Phytoconstituents from Sidastrum micranthum ( A . St .-Hil . ) Fryxell ( Malvaceae ) and antimicrobial activity of pheophytin a

Sidastrum micranthum (A. St.-Hil.) Fryxell, a member of the Malvaceae family, is called malva preta in Brazil. As this species is commonly used to treat bronchitis, cough, and asthma, better knowledge of its chemical compounds is important. The phytochemical study of its hexane extract, using chromatographic techniques, led to isolation of six compounds: the triterpene isoarborinol, a mixture of sitosterol and stigmasterol, sitosterol-3-O-β-D-glucopyranoside, pheophytin a, and 132-hydroxy-(132-S)-pheophytin a. Structural identification of these compounds was carried out using spectroscopic methods such as IR and 1D and 2D NMR (HOMOCOSY, HMQC, HMBC, and NOESY). Compounds isolated from S. micranthum were screened for their in vitro antifungal and antibacterial activity against twenty fungal and bacterial standard strains. Pheophytin a exhibited antimicrobial action against all microorganisms tested.


INTRODUCTION
Malvaceae is a widespread family comprising about 243 genera and 4225 species, distributed mainly in tropical areas (Costa et al., 2007).Mainly because of their anti-oxidant and anti-inflammatory activities, the natural compounds isolated from Malvaceae species are used worldwide to treat such diseases as asthma and gastritis, (Oliveira et al., 2012;Teles et al., 2014).
Sidastrum micranthum (A.St.-Hil.)Fryxell (Malvaceae), known as malva preta, is a small shrub commonly found in Cuba, Costa Rica, Venezuela, Guyana, and Brazil (Bovini, Carvalho-Okano, Vieira, 2001).The infusion prepared from its leaves has traditionally been used to treat bronchitis, cough, and asthma.The leaves are also used as cataplasms (poultices), with hot butter and olive oil, as a moisturizing agent (Agra et al., 2007).
The antimicrobial activity of Malvaceae species is well reported (Konaté et al., 2012;Silva et al., 2009).In recent decades, the emergence of microbial resistance to antibiotics has increased interest in exploring the potential of plant-derived antimicrobials (Silva et al., 2010).To increase our knowledge of S. micranthum phytoconstituents, this species was submitted to a phytochemical investigation.In addition, the antimicrobial activity of the compounds isolated was evaluated.

General procedures
Chromatographic columns were packed with silica gel 60 (ASTM, 230-400 mesh, Merck).Thin-layer chromatography (TLC) was performed on PF 254 plates, and the spots were visualized under ultraviolet light (244 and 366 nm) and by exposure to iodine vapor.Isolated compounds were identified by infrared (IR; Perkin-Elmer, FT-IR-1750, and Shimadzu, IR prestige 21) and extensive one-and two-dimensional nuclear magnetic resonance (NMR) analysis ( 1 H 200 MHz and 13 C 50 MHz, Varian-Mercury; or 1 H 500 MHz and 13 C 125 MHz, Bruker-AC) using deuterated solvents.

Plant material
The aerial parts of S. micranthum were collected in Monteiro City, Paraiba/Brazil, in June 2006.A voucher specimen (JPB 6865) was authenticated and deposited at the Professor Lauro Pires Xavier Herbarium, Federal University of Paraiba (CCEN/JPB/UFPB).

Extraction and isolation of compounds
Plant material was dehydrated in an oven at 40 ºC for 72 hours and then ground with a mechanical mill, yielding 6 kg of powder, which was macerated with 95% ethanol at room temperature.This process was repeated to maximize the extraction.The ethanolic extract was concentrated using a rotary evaporator, yielding 200 g of crude ethanol extract.This material was solubilized in ethanol:water (8:2) and submitted to liquid-liquid extraction with hexane, chloroform, ethyl acetate, and n-butanol, affording 46 g of hexane extract, 7 g of chloroform extract, 6 g of ethyl acetate extract, and 8 g of n-butanol extract.
Hexane extract (10 g) was chromatographed on silica gel (column A) eluted with hexane, ethyl acetate, and methanol.From this process, 139 fractions were obtained and combined by TLC.Compound 1 was the pure white powder (15 mg) from fractions 29-34.Recrystallization of fractions 39-64 with chloroform yielded 79 mg of colorless crystals, later identified as a mixture of compounds 2 and 3. Fractions 65-90 (400 mg) were chromatographed on a silica gel column under medium-pressure liquid chromatography (MPLC-Model BÜCHI 688), using hexane, ethyl acetate, and methanol and yielding 90 fractions.The pure fractions 60-64 (40 mg) and 71-83 (30 mg), both amorphous dark green solids, were named compounds 4 and 5, respectively.The combined fractions 95-129, from column A, yielded a white solid precipitated (48 mg) that was separated and identified as the compound 6 (Figure 1).

