Chemical Constituents from Branches of Maytenus gonoclada ( Celastraceae ) and Evaluation of Antimicrobial Activity

Seis triterpenos pentacíclicos isolados dos galhos de Maytenus gonoclada (Celastraceae), incluindo todos os dados de RMN do novo composto 3-oxo-12α,29-diidroxifriedelano são aqui relatados. A estereoquímica do novo friedelano foi estabelecida por dados de RMN bidimensional (HSQC, HMBC e NOESY), e sua massa molecular confirmada por espectrometria de massas (ESI). Testes de atividade antimicrobiana usando método de difusão em disco e de macrodiluição foram realizados contra as bactérias Escherichia coli, Citrobacter freundii e Bacillus cereus, e contra o fungo Candida albicans. O triterpeno 3-oxo-12α-hidroxifriedelano mostrou resultado positivo contra C. albicans.


Introduction
Celastraceae family contains many species that have been extensively studied in function of their use in traditional medicine.Some species of Maytenus genus are worldwide distributed and have been used by Africans to treat cancer, by Asian people as an insecticide, 1 and by the South American people on the treatment of gastrointestinal diseases. 2The biological activities associated to Maytenus species have been assigned to different classes of secondary metabolites such as phenolic glucosides, 3 flavonoids 4 and triterpenes. 5entacyclic triterpenes (PCTT) have been commonly isolated from species of the Celastraceae family, and some of them, like 3-oxofriedelane and 3β-hydroxyfriedelane, are considered taxonomic markers of Maytenus genus. 4 large number of pharmacological activities has been associated to triterpenes isolated from species of the Maytenus genus, such as, 3,15-dioxo-21αhydroxyfriedelane, isolated from Maytenus robusta, that showed antiulcerogenic activity, 6 and maytenfolic acid, isolated from Maytenus heterophylla, that showed growth inhibitory effect on Candida albicans. 7aytenus gonoclada Martius, popularly known as "tiuzinho", can be found in regions of "cerrado" and rupestrian fields of Southeastern and Northeastern Brazil.In our previous studies of the hexane extract from M. gonoclada leaves, the occurrence of five PCTT of the friedelane series was reported. 8he present paper reports the phytochemical study of hexane extract from branches of M. gonoclada, which was isolated five known triterpenes: 3-oxofriedelane (1), 3β-hydroxyfriedelane (2), 3-oxo-12α-hydroxyfriedelane (3), 3,11-dioxofriedelane (4) and 3,16-dioxofriedelane (5). 8In addition, a new compound of the friedelane series, 3-oxo-12α,29-dihydroxyfriedelane (6), was also isolated and its structure was developed by detailed 1 H and 13 C NMR analysis, including bidimensional (HSQC, HMBC and NOESY) spectral data.
The NMR data of compound 6, as well as the complete hydrogen chemical shifts assigned to compound 5, that have not been described in the literature yet, are herein reported for the first time.

General experimental procedures
Column chromatography (CC) was carried out using silica gel 60 (70-230 Mesh, Merck) and for thin layer chromatography (TLC) it was employed precoated silica gel plates.The detection of spots was made by spraying a mixture (1:1) of vanillin (ethanol solution, 1% m/v) and perchloric acid (aqueous solution, 3% v/v). 9A Mettler FP 80 HT apparatus was used to determine melting points (uncorrected).Elemental analyses were performed on a CHN Perkin-Elmer 2400 apparatus.Optical rotations were measured on a Perkin-Elmer model 341 polarimeter using a 100 mm, 1.0 mL cell tube capacity.Infrared spectra were recorded on a Perkin Elmer, Spectrum One spectrophotometer (ATR).Mass spectrometry was conducted in a LCQFleet (Thermo Scientific, San Jose, CA) bearing an electrospray ionization (ESI) source, operating in the positive mode.ESI source conditions were: heated capillary temperature of 290 °C, sheath gas (N 2 ) flow rate at 20 (arbitrary units), spray voltage of 4.8 kV, and capillary voltage of 2.0 V.
The 1 H and 13 C NMR spectra were measured on a Bruker DRX 400 Avance spectrometer at 400 and 100 MHz at 300 K, equipped with inverse detection 5 mm multinuclear head 1 H/ 13 C.Each compound was dissolved in CDCl 3 or in CDCl 3 with 2 drops of pyridine-d 5 , and transferred to a 5 mm o.d.NMR tube.TMS was used as internal standard (d H = d C = 0).Bi-dimensional (2D) NMR spectra were acquired under standard conditions.Data processing was carried out on SGI workstation using the Bruker (DRX 400) software.

