Complete 1 H and 13 C NMR assignments and antifungal activity of two 8-hydroxy flavonoids in mixture

A mixture of the two new flavonols 8-hydroxy-3,4’,5,6,7-pentamethoxyflavone (1) and 8-hydroxy-3,3’,4’,5,6,7hexamethoxyflavone (2) was isolated from a commercial sample of Citrus aurantifolia. An array of one(1HNMR, {1H}-13C NMR, and APT-13C NMR) and two-dimensional NMR techniques (COSY, NOESY, HMQC and HMBC) was used to achieve the structural elucidation and the complete 1H and 13C chemical shift assignments of these natural compounds. In addition, the antifungal activity of these compounds against phytopathogenic and human pathogenic fungi was investigated.

is increasingly used as a technique to provide insight into mixture of natural products belonging to the same or different chemical classes without previous separation of the individual components.The sample preparation for NMR is simpler and nondestructive.In this context, NMR methods have been used with success in the structural identifi cation of natural products in mixture such as alkanes (Loaiza et al. 1997), essential oil (Al-Burtamani et al. 2005), furanosesquiterpenes (Gaspar et al. 2005), diterpenoids (Appendino et al. 1992), triterpenoids (Olea and Roque 1990), sterols (Zollo et al. 1986), saponins (Young et al. 1997), anthocyanins (Kosir and Kidric 2002), glycerol esters (Gusntone 1991) and phenolic acids (Gerothanassis et al. 1998).
In this paper, we report the structural elucidation of two new 8-hydroxypolymethoxyflavonols (1 and 2) isolated from a commercial sample of Citrus auranti-folia.The structural determination of these compounds, as components of a mixture, was based on spectral data, including 2D NMR techniques, such as heteronuclear correlation 1 H-13 C-COSY-n J CH (n = 1, HMQC = 1 Hdetected Heteronuclear Multiple Quantum Coherence; n = 2 and 3, HMBC = 1 H-detected Heteronuclear Multiple Bond Connectivity) and gas chromatography mass spectrometry, together with chemical transformation and comparative analysis of chemical shifts described in the literature.In addition, the antifungal activity of these compounds against phytopathogenic and human pathogenic fungi was investigated.

GENERAL EXPERIMENTAL PROCEDURES
Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for 1 H and 100 MHz for 13 C on a JEOL Eclipse+ 400 spectrometer, using TMS as internal standard or by reference to solvent signals.GC-EIMS spectra were run at 70 eV on a Shimadzu QP-2000 spectrometer.

PLANT MATERIAL
The fruits of Citrus aurantifolia (" lime of Persia") were purchased from local supermarket in Florianópolis, Santa Catarina State, Brazil.

EXTRACTION AND PURIFICATION
The peels of the fruit were removed manually.The peels of C. aurantifolia (600 g) were extracted by maceration with hexane (1 L) at room temperature for 72 h.The peels were later removed by fi ltration and the hexane extracts were concentrated under reduced pressure.The addition of acetone to the extracts furnished the compounds 1 and 2 as precipitates.A mixture of 1 and 2 (26 mg) was treated with CH 2 N 2 as usual to yield 1a + 2a (26 mg). 1 H (400 MHz) and 13 C (100 MHz) NMR in benzene-d 6 : Tables I and II.

ANTIFUNGAL ACTIVITY
The assays were carried out with three phytopathogenic fungi (Penicillium digitatum, Colletotrichum sp. and Curvularia sp.) and two species of human pathogenic fungi (Trichophyton mentagrophytes and Microsporum canis).Fungal strains were maintained in potato dextrose agar at 4 • C and the inoculum was a suspension of each strain, in nutrient broth, containing approximately 5.10 4 spores/mL.BIOAUTOGRAPHY METHOD 50µL of each solution of extract or substance prepared in hexane-acetone (1:1) (100µg/mL) were applied on TLC plates (60F 254 ; Merck), as well as 50µL of amphotericin B (1.60µg/ml) (positive control).The plates were submerged on the fungal inoculum and incubated for 72 h at 30 • C in a humid camera.The plates inoculated with dermatophite fungi were then sprayed with p-iodonitrotetrazolium violet (INT) and once more incubated for 4 hour at 30 • C. The plates where fungal growth occurred, the INT changed from yellow to purple, while persistence of the yellow color indicated no a Number of hydrogens bound to carbon atoms deduced by comparative analysis of { 1 H}-and APT-13 C NMR spectra.Chemical shifts and coupling constants (J ) obtained from 1D 1 H NMR spectrum.Heteronuclear 1 H-13 C-COSY-n J CH (n = 2 and 3, HMBC, Table II) and homonuclear 1 H-1 H-COSY and 1 H-1 H-NOESY spectra were also used in these assignments.b AA'XX' system.
growth.The diameter of growth inhibition was expressed in millimeters.

MINIMAL INHIBITORY CONCENTRATION
The minimal inhibitory concentration was determined using the method described in the literature (Smânia et al. 1995, Pizzolatti et al. 2002) and the results are expressed in µL/mL.

