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

Abstracts

A mixture of the two new flavonols 8-hydroxy-3, 4', 5, 6, 7-pentamethoxyflavone (1) and 8-hydroxy-3, 3', 4', 5, 6, 7-hexamethoxyflavone (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.

polymethoxyflavones; antifungal activity; Citrus aurantifolia

CHEMICAL SCIENCES

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

Susana Johann^I; Artur Smânia-Jr^I; Moacir G. Pizzolatti^II; Jan Schripsema^III; Raimundo Braz-Filho^{III, *}; Alexsandro Branco^IV

^ILaboratório de Antibióticos, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, 88040-970 Florianópolis, SC, Brasil

^IIDepartamento de Química, Universidade Federal de Santa Catarina, 88040-970 Florianópolis, SC, Brasil

^IIISetor de Química de Produtos Naturais LCQUI-CCT, Universidade Estadual do Norte Fluminense, 28013-602 Campos dos Goitacazes, RJ, Brasil

^IVLaboratório de Fitoquímica, Departamento de Saúde, Universidade Estadual de Feira de Santana, 44031-460 Feira de Santana, BA, Brasil

^{Correspondence to}

ABSTRACT

A mixture of the two new flavonols 8-hydroxy-3, 4', 5, 6, 7-pentamethoxyflavone (1) and 8-hydroxy-3, 3', 4', 5, 6, 7-hexamethoxyflavone (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.

Key words: polymethoxyflavones, antifungal activity, Citrus aurantifolia.

RESUMO

Os flavonóis 8-hidroxi-3, 4', 5, 6, 7-pentametoxiflavona (1) e 8-hidroxi-3, 3', 4', 5, 6, 7-hexametoxiflavona (2) foram isolados em mistura a partir de uma amostra comercial de Citrus aurantifolia. A determinação estrutural e a inequívoca atribuição dos sinais de deslocamento químico dos átomos de hidrogênio e carbono destes compostos naturais foram realizadas através da análise dos espectros de RMN 1D e 2D, incluindo COSY, NOESY, HMQC e HMBC. Em adição, a atividade antifúngica destes compostos contra fungos patogênicos também foi investigada.

Palavras-chave: polimetoxiflavonas, atividade antifúngica, Citrus aurantifolia.

INTRODUCTION

Flavonoids constitute one of the most important classes of naturally occurring phenols with interesting properties, to the plants as protective agents and human health (Ezeonu et al. 2001, Del Río et al. 1998, Bohm 1998, Chen et al. 1997, Harborne et al. 1975, Mayer 1998). Many analytical procedures have been developed for flavonoids analysis in mixture, although the most successful are based on chromatographic techniques such as high-performance liquid chromatography (HPLC) and Gas Chromatography (GC) (Oliveira et al. 2001, Robards et al. 1997, Robards K. and Antolovich 1997, Branco et al. 1998, 2001, Stremple 1998).

Nuclear magnetic resonance (NMR) spectroscopy 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 identification 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 aurantifolia. The structural determination of these compounds, as components of a mixture, was based on spectral data, including 2D NMR techniques, such as heteronuclear correlation ¹H-¹³C-COSY-ⁿJ_CH (n = 1, HMQC = ¹H-detected Heteronuclear Multiple Quantum Coherence; n = 2 and 3, HMBC = 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 anti fungal activity of these compounds against phytopathogenic and human pathogenic fungi was investigated.

MATERIALS AND METHODS

GENERAL EXPERIMENTAL PROCEDURES

Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for ¹H and 100 MHz for ¹³C 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 filtration and the hexane extracts were concentrated under reduced pressure. The addition of acetone to the extracts furnished the compounds 1 and 2 as precipitates.