Plant material
Samples of M. gonoclada's branches were collected in Serra da Piedade, Caeté, Minas Gerais, Brazil, in October 2004.Botanical identification was provided by Dr. Rita Maria Carvalho-Okano.The voucher specimen (HBCB 60280) was deposited in the Herbarium of the Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Extraction and isolation
The branches of M. gonoclada were dried at room temperature, milled and the resulting powder (1000 g) was submitted to extraction with hexane in a Soxhlet apparatus.The formation of a white solid material was observed during the removal of the hexane in a rotatory evaporator.This solid was separated by filtration; the dried material (1.0 g) was submitted to silica gel (50 g) CC eluted with hexane, chloroform, ethyl acetate and ethanol in mixtures of increasing polarity.Fifty-seven fractions of 200 mL each were obtained and grouped according to the similar profiles observed in the chromatoplates.Fractions 22 to 37 were eluted with hexane-chloroform (1:1).After solvent evaporation, fraction (Fr.) 22 produced a white solid (5.0 mg), which was identified as 3-oxofriedelane (1).Fractions 23-27 provided a white solid (112.2 mg), which was rechromatographed furnishing additional amount (35.0 mg) of 3-oxofriedelane (1) and 3β-hydroxyfriedelane (2) (15.2 mg).These PCTTs were characterized by their respective 13 C NMR spectral data, being also compared to published data.Fractions 34-35 gave a white solid (34.4 mg) that was identified by NMR spectra analyses and comparison with published data as a mixture of 3,11-dioxofriedelane (4) and 3,16-dioxofriedelane (5).The group of fractions Fr.36-37 (234.0 mg) was determined as a mixture, by TLC, and named as material A. The group Fr.38-43 (173.2 mg) (eluted from hexane/chloroform 3:7) was submitted to flash CC (silica gel 230-400 Mesh, Merck, 5.8 g) eluted with hexane, chloroform, ethyl acetate and methanol in mixtures of increasing polarity.Forty-seven fractions of 10 mL each were obtained yielding the 3-oxo-12α-hydroxyfriedelane (3) (10.3 mg) and 3,16-dioxofriedelane (5) (12.0 mg).Using TLC, the fraction 56 (56.2 mg) (eluted with ethyl acetate) was also characterized as a mixture, and then named as material B.