Test-microorganisms
Among the strains (bacteria and yeast) selected for evaluation of the antimicrobial activity of the isolated compounds, six were obtained from the Mycology

Determination of minimum inhibitory concentration (MIC)
Chloramphenicol and ketoconazole (Sigma-Aldrich) were used as reference antibacterial and antimycotic controls.Antifungal and antibacterial activities were determined using microbroth dilution assays in 96-well microplates, in duplicate.
To each well was added 100 µL of doubleconcentrated liquid medium CSD or HCM.Ten microliters of each compound was then added to the wells of the first row of the plate, and by serial dilution, concentrations from 300 to 9 µg/mL were obtained.Later, 10 µL of the microorganism inoculum was added to the wells.The plate was incubated at 35 °C for 24 h.Afterward, 20 µL of resazurin sodium 0.01% (w/v) (Sigma-Aldrich) was added.Resazurin is a colorimetric indicator of oxidationreduction for bacteria.As an indicator for yeast, 20 μL of 1% triphenyltetrazolium chloride (TTC) (Sigma-Aldrich) was used.The assay was incubated at 35 °C.Results were read by viewing the color change from blue to pink for bacteria, and from colorless to pink for yeast, indicating growth of the microorganism.
The MIC of the active compound was defined as the lowest concentration able to inhibit growth (Deswal, Chand, 1997).Compounds were considered either active or nonactive according to the following criteria: Compounds with MICs between 50 and 500 μg/mL were classified as having strong/great antimicrobial activity; those with MICs from 500 to 1500 μg/mL had moderate activity; and those with MICs higher than 1500 μg/mL were considered to have weak antimicrobial activity (Houghton et al., 2007;Sartoratto et al., 2004).
Compounds 4 and 5 appeared similar (dark green amorphous solids) and had similar IR bands.On the basis of 1 H and 13 C NMR spectra, we confirmed that compounds  4 and 5 are structurally related, both being formed by porphyrin rings.Extensive analyses of spectral data and comparisons with the literature led to identification of compounds 4 and 5 as pheophytin a and 13 2 -hydroxy-(13 2 -S)-pheophytin a, respectively, previously isolated from the Malvaceae species W. periplocifolia, S. rhombifolia, and S. galheirensis (Teles et al., 2014;Chaves et al., 2013;Silva et al., 2006).
The results obtained indicate that the mixture of phytosterols 2/3 and the flavones acacetin (7) and 7,4′-di-O-methylisoescutelarein (8) did not have an inhibitory effect on the test strains.In contrast, pheophytin a (4) exhibited strong antimicrobial activity against all test strains (Table I).
Two yeast (C.albicans ATCC -90028 and C. albicans ATCC -76615) were sensitive to pheophytin a at 38 µg/mL and were the most sensitive test strains.At 75 µg/mL, pheophytin a was able to inhibit growth of 85% of all strains, indicating strong antimicrobial activity for this compound, according to the criteria reported by Sartoratto et al. (2004) and Houghton et al. (2007).
Pheophytins are formed by the degradation of chlorophyll by Mg-dechelatase and chlorophyllase enzymes and have been previously been reported to have anti-leishmanial and cytotoxic activity (Hörtensteiner et al., 1998;Cheng et al., 2001;Sakata et al., 1990).The results obtained are in agreement with the antimicrobial effect of some chlorophyll derivatives, including pheophorbide, as determined by Gerola et al. (2011).Thus, the antimicrobial activity shown by these compounds may be evidence of their additional role protecting plants from microorganisms in their environment.

TABLE I -
MIC values (µg/mL) of compounds isolated from Sidastrum micranthum Presence of microbial growth (compound not active), − = Absence of microbial growth