RESULTS AND DISCUSSION
1 H and 13 C NMR spectral data of the polymethoxylated flavonoids 1 and 2, recorded in CDCl 3 (See Experimental), are in good agreement with those described in the literature (Calvert et al. 1979, Chen et al. 1997).
The resonances of the aromatic methoxyl groups attached to ortho-disubstituted carbons occur considerably downfi eld (ca δ C 60 ppm) when compared with aromatic methoxyl groups attached to carbons bearing only  a Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD-and APT-13 C NMR spectra.Heteronuclear 1 H-13 C-COSY-1 J CH (HMQC, Table I) and homonuclear 1 H-1 H-COSY and 1 H-1 H-NOESY spectra were also used in these assignments.
one or no ortho substituent (ca δ C 55 ppm).Unequivocal 1 H and 13 C chemical shift assignments of these natural compounds were also carried out by 2D 1 H-13 C correlation techniques (HMQC and HMBC) involving comparison with literature data (Calvert et al. 1979, Chen et al. 1997).The mass spectra of these flavonoids, obtained by high-resolution gas chromatography (HRGC) mass spectral analysis, showed intense molecular ions and [M-15] + fragments characteristic of flavonoids with methoxyl groups at C-6 and/or C-8 (Bohm 1998).The ionic fragments attributed to principal peaks observed in mass spectra of the flavonoids 1 and 2 are showed in Figure 2. Exhaustive analysis of 1D and 2D NMR spectra (CDCl 3 , Experimental; benzene-d 6 , Tables I and II) of the mixture of 1 and 2 and of their O-methyl ether derivatives 1a and 2a obtained by methylation with CH 2 N 2 , recorded in benzene-d 6 (Tables I and II), was used to confi rm the presence of a hydroxyl group at carbon C-8.This analysis was facilitated by consid-An Acad Bras Cienc (2007) 79 (2)  ering electronic effects (inductive and mesomeric) and anisotropic effects as responsible for usual shift-parameters (Günther 1995), solvent effects induced by benzene-d 6 (Horie et al. 1998) and comparison with model compounds, e.g. 3 (Agrawal et al. 1989).In benzened 6 as solvent, all the methoxyl signals in the 1 H NMR spectra of the mixtures of 1 + 2 and 1a + 2a appeared clearly separated, which in combination with the relative intensities (1 in major percentage than 2) allowed to assign methoxyl groups of each component (Table I).The 1 H-1 H-NOESY spectra of mixtures of 1 + 2 and 1a + 2a showed dipolar interactions (NOE effect) between: MeO-3 and H-2'/H-6', and MeO-4' and H-3'/H-5' of 1 and 2a; ' of 2 and 2a.Consequently, these results allowed unequivocally to assign the 1 H chemical shifts of the signals corresponding to MeO-3 and MeO-4' of 1 and 1a as well as those of MeO-3' and MeO-4' of 2 and 2a (Table I).Subsequently, the assignments of the 13 C signals attributed to quaternary carbon atoms C-2, C-3 and C-4' of 1 were based on the heteronuclear long-range coupling with hydrogens H-2'/H-6', MeO-3 and MeO-4', respectively, observed in the HMBC spectrum, which also revealed couplings ( 3 J CH ) of C-3' with both H-5' and ' of 2 (Table II).Comparative analysis of the 1 H NMR spectra (in C 6 D 6 ) of the mixtures 1 + 2 and 1a + 2a showed additional singlet signals for MeO-8 at δ H 3.85 (1a) and 3.88 (2a), which revealed correlation with the 13 C signals corresponding to C-8 at δ C 141.3 (1a) and 141.4 (2a) in the HMBC spectrum (Table II).These 13 C chemical shifts compared with the remaining signals corresponding to quaternary oxygenated carbon atoms of the A ring of flavonoids may be attributed only to C-8, as anticipated by a mesomeric effect ( 13 C chemical shifts: C-7C-5C-9, conjugated with carbonyl group C-4) and also comparison with model flavonol 3 (e.g.) showing C-8 with a smaller chemical shift than C-6 (Agrawal et al. 1989).Finally, our attention was drawn to the signifi cant 13 C chemical shift difference ( δ C : 9.8 ppm) observed between the signals corresponding to C-2 of 8-hydroxy-(1 and 2: δ C 143.1) and 8-methoxy-(1a and 2a: δ C 152.9) derivatives.These assignments are summarized in Tables I and II.Most probably, this difference may be attributed to an intramolecular hydrogen bond involv-ing the pyran oxygen (1b and 2b), containing unpaired electrons conjugated with the carbonyl group C-4.A hydrogen bond may be used to justify an attenuation of delocalization (mesomeric effect) of the unpaired electrons of the heterocyclic oxygen atom, which results in a smaller contribution of the canonical structure shown in 1c and 2c.Thus, presence of a methoxyl group at C-8 allows major contribution of the corresponding canonical structures (1c and 2c) and, consequently, the partial positive charge at the oxygen atom reduces the electronic density at C-2 by a major inductive electron attracting effect (deshielding).
The fragments 1d-f and 1e-2f (Figure 2) attributed to main peaks observed in EIMS are consistent with these structural deductions.
Thus, the new polymethoxylated flavonols isolated as a mixture from commercial C. aurantifolia were characterized as 8-hydroxy-3,4',5,6,7-pentamethoxyflavone (1) and 8-hydroxy-3,3',4',5,6,7-hexamethoxyflavone (2).The antifungal activity of mixture of the flavonols 1 and 2, against phytopathogenic and human pathogenic fungi was studied by two different methods.A preliminary analysis was carried out by a bioautography method.The mixture contends 1 and 2 was active against all tested organisms (Table III).However, when these compounds were assayed by a micro dilution method, only a discrete activity was observed.As can be observed in Table IV, the phytopathogenic fungi are more resistant than the An Acad Bras Cienc (2007) 79 (2) 1 H AND 13 C NMR ASSIGNMENTS OF TWO 8-HYDROXY FLAVONOLS 221 human pathogenic fungi.These results could already be expected because these phytopatogenics were isolated from the same it Citrus species from which the flavonoids were originally obtained.