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The Journal of Argentine Chemical Society

On-line version ISSN 1852-1428

J. Argent. Chem. Soc. vol.95 no.1-2 Ciudad Autónoma de Buenos Aires Jan./Dec. 2007

 

REGULAR PAPERS

Flavonoids from Gutierrezia Repens (Asteraceae)

Alarcón, S. R.1, Ábalos, M.1, Colloca, C. B.2, Pacciaroni, A.2, Sosa, V. E.2

1 Facultad de Ciencias Naturales, Universidad Nacional de Salta (UNSa), 4400 Salta, Argentina.
2 Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Instituto Multidisciplinario de Biología Vegetal- IMBIV (CONICET-UNC), 5000 Córdoba, Argentina.
Fax: +54-3874255455     E-mail: ralarcon@unsa.edu.ar

Received September 18th, 2007.
In final form November 23th, 2007

Abstract
7,3'-dimethylquercetin 1, 7,3,3'-trimethylquercetin 2, 7,3,4'-trimethylquercetin 3 and quercetin 4 were isolated from aerial parts of Gutierrezia repens (Asteraceae). The structures of 1, 2 and 3 were determined mainly on the basis of 2D NMR data. Their 1H NMR spectra in CDCl3 and Me2CO-d6 are compared and discussed. The 13C NMR spectra of these compounds are given here for the first time.

Keywords: Gutierrezia repens; Asteraceae; Photochemistry; Flavonoids

Resumen
7,3'-dimetilquercetina 1, 7,3,3'-trimetilquercetina 2, 7,3,4'-trimetilquercetina 3 y quercetina 4 fueron aislados de las partes aéreas de Gutierrezia repens (Asteraceae). Las estructuras de 1, 2 y 3 fueron determinadas principalmente por espectroscopía 2D RMN. Sus espectros de RMN 1H  en CDCl3 y Me2CO-d6 son comparados y discutidos. En este trabajo informamos por primera vez, los espectros de RMN 13C de los flavonoides metilados.

Palabras clave: Gutierrezia repens; Asteraceae; Fitoquímica; Flavonoides.

Introduction
The Asteraceae is the second largest family in the Magnoliophyta Division with around 1100 genera and over 20000 recognized species. Cabrera, reported the occurrence of 197 genera and about 1400 species in Argentina [1].
As part of our phytochemical study on Asteraceae species growing in Argentina, we investigated the aerial parts of Gutierrezia repens Grisebach. There is no information about chemical and biological studies carried out on G. repens. Plant specimens were collected from their natural habitat in the northwest of Argentina, in Salta Province.
The genus Gutierrezia (tribe Eupatorieae) includes approximately 25 species which occur exclusively in the arid areas of America [1]. Earlier work on this genus revealed that diterpenes [2-8] and flavonoids [9-15] are the main classes of substances representative of the Gutierrezia genus.
In this paper, we report for the first time on a phytochemical investigation of G. repens
.

Experimental
General
The NMR spectra were recorded on a Bruker  AC 200 (1H at 200 MHz and 13C at 50 MHz) or a Bruker Avance 400 (1H at 400 MHz and 13C at 100 MHz) spectrometer with TMS as internal reference. CC were performed on silica-gel 230-400 mesh, RPCC on C-18 silica gel, TLC was carried out on precoated Silica gel 60 F254 plates (Fluka). Detection was achieved by UV light and spraying with vanillin reagent followed by heating.

Plant Material
G. repens was collected during the flowering period in Valle Encantado, Province of Salta, Argentina, on February 2004. The identification was carried out by Ing. Julio Tolaba. A voucher specimen (nº 3464) is deposited at the Museo de la Facultad de Ciencias Naturales, Universidad Nacional de Salta.