Antimicrobial bioassays
The bacteria strains, E. coli ATCC 25723, B. cereus ATCC 11778, C. freundii ATCC 29935 and the yeast C. albicans ATCC 18804 used in this study were obtained from American Type Culture Collection.The media broth heart infusion (BHI) was purchased from Merck (Darmstadt, Germany) and Biobrás (Montes Claros, Brazil).The strains were maintained on BHI medium and refrigerated at 7 °C.
Antimicrobial activity was evaluated using the disk diffusion method, according to the literature. 10The microorganisms were cultivated in medium BHI and incubated for 18 h at 37 °C.Cells were suspended, according to the McFarland protocol, in saline solution to produce a suspension containing approximately 5×10 5 CFU mL -1 .An aliquot (10 mL) of this suspension was added to 10 mL of sterile antibiotic agar at 40 °C and then, inside a laminar flow cabinet, this mixture was poured onto an agar plate.Each tested compound (100 mg) was dissolved in chloroform and put on a paper disk (6 mm diameter), that was dried and placed on the agar plate.Each plate was constituted by 5 sample/disk, together with a disk containing chloramphenicol (30 mg) and another containing only chloroform that were used as positive and negative controls, respectively.The susceptibility of the bacteria and the yeast was determined by the formation of a growth inhibitory zone (mm) of each extract and tested compounds, observed after 18 h of incubation at 37 °C.Experiments were run in triplicate, and the results are presented as mean values of the three measurements.Chloramphenicol and miconazole were purchased from Sigma Chemical Co.(St.Louis, MO).The broth dilution test was used to evaluate the minimum inhibitory concentration (MIC) of growth and the initial inoculums contained 5×10 5 CFU mL -1 . 11Tested compounds were dissolved in dimethyl sulfoxide (DMSO).Sequential dilutions provided the final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 mg/mL of tested compounds in BHI medium.Then, 100 mL of the inoculum was added to each tube.After an incubation time of 18 h at 37 °C, the lowest concentration of the tested compounds that inhibited the microorganisms growth (MIC) was visually determined.Tests using DMSO as negative control and chloramphenicol (for bacteria) and miconazole (for Candida) as positive controls were carried out in parallel.MIC tests were performed in duplicate with full agreement between both results.
Compound 5 was isolated from hexane extract as a white amorphous solid, mp 218-220 °C, and showed positive Liebermann-Burchard (LB) test for triterpenes. 17he carbon signals attributed to compound 5 were in accordance with the reported data. 12,16Through 2D NMR spectral data (HSQC, HMBC and NOESY), the chemical shifts of all hydrogens assigned to compound 5 as well as its correlations were fully established (Figure 2 and Table 1).To the best of our knowledge, it is the first time that these data are reported.
Compound 6 (Figure 2) was obtained as an amorphous white solid, mp 250-254 °C, [α] D 20 -23, CHCl 3 ], and showed positive LB test for triterpenes. 17The elemental analysis of compound 6 presented C 78.54% and H 10.97%, compatible with the molecular formula C 30 H 50 O 3 (MW 458 g mol -1 ; calculated: C 78.55%; H 10.99%).ESI mass spectrometry confirmed this molecular weight, presenting a fragment m/z 423.35 (24%) [M + H -2H 2 O].This type of fragmentation is described for PCTT.According to Rhourri-Frih, 18 in PCTTs like betulin, the loss of two molecules of water occurs due to the presence of two hydroxyl groups, as observed for compound 6.The IR spectra of 6 showed absorption bands: at 3327 cm -1 , compatible with the presence of hydroxyl group; at 2920 and 2850 cm -1 , characteristic of the alicyclic hydrocarbon; and also at 1712 cm -1 , which was attributed to a carbonyl group.The 13 C NMR and DEPT 135 spectra showed 30 signals: seven primary, eleven secondary, five tertiary and seven quaternary carbons, which were according to the PCTT skeleton.The 1 H and 13 C NMR data indicated the compound 6 as friedelane derivative, 8     was consistent with a friedelane type triterpene skeleton. 12,14,16,19he stereochemistry of triterpene 6 was established by means of data obtained from NOESY spectrum.It was possible to observe nOe correlations between H-23 and H-4ax, H-2eq, H-6eq and H-24.nOe effects were also observed between H-24 and H-1ax, H-6eq and H-25, between H-25 and H-11eq and H-12ax.By these NOESY correlations the establishment of a chair conformation for rings B and C (Figure 3) was enabled.In the NOESY contour map, it was observed that the signal of H-  1.75).The NOESY data allowed determining that ring E also has chair conformation (Figure 3).Antimicrobial activities have been described for pentacyclic triterpenes, such as oleananes, 20 ursanes, 20 friedelanes, 21 and lupanes. 22It is speculated that the mechanism of action of triterpenes is due to a disruption on the microorganism's cellular membrane. 20,23For this reason, the hexane extract and triterpenes 3-oxofriedelane (1), 3-oxo-12α-hydroxyfriedelane (3), 3,16-dioxofriedelane (5) and 3-oxo-12α,29-dihydroxyfriedelane (6) were tested against standard bacteria strains of Escherichia coli, Citrobacter freundii, Bacillus cereus and against the yeast Candida albicans, using disk diffusion test.The hexane extract was moderately active on disk diffusion test as well as in the broth dilution test, presenting a MIC value of 512 mg/mL.From the tested triterpenes, 3-oxo-12αhydroxyfriedelane (3) was active in the macrodilution test, presenting a MIC = 512 mg/mL against C. albicans.The activity of 3 was lower in comparison to that was produced by miconazole (16 mg/mL), and was not detected in the disk diffusion test, probably because of low polarity of this compound. 24able S1.Antimicrobial activities of hexane extract and compounds 1, 3, 5 and 6 (each concentration at 100 mg/mL)

Samples
Inhibition zone diameter (mm)

Figure 1 .
Figure 1.Chemical structures of the triterpenes isolated from branches of M.gonoclada.

a
Negative control; b Positive control (bacteria); c Positive control (fungus); ND (Not Detected).
containing two hydroxyl groups.The 1 H NMR spectrum of 6 showed hydrogen signals at d H 0.70 (s), d H 0.93 (s), d H 0.97 (s), d H 1.01 (s), d H 1.17 (s), d H 1.18 (s) and d H 0.87 (d; J 7.0 Hz) that were associated to seven methyl groups.According to the literature, 8 the doublet at d H 0.87 is consistent with methyl group H-23 of members of the friedelane series.In the 1 H NMR spectrum, the hydrogen signals at d H 3.12 (d; J 11.2 Hz) and d H 3.64 (d; J 11.2 Hz) were observed.Correlations of these signals with the signal of C-29 (d C 71.6) (CH 2 ) were observed in the HSQC contour map.The chemical shifts in this NMR region are typical of carbons attached to hydroxyl group, 14,16,19 suggesting the existence of hydroxyl group attached to C-29.Correlations observed in the HMBC contour map, among the signal of H-29 with signals of C-19 and C-30, confirmed the presence

Table 1 .
13 and13C NMR spectral data of 3,16-dioxofriedelane (5) and 3-oxo-12α,29-dihydroxyfriedelane(6) 18 (d H 1.94) is correlated with the signals of H-12ax (d H 3.94) and H-26 (d H 0.97).The H-12 (d H 3.94) showed correlations between the signals of H-25 (d H 0.93) and H-26 (d H 0.97), indicating that H-12 is in axial position, and consequently the ring D of this new friedelane structure is in a chair conformation.Other evidence that confirms this hypothesis is the correlation between H-8 (d H 1.