 100%.

Determination of Antimicrobial Activity by Bio-Autographic Assay and Minimum Inhibitory Concentration (MIC)
The Croton antisyphiliticus crude extract CHCl 3 and its sub-fractions were subjected to bio-autographic assay. The samples (2.0 mg) were diluted in 1 mL chloroform and applied on glass plates with silica gel F254. The plates were developed in mobile hexane/ethyl acetate phase (8:2) and then covered with Agar BHI containing 3 × 10 6 cfu/mL of each microorganism in suspension separately. This procedure was duplicated. After 24 h at 35 °C, each bio-autogram was stained with 2,3,5-triphenyltetrazolium chloride (TTC) aqueous solution in order to observe inhibition areas. To determine the chemical profile of the samples, replication of chromatographic plate was performed simultaneously and revealed with vanillin/sulphuric acid color reagent, followed by heating.
The bio-autography confirmed the antimicrobial activity of crude extract and fractions. MIC values of crude root extracts and fractions were determined against the bacterial strains according to the CLSI M27-A2 (2003) guidelines [21] Stock cultures were submitted to serial dilutions ranging from 15.6-1000 µg.

Microbial Strains
The antimicrobial assay was carried out against four selected microorganisms: Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 purchased from the American Type Culture Collection (Manassas, VA, USA) and two clinical isolates, Escherichia coli and Staphylococcus aureus, obtained from urinary tract and oropharynx, respectively. The final concentration of DMSO in each medium was 1.25%, which did not affect the growth of the test strains. The microorganisms were reactivated before the assay in Müller Hinton medium, cultured with no agitation at 37 °C for 1 day. The antimicrobial assay was performed by using the serial dilution method according to the CLSI M7-A8 (2009) [22]. The lowest concentration of the tested compounds in which no growth was observed was defined as MIC. The assays were performed in triplicate.

Conclusions
The isolation of ent-kaur-16-en-18-oic acid and validation of the antibacterial activity of this compound against Staphylococcus aureus corroborates other ethnopharmacological studies.