Flavonoids from Chiococca braquiata ( Rubiaceae )

The genus Chiococca (Rubiaceae) with 22 species is endemic of the American Continent, and occurs from North America to Brazil. Several Chiococca species have been traditionally used in these regions for the treatment of numerous human ailments including inflammation, antivirus, anti-edema and as aphrodisiac. Other plants belonging to the Rubiaceae family have yielded a number of interesting biologically active compounds, including seco-iridoids with mild DNA-activity isolated from Chiococca alba. C. braquiata, however, has not been subjected to phytochemical analysis or assayed for any biological activity. In an ongoing quest to identify biologically active compounds from the Brazilian Rubiaceae plant species, antifungal evaluation using Cladosporium sphaerospermum and C. cladosporioides was performed on CH 2 Cl 2 -MeOH (2:1, v/v) extract from C. braquiata leaves, which exhibited strong antifungal activity. Bioassay-guided fractionation of the bioactive extract led to the isolation of the inactive flavonoids 4’methoxykaempferol-7-(acetyloxy)-3,5-O-L-rhamnoside (1), apigenin, 7-O-methoxyquercetrin and quercetrin. The triterpenes -amirin, -amirin, ursolic and oleanolic acids were also isolated. The structure of the new derivative 1 and the known compounds were elucidated by spectroscopic methods, mainly 2D NMR and MS.


Introduction
The genus Chiococca (Rubiaceae) with 22 species is endemic of the American Continent, and occurs from North America to Brazil.Several Chiococca species have been traditionally used in these regions for the treatment of numerous human ailments including inflammation, antivirus, anti-edema and as aphrodisiac. 1Other plants belonging to the Rubiaceae family have yielded a number of interesting biologically active compounds, 2,3 including seco-iridoids with mild DNA-activity isolated from Chiococca alba. 4 C. braquiata, however, has not been subjected to phytochemical analysis or assayed for any biological activity.In an ongoing quest to identify biologically active compounds from the Brazilian Rubiaceae plant species, antifungal evaluation using Cladosporium sphaerospermum and C. cladosporioides was performed on CH 2 Cl 2 -MeOH (2:1, v/v) extract from C. braquiata leaves, which exhibited strong antifungal activity.Bioassay-guided fractionation of the bioactive extract led to the isolation of the inactive flavonoids 4'methoxykaempferol-7-(acetyloxy)-3,5-O--L-rhamnoside (1), apigenin, 7-O-methoxyquercetrin and quercetrin.The triterpenes -amirin, -amirin, ursolic and oleanolic acids were also isolated.The structure of the new derivative 1 and the known compounds were elucidated by spectroscopic methods, mainly 2D NMR and MS.

General procedures
For column chromatography silica gel 60 (Merck 230-400 mesh) and Sephadex LH-20 were used.TLC analysis carried out on silica gel 60 F 254 .IR spectrum was recorded on a Nicolet Spectrometer.UV spectra were recorded on a Perkin-Elmer UV/Vis Spectrometer Lambda 14P.The ES-MS spectra were obtained on a VG Platform II Spectrometer.NMR spectra were recorded in DMSO-d 6 or CDCl 3 on a Varian Unit 500 instrument at 500 MHz for 1 H and 125 MHz for 13 C, using TMS as internal standard.The DEPT experiments were performed using polarization transfer pulses of 90 and 135 o .

Plant material
Leaves of Chiococca braquiata Ruiz & Pav.(now Chiococca alba (L.) Hitchc.) were collected around Lagoa do Abaeté, BA, Brazil and identified in the Botanical Institute, SMA, SP.A voucher no.1934 specimen has been deposited in the herbarium Maria Eneida P. K. Fidalgo, SP, Brazil.

Bioassay
The antifungal activity against C. cladosporioides was performed using direct bioautography with 10 L of the solutions of crude extracts and pure compounds, which were prepared in different concentrations ranging from 300 to 10 g respectively, as described elsewhere. 5

