Chemical Constituents of Cordia piauhiensis – Boraginaceae

Uma nova saponina triterpênica monodesmosídica, caracterizada como ácido 3 -O-Lramnopiranosil-(1→2)-D-glicopiranosil pomólico, foi isolada de Cordia piauhiensis Fresen (Boraginaceae). Sua estrutura foi determinada através de extensiva análise de métodos de RMN, incluindo os experimentos H, H-COSY, HMQC, HMBC e NOESY. Os triterpenóides ácido quinóvico, ácido cinchólico, ácido 3 -O-6-deoxi-D-glicopiranosídeo cinchólico e 3 -O-Dglicopiranosídeo quinóvico foram também isolados.


Introduction
The genus Cordia (Boraginaceae), a known source of benzoquinones, 1 naphthoquinones, 2 hydroquinones, cromenes, 3 triterpenes, 4 sesquiterpenes, 5 polyphenols, 6 and flavonoids 7 comprises about 250 species distributed throughout the New World. 8Many compounds originally isolated from Cordia species have been reported as presenting several biological activities such as antifungal, larvicidal, anti-inflammatory and anti-androgenic. 2,4,6,7s part of our current interest in Cordia species from Northeastern Brazil flora we have investigated C. piauhiensis Fresen (syn.: Cordia rufescens A. DC.), an endemic shrub distributed in the South, Southeast and Northeast regions of Brazil. 8In a previous paper we reported the isolation and characterization of a triterpenoid bidesmoside saponin from the stems of this plant. 9ontinuing with our phytochemical research we now report the isolation and structural elucidation of 3 -O--Lrhamnopyranosyl-(1→2)--D-glucopyranosyl pomolic acid (1), a novel monodesmoside saponin, along with four triterpenoids and other known compounds.

Results and Discussion
Compound 1 was isolated as an amorphous solid, mp 218-220 °C.The ESIMS (negative ion mode) showed a molecular ion peak at m/z 779 ([M] -), consistent with a molecular formula C 42 H 68 O 12 , which was confirmed by its 1 H and 13 C NMR spectral data (Tables 1).Its IR spectrum showed strong absorption bands at max 3428 (hydroxyl) and 1695 cm -1 (carboxyl).The 13 C NMR BB and DEPT spectra of 1 (Table 1) indicated 42 carbon atoms, related to 8 methyls, 10 methylenes, 16 methines and 8 nonhydrogenated carbons.Besides the 30 carbons assigned to the aglycone, 12 carbons were assigned to two sugar units.The 13 C NMR spectra demonstrated the presence of a trisubstituted double bond ( 129.6 and 140.1), a carboxylic acid group ( 182.6), an oxymethine carbon ( 90.5), an oxygenated non-hydrogenated carbon ( 73.9) and seven methyl groups between 16.2 and 28.7, consistent with an aglycone, which was established to be the 3 ,19 -dihydroxyurs-12-en-28-oic acid, also known as pomolic acid, after appropriate NMR and mass spectrometry (negative ion peak at m/z 471) analysis, and comparison with literature data. 10The two sugar units were evidenced by two anomeric carbon signals at 105.7 and 102.0 correlated with the proton signals at 4.41 (d, J 7.2 Hz) and 5.36 (d, J 1.3 Hz), respectively, in the HMQC spectrum.To determine the identity of each monosaccharide interglycoside linkage, as well as to establish the linkage of the disaccharide chain to the aglycone, 1 H and 13 C NMR assignments were unambiguously made by a combination of several 2D experiments such as 1 H, 1 H-COSY, HMQC, HMBC and NOESY.In the 1 H NMR spectrum of 1 the presence of a glucose unit was supported by the anomeric proton signal at 4.41 and two double doublets for the oxymethylene protons at 3.66 and 3.83.Similarly, the presence of a rhamnose unit was readily supported by the characteristic methyl doublet at 1.21 and by the anomeric proton signal at 5.36. 11The anomeric configurations for the sugar moieties were defined as for the glucose and for the rhamnose from their coupling constants of 7.2 and 1.3 Hz, respectively and by comparison with data from literature. 11he linkage between the two sugar units was established from the HMBC correlation between the signals of the anomeric proton of the rhamnose at 5.36 and the carbon signal of the glucose at 79.1 (C-2).The attachment of the disaccharide chain was particularly determined based on the relevant correlation between C-1' ( 105.7) and H-3 ( 3.18) in the HMBC spectrum, and by NOE correlation between H-1'( 4.41) and H-3 ( 3.18), observed in the NOESY experiment as shows in Figure 1.This also was confirmed by the conspicuous deshielding of C-3 ( 90.5) as well as by analogy with the NMR data of the known saponins. 9,12Thus, the structure of 1 was determined as the 3 -O--L-rhamnopyranosyl-(1→2)--D-glucopyranosyl pomolic acid.

General experimental procedures
Melting points were determined using a digital Mettler Toledo FP90 apparatus.The optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.IR spectra (KBr pellets) were recorded using a Perkin-Elmer FT-IR 1000 spectrometer.ESIMS were measured on a Micromass Quatro LC instrument.NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for 1 H and 125 MHz for 13 C) or DPX-300 (300 MHz for 1 H and 75 MHz for 13 C) spectrometers using pyridine-d 5 , CD 3 OD or D 2 O as solvents.Chemical shifts, given on the scale, were referenced to internal DSS for D 2 O solution, and to the residual undeuterated portion of the deuterated organic solvent, for proton (pyridine, H 8.74, 7.58, 7.22; CD 3 OD, H 2.31), and the center peak of the deuterated solvent (pyridine, C 150.35, 135.91, 123.87;CD 3 OD, C 49.15).Column chromatography was run using silica gel 60 (70 -230 mesh, Vetec) and Sephadex LH-20 (Pharmacia).TLC was performed on precoated silica gel poliester sheets (kieselgel 60 F 254 , 0.20 mm, Merck).Saponins were detected by spraying with vanillin/perchloric acid/EtOH solution followed by heating at 120 °C, while the sugar was detected by spraying the orcinol reagent.

Plant material
Cordia piauhiensis was collected in August 1999, from Barreiro Grande -Crato County, State of Ceará, and identified by Prof. Edson Paula Nunes.A herborized specimen (# 29.104) has been stored at the Herbario Prisco Bezerra (EAC) of the Departamento de Biologia, Universidade Federal do Ceará.

Extraction and isolation
Air-dried and powdered roots (2.8 Kg) and stems (1.7 Kg) were individually extracted exhaustively with EtOH at room temperature.After evaporation of the solvents under reduced pressure the crude extracts were obtained.Upon concentration of the EtOH extract from roots a precipitate was obtained, which was filtered and washed successively with acetone and EtOH to yield 2 (15 g).The remaining EtOH liquors were evaporated (198 g) and coarsely fractionated over silica gel by elution with nhexane, CHCl 3 followed by EtOAc and finally MeOH.The  ) correlations for 1.