( R )-(-)-Carvone and ( 1 R , 4 R )-trans-( + )-Dihydrocarvone from Poiretia latifolia Vogel

Os óleos essenciais de folhas de Poiretia latifolia Vogel nativas e cultivadas (amostras A e B, respectivamente) e de flores nativas (amostra C), foram obtidos por hidrodestilação e analisados por CG, CG/EM, e através de cromatografia gasosa em fase quiral (CGFQ). Vinte e quatro componentes foram identificados, representando 99,25, 99,26 e 99,23% dos óleos, respectivamente. Os maiores constituintes dos óleos foram os monoterpenos (S)-(-)-limoneno (16,05, 27,60, 15,60%, respectivamente), (1R, 4R)-trans-(+)-diidrocarvona (18,05, 0,66, 77,80%, respectivamente) e (R)-(-)-carvona (61,05, 64,20, 4,50%, respectivamente). Os óleos essenciais apresentaram uma atividade antimicrobiana moderada quando avaliados frente a bactérias e Gram-positivas, Gramnegativas e fungos.


Introduction
Poiretia, a genus of the Papilionoidae-Leguminosae family, is found throughout the equatorial and subtropical regions of the Americas.In Brazil, it comprises about 12 species found in the Southwestern region. 1 Poiretia latifolia Vogel, locally called "erva-de-touro", is native to South America (Southern Brazil, Uruguay, Paraguay, and Argentina).In Brazil, Poiretia latifolia is generally used in popular medicine for the treatment of hemorrhoids, renal diseases, dysentery, and as an aphrodisiac. 2As a continuation of our research on the essential oils of aromatic plants from the state of Rio Grande do Sul, we now report on the chemical constituents of the essential oils obtained from leaves and flowers of native and cultivated P. latifolia.
The identification of the chemical constituents was based on comparison of their relative retention times and mass spectra with those obtained from authentic samples and/ or the Wiley/Nist libraries and those published by Adams. 3 The stereochemistry of the main chiral compounds present in the oils (limonene, carvone and dihydrocarvone) was determined by chiral phase gas chromatography (CPGC), and by chemical reactions.
The oxygenated monoterpenes carvone and dihydrocarvone are potential inhibitors of bacterial, [4][5][6] fungal growth, 7 as well as prospective insect repellents. 8The most important technical application of carvone is its use as a reversible suppressant of sprouting in stored potatoes or flower bulbs. 5Carvone and dihydrocarvone have shown insecticidal activities against the rice weevil Sitophilus oryzae (L), one of the most widespread insect pests of stored cereals. 91][12] (R)-(-)-carvone is the principal constituent in spearmint oil (Mentha spicata) and some oils, such as gingergrass oil, contain racemic carvone. 13S)-(+)-carvone is usually obtained from caraway seeds, while (R)-(-)-carvone is obtained from spearmint oil.

Plant material
Native Poiretia latifolia was collected in the town of São Pedro do Sul, RS, Brazil (29º 37' 14" S, 54º 10' 44'' W).Cultivated P. latifolia was obtained from the botanical garden of the University of Santa Maria, RS, Brazil (29° 42' 39" S, 53° 41' 32" W).Leaves and flowers of native and cultivated P. latifolia were collected in the flowering stage (November-December 2005) from the same population, respectively, and identified by a single author (T.C. D.).Voucher specimens (SMDB 952-954) have been deposited at the Herbarium of the Federal University of Santa Maria.
The oils were analyzed by GC and GC-MS.GC analyses were performed using a Varian CP-3800 gas chromatograph with a data handling system, FID detector and SE-54 fused-silica column (25 m x 0.25 mm i.d., film thickness 0.25 mm).Operation conditions were as follow: injector and detector temperatures, 220 and 280 o C, respectively; carrier gas, H 2 ; oven temperature program from 50 o C to 250 o C at 4 o C min -1 .GC-MS analyses were performed using a VARIAN model 3800 Saturn system operating in the EI mode at 70 eV equipped with CP-SIL cross-linked capillary columns (25 m x 0.25 mm i.d., film thickness 0.25 mm).The identity of the oil components was established from their GC retention times and comparison of their MS spectra with those reported in the literature, 3 and by computer matching with the Wiley 5 mass spectra library, and as well as by co-injection with standards available in our laboratories whenever possible.