MIXTURE OF 8-HYDROXY FLAVONOLS 1 AND 2

IR _max/cm^-1: 3408 (OH), 1651, 1602, 1559 (KBr). GCEIMS 70 eV m/z (rel. int.): 1 [Rt 17.1 min], 388 ([M]⁺, 100), 373 ([M-CH₃]⁺, 1d, 98), 345 ([M-CH₃-CO]⁺, 1e, 12), 327 (39), 135 (1f, 58); 2 [Rt 19.8 min], 418 ([M]⁺, 100), 403 ([M-CH₃]⁺, 2d, 89), 375 ([MCH₃-CO]⁺, 2e, 9), 357 (24), 165 (2f, 25). ¹H NMR (400 MHz, CDCl3): 1) ^dH 8.24 (d, J 9.2 Hz, H-2' and 6'), 7.05 (d, J 9.2, H-3'and 5'), 4.12 (s, CH₃O-7), 4.03 (s, CH₃O-3), 3.99 (s, CH₃O-6), 3.96 (s, CH₃O-5), 3.89 (s, CH₃O-4'); 2) 7.90 (dd, J 1.8 Hz, H-2'), 4.04 (s, CH₃O-3), 3.99 (s, CH₃O-3'), 3.96 (s, CH₃O-4'). ¹³C NMR (100 MHz, CDCl3): 1) ^dC 143.21 (C-2), 137.89 (C-3), 171.80 (C-4), 147.51 (C-5), 143.47 (C-6), 151.51 (C-7), 137.23 (C-8), 146.85 (C-9), 111.73 (C-10), 123.61 (C-1'), 160.88 (C-4'), 129.04 (CH-2'), 114.09 (CH-3'), 114.09 (CH-5'), 129.04 (CH-6'), 61.94 (CH₃O-3), 62.25 (CH₃O-5), 61.76 (CH₃O-6), 61.61 (CH₃O-7), 55.36 (CH₃O-4'); 2) 142.75 (C-2), 137.81 (C-3), 171.80 (C-4), 147.57 (C-5), 143.47 (C-6), 151.60 (C-7), 137.36 (C-8), 146.80 (C-9), 111.67 (C-10), 123.83 (C-1'), 148.88 (C-3'), 150.53 (C-4'), 110.27 (C-2'), 111.07 (C-5'), 120.97 (C-6'), 61.86 (CH₃O-3), 62.25 (CH₃O-5), 61.76 (CH₃O-6), 61.61 (CH₃O-7), 55.90 (CH₃O-3'), 55.81 (CH₃O-4'). ¹H (400 MHz) and ¹³C (100 MHz) NMR in benzene-d₆: Tables I and ^II.

MIXTURE OF 8-O-METHYL DERIVATIVES 1a AND 2a

A mixture of 1 and 2 (26 mg) was treated with CH₂N₂ as usual to yield 1a + 2a (26 mg). ¹H (400 MHz) and ¹³C (100 MHz) NMR in benzene-d₆: 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 4ºC and the inoculum was a suspension of each strain, in nutrient broth, containing approximately 5.10⁴ spores/mL.

BIOAUTOGRAPHY METHOD

50 µL of each solution of extract or substance prepared in hexane-acetone (1:1) (100µg/mL) were applied on TLC plates (60F₂₅₄; 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 30ºC 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 30ºC. The plates where fungal growth occurred, the INT changed from yellow to purple, while persistence of the yellow color indicated no 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

¹H and ¹³C NMR spectral data of the polymethoxylated flavonoids 1 and 2, recorded in CDCl₃ (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 down field (ca d_C 60 ppm) when compared with aromatic methoxyl groups attached to carbons bearing only one or no ortho substituent (ca d_C 55 ppm). Unequivocal ¹H and ¹³C chemical shift assignments of these natural compounds were also carried out by 2D H-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 showedin Figure 2.

