First Chemical Constituents from Cordia exaltata Lam and Antimicrobial Activity of Two Neolignans

The phytochemical study of Cordia exaltata Lam. (Boraginaceae) led to the isolation, through chromatographic techniques, of nineteen secondary metabolites: 8,8'dimethyl-3,4,3',4'-dimethylenedioxy-7-oxo-2,7'cyclolignan (1), 8,8'-dimethyl-4,5-dimethoxy-3',4'-methylenodioxy-7-oxo-2,7'cyclolignan (2), sitosterol (3a), stigmasterol (3b), sitosterol-3-O-β-d-glucopyranoside (4a), stigmasterol-3-O-β-d-glucopyranoside (4b), phaeophytin A (5), 132-hydroxyphaeophytin A (6), 173-ethoxypheophorbide A (7), 132-hydroxy-173-ethoxypheophorbide A (8), m-methoxy-p-hydroxybenzaldehyde (9), (E)-7-(3,4-dihydroxyphenyl)-7-propenoic acid (10), 1-benzopyran-2-one (11), 7-hydroxy-1-benzopyran-2-one (12), 2,5-bis-(3',4'-methylenedioxiphenyl)-3,4-dimethyltetrahydrofuran (13), 3,4,5,3',5'-pentamethoxy-1'-allyl-8.O.4'-neolignan (14), 3,5,7,3',4'-pentahydroxyflavonol (15),5,7-dihydroxy-4'-methoxyflavone (16), 5,8-dihydroxy-7,4’-dimethoxyflavone (17), kaempherol 3-O-β-d-glucosyl-6''-α-L-ramnopyranoside (18) and kaempherol 3,7-di-O-α-l-ramnopyranoside (19). Their structures were identified by 1H and 13C-NMR using one and two-dimensional techniques. In addition, the antimicrobial activity of compounds 1, 2, 13 and 14 against bacteria and fungi are reported here for the first time.


Figure 2. Constituents isolated from Cordia exaltata.
The MIC data of the compound 2 are also summarized in Table 1. It was observed that the MIC was 300 for 35% of the strains. The percentage of strains resistant to the compound 2 to the highest concentration was 40%, therefore higher than that found with the compound 1 (10%). As for the antibiotics tested, they showed inhibitory effect on 60% of the strains, similar to compound 2.
Thus, one can conclude that both the tested compounds have detectable antimicrobial activity against a variety of pathogens, including bacteria and yeasts, in infectious processes relevant from a clinical point of view. It also may be noted the compound 1 had the strongest antimicrobial effect, which could be explained by the lower MIC values against microorganisms [31][32][33][34]. Compounds 13 and 14 were also tested against the same strains and following the same protocol described for 1 and 2, but did not show any significant activity.

General
Silica gel 60 (Merck) 7734 (0.063-0.2 mm particle, 70-230 mesh), flash silica (0.04-0.063 mm particles, 230-400 mesh) and Sephadex LH-20 were used for the fractionation and isolation of the secondary metabolites from C. exaltata. TLC was used to analyse and compare the fractions obtained from chromatographic column procedures. Solvents used in TLC were basically mixtures of the same solvents of the chromatographic experiments. For pure compounds analysed by TLC, three different systems of solvents were tested, to make sure that these compounds were free of impurities. The melting point of the constituents was recorded on a MQAPF-302 apparatus. IR spectra were recorded on a FT-IR-1750 Perkin-Elmer spectrometer. 1 H and 13 C-NMR spectra were recorded on a Varian Oxford 200 NMR spectrometer (200/50 MHz) and on a Varian 500 NMR spectrometer (500/125 MHz).