Extraction and isolation
Air-dried and powdered aerial parts of G. repens (260 g) were macerated with EtOH at room temperature for 7 days to give 13.10 g of crude extract which was suspended in EtOH:H2O (1:1) and extracted successively with hexane (3x150 mL), CH2Cl2 (3x150 mL) and EtOAc (3x100 mL). Evaporation of the CH2Cl2 extract in vacuo furnished 5.37 g of residue which was divided into 3 fractions by chromatography on reversed-phase silica gel flash column, eluting with MeOH-H2O (8:2), MeOH and Me2CO. The fraction 1 (2.0 g) was chromatographed on a 230-400 mesh silica gel column using hexane containing increasing amounts of EtOAc (0-100 %), seven fractions being collected (F1 to F7). Fraction F5 (269 mg, hexane-EtOAc 3:7), was first purified by column chromatography on silica gel eluting with a gradient of hexane-Et2O followed by preparative TLC (hexane-Me2CO 7:3) affording 2.5 mg of 7,3`-dimethylquercetin 1 (Rf= 0.30) [16], 3.0 mg of 7,3,3`-trimethylquercetin 2 (Rf= 0.36) [16, 17] and 3.5 mg of 7,3,4`-trimethylquercetin 3 (Rf= 0.33) [18]. Column chromatography of Fraction F6 (230 mg, hexane-EtOAc 1:9) on silica gel and benzene-EtOAc gradient system followed by preparative TLC (hexane-Me2CO, 1:1) afforded 7.0 mg of quercetin 4 (Rf= 0.48) [19].
7,3`-dimethylquercetin1. Amorphous solid, UV (MeOH) λmax nm: 260, 270, 370; +NaOMe: 270, 300, 330, 430 (dec); +NaOAc: 260, 335, 375; +AlCl3: 275, 430; +AlCl3/HCl: 275, 430.
7,3,3`-trimethylquercetin2. Amorphous solid, UV (MeOH) λmax nm: 270, 348, 355; +NaOMe: 265, 405; +NaOAc: 270, 355; +AlCl3: 270, 302, 406; +AlCl3/HCl: 348, 406.
7,3,4`-trimethylquercetin3. Amorphous solid, UV (MeOH) λmax nm: 270, 300, 348; +NaOMe: 265, 375; +NaOAc: 265, 355; +AlCl3: 270, 300, 362, 400; +AlCl3/HCl: 270, 300, 362, 400.
quercetin 4. Yellow solid, UV (MeOH) λmax nm: 255, 300, 370, 385; +NaOMe: 330 sh; +NaOAc: 274, 394; +AlCl3: 266, 300, 358, 430; +AlCl3/HCl: 266, 300, 358, 430.

Discussion
The CH2Cl2 soluble extract of the aerial parts of G. repens Griseb. yielded four known flavonoids 7,3'-dimethylquercetin 1 [16], 7,3,3'-trimethylquercetin 2 [16, 17], 7,3,4'-trimethylquercetin 3 [18] and quercetin 4 [19].


Bathocromic shifts upon addition of AlCl3 and AlCl3/HCl (see experimental) together with the presence of a chelated hydroxyl group in the 1H NMR spectrum (Table 1 and Table 2), indicated 5-hydroxy substitution for all four compounds

 

Table 1. Spectroscopic data of flavonoid 1 and 4* (Me2CO-d6, TMS as internal standard).

* At 400 MHz for 1H NMR and 100 MHz for 13C NMR.
† At 200 MHz in CDCl3.
δ (H)  values are followed by multiplicity and below, in parentheses, coupling constants in Hz.

Table 2. Spectroscopic data of flavonoid 2 and 3* (Me2CO-d6, TMS as internal standard)

* At 400 MHz for 1H NMR and 100 MHz for 13C NMR. 
† At 200 MHz in CDCl3.
δ (H) values are followed by multiplicity and below, in parentheses, coupling constants in Hz.
‡ Overlapped signals.

In the 1H NMR spectra of all the compounds three aromatic protons formed the characteristic pattern for a 3',4'-disubstituted B ring. Additionally, the UV spectra recorded with NaOMe indicated 4'-hydroxy substitution for 1, 2 and 4.
Flavonoids 1, 2 and 3 also showed 1H NMR signals indicative of O-methyl substituents. Their UV spectra were unchanged upon addition of NaOAc, indicating that one of the methoxyl groups was at the C-7 position. The structures of these compounds were deduced on the basis of their HSQC, HMBC and NOESY spectra.
The 1H NMR data of known compounds 1, 2 and 3 were previously measured using low resolution instrument. As far as we know, the 13C NMR spectra of flavonoids 1, 2 and 3 have not been described in the literature so far (Tables 1 and 2).
The 1H NMR data of 3, indicate that 4`-O-methylation induces a downfield shift of ca. 0.15 ppm in the signal of H-5', in the spectrum measured in Me2CO-d6 (Table 2). In the spectrum measured in CDCl3 this effect is clearly smaller (less than 0.1 ppm) (Table 2). On the other hand, in all the compounds with 7-O-methylation (1, 2 and 3), we always observed a downfield shift of 0.15-0.30 ppm in the signal of H-8, in spectra measured in Me2CO-d6 (Tables 1 and 2).
The A-Ring carbon signals are similar in 1, 2 and 3. B-ring signals show that 4'-O-methylation in 3 induces an upfield shift of ca. 4.0 ppm in the chemical resonance of C-5' (Table 2).

Conclusions
7,3'-dimethylquercetin 1 and 7,3,3'-trimehtylquercetin 2 are now reported for the first time in the genus, while 7,3,4'-trimethylquercetin 3 was isolated before from G. alamanii [13] and quercetin 4 from G. grandis [12], G. alamanii [13] , G. wrightii [14]and G. microcephala [15].
The isolation of compounds 1-4 from G. repens is completely in accordance with the typical chemical profile of the Gutierrezia genus.

Acknowledgments
Thanks are due to the Consejo de Investigación de la Universidad Nacional de Salta for financial supports.

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