Exhaustive analysis of 1D and 2D NMR spectra (CDCl₃, Experimental; benzene-d₆, Tables I and ^II) of the mixture of 1 and 2 and of their O-methyl ether derivatives1a and 2a obtained by methylation with CHN, recorded in benzene-d₆ (Tables I and ^II), was used to confirm the presence of a hydroxyl group at carbon C-8. This analysis was facilitated by considering electronic effects (inductive and mesomeric) and an isotropic effects as responsible for usual shift-parameters (Günther 1995), solvent effects induced by benzene-d₆ (Horie et al. 1998) and comparison with model compounds, e.g. 3 (Agrawal et al. 1989). In benzene-d₆ as solvent, all the methoxyl signals in the ¹H 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 ¹H-¹H-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; MeO-3' and H-2' and MeO-4' and H-5' of 2 and2a. Consequently, these results allowed unequivocally to assign the ¹H 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 ¹³C 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 (³J_CH) of C-3' with both H-5' and MeO-3' and C-4' with H-2', H-6' and MeO-4' of 2 (^{Table II}). Comparative analysis of the H NMR spectra (in C₆D₆) of the mixtures 1 + 2 and 1a + 2a showed additional singlet signals for MeO-8 at d_H 3.85 (1a) and 3.88 (2a), which revealed correlation with the ¹³C signals corresponding to C-8 at d_C 141.3 ( 1a) and 141.4 (2a) in the HMBC spectrum (^{Table II}). These ¹³C 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 (¹³C chemical shifts: C-7 C-5 C-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 significant C chemical shift difference (Dd_C: 9.8 ppm) observed between the signals corresponding to C-2 of 8-hydroxy- (1 and 2: d_C 143.1) and 8-methoxy- (1a and 2a: d_C 152.9) derivatives. These assignments are summarized in Tables I and ^II. Most probably, this difference may be attributed to an intra molecular hydrogen bond involving 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).

ANTIFUNGAL ACTIVITY OF 8-HYDROXY FLAVONOLS 1 AND 2

The anti fungal 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 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.

ACKNOWLEDGMENTS

We would like to thank Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro(FAPERJ, Rio de Janeiro, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for financial support.

Manuscript received on March 23, 2006; accepted for publication on December 28, 2006; contributed by RAIMUNDO BRAZ-FILHO*