Results and Discussion
The bioactive soluble EtOAc part of a CH 2 Cl 2 :MeOH (2:1, v/v) extract, prepared from the leaves of C. braquiata, was chromatographed over Sephadex LH-20 column and preparative TLC to afford a new flavonoid 1, along with the know compounds apigenin (2), 6 7-O-methoxyquercetrin (3), 7 quercetrin (4), 8 -amirin, -amirin, ursolic and oleanolic acids. 9Compound 1 was isolated as a white amorphous powder, with molecular formula C 30 H 34 O 15 deduced from the [M+Na] + peak at m/z 657 in the ES-MS and supported by 13 C and 1 H NMR data.Its UV spectrum exhibited characteristic absorbance bands of flavonols 10 at 259 and 341 nm.The 1 H and 13 C NMR spectra (Table 1) revealed two set signals, which features indicated a flavonoid with glycosidic and acetyl groups.The 1 H NMR signals attributed to the aglycone at d 7.75 (2H, d, J 8.0 Hz), 6.89 (2H, d, J 8.0 Hz), identified as a 2H AA' and a 2H XX'system, 6.63 (1H, d, J 1.25 Hz), 6.32 (1H, d, J 1.25 Hz) showed characteristic pattern of kaempferol. 5The 1 H NMR spectra also showed signals at 5.30 (2H, br s), 3.92 (2H, br, dd, J 9.0, 11.0 Hz), 3.48 (2H, dd, J 9.0, 10.8 Hz), 3.28 (2H, m), 3.08 (2H, m) and 0.87 (6H, d, J 6.5 Hz), which revealed the presence of at least two glycosyl moieties, clearly evidenced by integration area of the peaks corresponding to these signal values.In addition, a methoxyl and an acetyl groups were also confirmed by two singlets at 3.79 (56.0 q) and 1.87 (24.3 q) and 174.7 (s), respectively.The identification of the sugar moieties as two -rhamnopyranosides was determined from the chemical shifts, multiplicity of the signals, and absolute values of the coupling constants in the 1 H NMR and 1 H-1 H COSY spectra as well as 13 C NMR data.The signals observed in the 13 C NMR spectrum (Table 1) at 101.The first indication of the positions C-3 and C-5 as substitution sites in 1 was evidenced from the 1 H NMR spectrum, which do not showed the typical signals assigned to H-3 and the HO-C-5 quelate.The HMBC spectrum (Table 1) confirmed these positions as glycosylation sites due to the connectivities that were observed between 5.30 (1H, br s, H-1") and 134.2 (C-3) and the correlation between 5.30 (1H, br s, H-1"') and 161.7 (C-5).The correlation between the signal corresponding to -OCH 3 at 3.79 with C-4' at d 161.7 also was important to attribute all substitutions in 1, if we consider that the only remain place linkage for the acetyl group should be at hydroxyl group in C-7.In fact, by the NOESY experiments (Figure 1) was observed a weak spatial correlation between the signal at 6.89 (H-3' and/or H-5' with the resonance at 3.79 corresponding to -OMe.The absence of cross peaks correlation between H-6 and H-8 with the CO of the acetyl group could suggested that this group was not attached to the hydroxyl at C-7.However, a correlation observed in the HMBC spectrum (Table 1) between the anomeric hydrogens at 5.30 and C-5 (161.7) and C-3 (134.2) indicated that the second sugar unit was located at C-3, and thus corroborated the acetylation site at C-7.These findings confirmed the substitution pattern for compound 1.The absence of a bathochromic shift in the UV (methanol) spectrum after the addition of AlCl 3 was other important evidence for the substitution of the 5-OH, as well as the substitution at C-7 being indicated by the absence of a bathochromic shift in band II (ring A) upon addition of NaOAc.To our knowledge, 1 is a new flavonol derivative assigned as 4'-methoxykaempferol-7-(acetyloxy)-3,5-O--L-rhamnoside (1).
The strong antifungal activity detected in the crude extract (minimum amount required for the inhibition of fungal growth on TLC plates = 10 g for C. cladosporioides, standard nystatin = 1.0 g) was decreased proportionally during fractionation procedures.The detection limit values of the CHCl 3 , and EtOAc bioactive solubles (50 and 30 g, respectively) suggested that the antifungal activity was substantially lost during guidedfractionating of the EtOAc extract with fungus C. cladosporioides.The very weak activity detected for pure flavonols 1-4 (detection limit values higher than 300 g) indicated that their individual activities are not potent enough to be considered for practical use or to justify the strong activity detected in the crude extract.The antifungal activity against C. cladosporioides was significantly enhanced when a combination of the flavonols 1, 2, 3 and 4 where tested.The detection limit of compound 1 against C. cladosporioides was reduced from 350 to 100 g when it was combined with compounds 2 (300), 3 (400), and 4 (250).Based on these observations, the synergistic activity of these compounds, and probably of others not isolated in this study, against this fungus, could be inferred.