Antimicrobial bioassay
The MICs of samples A and B and limonene, carvone, and trans-dihydrocarvone, were determined on 96-well culture plates by a micro dilution method using a microorganism suspension at a density of 10 5 CFU mL -1 with Casein Soy Broth incubated for 24 h at 37 o C for bacteria, and Sabouraud Broth incubated for 72 h at 25 o C for yeasts.The cultures that did not present growth were used to inoculate plates of Standard strains of microorganisms were obtained from American Type Culture Collection (ATCC), and standard antibiotics, chloramphenicol and nystatin, were used in order to control the sensitivity of the microbial test. 21Proper blanks were assayed simultaneously and samples were tested in triplicate.[24]

Results and Discussion
As shown in Table 1, the qualitative and quantitative composition of samples A, B and C, displayed significant differences.More than 20 components were detected in the essential oils, making up 99.25, 99.26, and 99.23% of the total oil of samples A, B and C, respectively.Monoterpenes (ca.99%), mainly limonene, carvone, and trans-dihydrocarvone, were predominant in the essential oils of all the leaves and flowers studied.In leaves of cultivated P. latifolia (sample B), trans-dihydrocarvone was found in lower abundance (0.66%), while in flowers of native P. latifolia (sample C), trans-dihydrocarvone was the main component (77.80%) and there was a lower abundance of carvone (4.50%).These monoterpenes are very important to the flavor and fragrance industry.Because the biological activity, smell and organoleptic properties of these compounds are determined by their stereochemistry, 25 in this study the absolute configuration of a-pinene, limonene and carvone was determined by enantioselective capillary gas chromatography using heptakis (3-O-pentafluoropropionyl-2,6-di-O-pentyl)-bcyclodextrin 17 as the chiral stationary phase (Figure S1).The absolute configuration of dihydrocarvone was determined by enantioselective chemical transformation of carvone to dihydrocarvone.For this purpose, (S)-(+) and (R)-(-)-carvones were submitted to reduction with Zn in methanol-water 19,20 to afford a mixture of diastereoisomers of cis-and trans-dihydrocarvones in a 1:4.5 ratio, respectively (Figure 1), by GC experiment.The four stereoisomers of dihydrocarvone were used as CPGC standards.By comparison of the chromatograms of the standards with the chromatograms of the oils and by co-injection, it was possible to assign unambiguously the absolute configuration of dihydrocarvone as (1R, 4R)-trans.To conduct this study, octakis(3-O-butiryl-2,6di-O-pentyl)-g-CD (Lipodex-E) 18 was used as the chiral stationary phase.(Figure S2).All monoterpenes analyzed were present in the oils as only one isomer (ee > 99%).Thus, the absolute configuration of the monoterpenes was determined as (S)-(-)-a-pinene, (S)-(-)-limonene, (R)-(-)carvone, and (1R, 4R)-trans-(+)-dihydrocarvone.
The antimicrobial activity of the oils and of the isolated (R)-(-)-carvone and (1R, 4R)-trans-(+)dihydrocarvone, was evaluated by determining the minimal inhibitory concentration (MIC).The results, between 1.25-10.0mg mL -1 ), showed that the oils of samples A and B, (R)-(-)-carvone and (1R, 4R)-trans-(+)-dihydrocarvone have only a modest antimicrobial activity against the tested microorganisms compared to chloramphenicol for bacteria and nystatin for yeasts (Table 2).Because of the small amounts of sample C available, we were unable to obtain information about their antimicrobial activity.

Table 1 .
Percentage composition of the essential oils of Poiretia latifolia Vogel a Compounds listed in order of elution from SE-54 column.b Kovats Indices determined on an apolar SE-54.c Kovats Indices determined on a polar PEG-20M column.nd: not identified.Co: peak identifications are based on standard comparison with relative retention time.A: leaves from native P. latifolia.B: leaves from cultivated P. latifolia.C: flowers from native P. latifolia.

Table 2 .
Antimicrobial activity (MIC and MLC, in mg mL -1 ) of the oils of Poiretia latifolia and of carvone and dihydrocarvone