• AGRAWAL PK, TKAKUR RS AND BANSAL MC. 1989. Carbon-13 NMR of Flavonoids, Elsevier, Amsterdam, p. 95182.
• AL-BURTAMANI SKS, FATOPE MO, MARWAH RG, ONIFADE AK AND AL-SAIDI SH. 2005. Chemical composition, antibacterial and anti fungal activities of the esencial oil of Haplophyllum tuberculatum from Oman. J Ethnopharmacol 96: 107112.
• APPENDINO G, GARIBOLDI P, PISETTA A, BOMBARDELLI E AND GABETTA B. 1992. Taxanes from Taxus baccata Phytochemistry 31: 42534257.
• BOHM BA. 1998. Chemistry and biochemistry of organic natural products in Introduction to Flavonoids, Harwood Academic Press: Netherlands, p. 339365.
• BRANCO A, BRAZ-FILHO R, KAISER CR AND PINTO AC. 1998. Two monoisoprenylated flavonoids from Vellozia graminifolia Phytochemistry 47: 471474.
• BRANCO A, PEREIRA AS, CARDOSO JN, AQUINO NETO FR, PINTO AC AND BRAZ-FILHO R. 2001. Further lipophilic flavonols in Vellozia graminifolia (Velloziaceae) by high temperature gas chromatography: quick detection of new compounds. Phytochem Anal 12: 266270.
• CALVERT DJ, CAMBIE RC AND DAVIS BR. 1979. 13CNMR spectra of polymethoxy- and methylenedioxyflavonols. Org Mag Reson 12: 583586.
• CHEN J, MONTANARI M AND WIDMER WW. 1997. Two new polymethoxylated flavones, a class of compounds with potencial anticancer activity isolated from cold pressed Dancy Tangerina peel oil solids. J Agric Food Chem 45: 364368.
• DEL RÍO JA, ARCAS MC, BENAVENTE-GARCÍA O AND ORTUÑO A. 1998. Citrus polymethoxylated flavones can confer resistence against Phytophthora citrophthora, Penicillium digitatum and Geotrichum species. J Agric Food Chem 46: 44234428.
• EZEONU FC, CHIDUME GI AND UDEDI SC. 2001. Inseticidal properties of volatile extracts of orange peels. Bioresour Technol 76: 273274.
• GASPAR H, GAVAGNIN M, CALADO G, CASTELLUCCIO F, MOLLO E AND CIMINO G. 2005. Pelseneeriol-1 and 2: new furanosesquiterpene alcohols from porostome nudibranch Doriopsilla pelseneeri Tetrahedron 61: 1103211037.
• GEROTHANASSIS IP, EXARCHOU V, LAGOURI V, TROGANIS A, TSIMIDOU M AND BOSKOU D. 1998. Methodology for identification of phenolic acids in complex phenolic mixtures by high-resolution two-dimensional nuclear magnetic resonance. Aplication to methanolic extracts of two oregano species. J Agric Food Chem 46: 41854192.
• GÜNTHER H. 1995. NMR Spectroscopy Basic Principles, Concepts, and Applicatons in Chemistry, 2^nd ed., J Wiley & Sons, New York, USA.
• GUSNTONE FD. 1991. ¹³C NMR studies of mono-, di-and triacylglycerols leading to qualitative and semi quantitative information about mixtures of these glycerol esters. Chem Phys Lipids 58: 219224.
• HARBORNE JB, MABRY TJ AND MABRY H. 1975. The Flavonoids, Academic press, New York, Part I.
• HORIE T, OHTSURU Y, SHIBATA K, YAMASHITA K, TSUKAYAMA M AND KAWAMURA Y. 1998. 13C NMR spectral assignment of the A-ring of poly oxygenated flavones. Phytochemistry 47: 865874.
• KOSIR IJ AND KIDRIC J. 2002. Use of modern NMR in wine analysis: determination of minor compounds. Anal Chim Acta 458: 7784.
• LOAIZA A, BORCHARDT D AND ZAERA F. 1997. A NMR method for the analysis of mixtures of alkanes with different deuterium substitutions. Spectrochim Acta Part A 53: 24812493.
• MAYER AM. 1998. Plant-fungal interactions: a plant physiologists viewpoint. Phytochemistry 28: 311317.
• OLEA RSG AND ROQUE NF. 1990. Análise de misturas de triterpenos por RMN de ¹³C. Quím Nova 13: 278281.
• OLIVEIRA BH, NAKASHIMA T, SOUZA FILHO JD AND FREHSE FL. 2001. HPLC analysis of flavonoids in Eupatorium littorale J Braz Chem Soc 12: 243246.
• PIZZOLATTI MG, VENSON AF, SMÂNIA JR A, SMÂNIA EFA AND BRAZ-FILHO R. 2002. Two epimeric flavolignans from Trichilia catigua (Meliaceae) with antimicrobial activity. Z Naturforsch 57c: 483488.
• ROBARDS K AND ANTOLOVICH M. 1997. Analytical chemistry of fruit bioflavonoids: a review. Analyst 112: 11R34R.
• ROBARDS K, LI X, ANTOLOVICH M AND BOYD S. 1997. Characterization of Citrus by chromatographic analysis of flavonoids. J Sci Food Agric 75: 87101.
• SMÂNIA JR A, DELLE MONACHE F, SMÂNIA EF, GIL ML, BENCHETRIT LC AND CRUZ FS. 1995. Antibacterial activity of a substance produced by the fungus Pycnosporus sanguíneus (Fr.) Murr. J Ethnopharmacol 45: 177181.
• STREMPLE P. 1998. GC/MS analysis of polymethoxylated flavones in Citrus oils. J High Resol Chromatogr 21: 587591.
• YOUNG MCM, POTOMATI A, CHU EP, HARAGUCHI M, YAMAMOTO M AND KAWANO T. 1997. ¹³CNMRanalysis of monodesmosidic saponins from Gomphrena macrocephala Phytochemistry 46: 12671270.
• ZOLLO F, FINAMORE E, GARGIULO D, RICCIO R AND MINALE L. 1986. Marine sterols. Coprostanols and 4a-methyl sterols from Mediterranean tunicates. Comp Biochem Physiol Part B 85: 559560.

*

Member Academia Brasileira de Ciências

Publication Dates

• Publication in this collection
  27 June 2007
• Date of issue
  June 2007

History

• Accepted
  28 Dec 2006
• Received
  23 Mar 2003