Collection, Extraction and Isolation
The leaves, stems, fruits and stem bark of Cordia exaltata Lam, were collected near the city of Porto Grande-AP in July 2006 and were identified by Prof. Dr. Rosangela do Socorro Ferreira Rodrigues Sarquis-IEPA (Instituto de Pesquisa Científica e Tecnológica do Estado do Amapá/BR-Institute for Scientific and Technological Research of Amapá). A voucher specimen is deposited in the Herbarium Amapaense/AP/BR (HAMAP), under the code 2528.
Leaves, stems, fruits and stem bark of Cordia exaltata Lam were dehydrated in an oven at 40 °C for 72 h and ground separately in a mechanical mill to yield 847.5 g, 1000.5 g, 281.0 g and 2.310.0 g of a powder, respectively. Each powdered part of the plant was submitted to maceration with methanol (2.5 L, 3.0 L, 1.0 L and 4 L, respectively) for three consecutive days at room temperature and this process was repeated until the maximum amount of chemical constituents had been extracted. The obtained methanol extract solutions were concentrated in a rotatory evaporator, yielding 150.0 g, 150.0 g, 11.0 g and 211.0 g of the respective crude extracts.
An amount of the crude methanolic extract from fruits (12.0 g) was subjected to filtration under reduced pressure using silica gel 60 as stationary phase and eluted with hexane (hex.), ethyl acetate (EtOAc) and methanol (MeOH) alone or in binary mixtures following an increasing gradient polarity. After that, the extracts were concentrated under pressure, leading to the respective phases. The hexane phase (1695.0 mg) yielded 847.8 mg of a precipitate, which was chromatographed on a silica gel column and eluted with hex., EtOAc and MeOH. This procedure led to 28  The methanolic crude extract from the stem bark (100 g) was solubilized in methanol-H 2 O (7:3) and successively partitioned under vacuum using silica gel with hex., CHCl 3 , EtOAc and n-butanol as described in the previous procedures. The hexane phase (5.0 g) was submitted to column chromatography with silica gel using hexane (hex.), ethyl acetate (EtOAc) and methanol (MeOH) alone or in binary mixtures with increasing polarity, leading to 93 fractions, which were further combined according to TLC (hex., AcOEt and MeOH, in three different mixtures at least). Using this technique fractions 16/24, 37/47 and 57/70 were pure and gave 25.0 mg of compound 8, 18.0 mg of compound 9 and 40.0 mg of compound 10. Through column chromatography on Sephadex LH-20, the EtOAc (2.9 g) and n-butanol (2.6) phases yielded compound 17 (15.0 mg) and compound 18 (30.0 mg), respectively.

Antimicrobial Activity Experiments
The microorganisms used througout the tests were: The stock bacterial strains were maintained in Muller Hinton agar (AMH) and the yeast on Sabouraud dextrose agar (SDA) under refrigeration (8 °C). The inoculum of microorganisms was prepared and standardized in sterilized saline (0.85%), containing Tween 80 (1%). Turbidity was visually compared and adjusted to the range of 0.5 tube McFarland, which corresponds to an inoculum of approximately 10 6 CFU/mL (CFU/mL) [37][38][39]. The MIC determination was made by the microdilution method using 96 well plates (INLAB/Brazilian Industry) as protocol [39,40]. In each well of the plate was added 100 µL of liquid medium CSD or HCM doubly concentrated. Then was added 100 µL of each compound in the cavities of the first row of the plate, and by serial dilution concentrations were obtained 300 µg/mL to 9 µg/mL. Then was added 10 µL of the inoculum of microorganisms into the wells. Controls were: Tween 80 (10% in distilled water), chloramphenicol microbial growth control (30 µg/mL, Sigma-Aldrich ® ) for the bacteria and ketoconazole (50 µg/mL, Sigma-Aldrich ® ) for yeasts.
The assay was performed in duplicate and incubated at 35 °C/24 h. After the incubation time was added 20 µL of resazurin sodium 0.01% (w/v) (Sigma-Aldrich ® ) recognized as an indicator of oxidation-reduction colorimetric for bacteria. And in parallel, 20 μL of triphenyltetrazolium chloride (TTC) 1% (Sigma-Aldrich ® ), a colorimetric indicator for the redox yeast [41][42][43]. The assay was incubated at 35 °C. The reading of the test was performed by viewing the color change from blue to pink cavities tests of bactéria, and colorless to pink tests of yeast, indicating growth of the microorganism. MIC for each product was defined as the lowest concentration able to inhibit fungal growth visually, checked by the permanence of the color of the growth indicator.
Biological properties of the compounds were considered either active or non-active according to the criteria reported [31][32][33][34], in which natural compounds showing MIC between 50 and 500 μg/mL are classified as possessing strong antimicrobial activity, those with MIC from 500 to 1,500 μg/mL possess moderate activity and those with MIC larger than 1,500 μg/mL are considered with weak antimicrobial activity.