www.elsevier.com/locate/biochemsysecoBiochemical Systematics and Ecology 35 (2007) 809e811

Polyphenols from the hydroalcoholic extract of Arbutus unedoliving in a monospecific Mediterranean woodland

Antonio Fiorentino a,*, Simona Castaldi b, Brigida D’Abrosca a, Angela Natale a,Anna Carfora b, Anna Messere b, Pietro Monaco a

a Dipartimento di Scienze della Vita, Seconda Universita degli Studi di Napoli, via Vivaldi 43, 81100 Caserta, Italyb Dipartimento di Scienze Ambientali, Seconda Universita degli Studi di Napoli, via Vivaldi 43, 81100 Caserta, Italy

Received 22 February 2007; accepted 15 April 2007

Keywords: Arbutus unedo L.; Phenol constituents; Monospecific woodland; NMR data

1. Subject and source

Arbutus unedo L. known as strawberry tree is an evergreen shrub belonging to the Ericaceae family. Leaves ofA. unedo were collected in a Mediterranean woodland coppice, located in the area of Tolfa hills in Central Italy(42�110 N, 11�560 E), dominated by A. unedo (65e90% plant cover), with occurrence of Erica arborea L. (max13% plant cover), Fraxinus ornus L. (max 8% plant cover), Quercus pubescens Willd. (max 5% plant cover) andQuercus cerris L. (max 4% plant cover). Soil is acidic (pH 4.3) and is classified, according to the USDA system,as Andisol (USDA-NCRS, 1998). A voucher specimen (CE165) has been deposited at the Herbarium of the Diparti-mento di Scienze della Vita of the Second University of Naples.

2. Previous works

Previous phytochemical study of A. unedo isolated a number of compounds, such as flavan, steroid and terpenoids(Karikas and Giannitsaros, 1990). The leaves of strawberry tree were investigated to determine their flavonoidcomposition, and five flavonol glycosides were isolated and characterized: afzelin (kaempferol 3-rhamnoside), jugla-nin (kaempferol 3-arabinoside), avicularin (quercetin 3-arabinofuranoside), quercitrin (quercetin 3-rhamnoside), andhyperin (quercetin 3-galactoside) (Dauguet and Foucher, 1982). Furthermore, some iridoid glycosides have also beenisolated from the organic extract of the plant (Davini et al., 1981a,b).

* Corresponding author. Tel./fax: þ39(0)823274576.

E-mail address: antonio.fiorentino@unina2.it (A. Fiorentino).

0305-1978/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2007.04.005

810 A. Fiorentino et al. / Biochemical Systematics and Ecology 35 (2007) 809e811

3. Present study

The phytochemical study of the hydroalcoholic extract of the strawberry tree, collected in July 2005 in a woodlanddominated by A. unedo, led to the isolation of 12 hydrophilic compounds identified as ethyl gallate 1, arbutin 2, twoarbutin derivatives (3 and 4) and eight flavonoids (5e12). All of the structures were elucidated on the basis of theirspectral data (MS, 1H-NMR, 13C-NMR, 2D-NMR), as well as by comparison of their spectral data with those reportedin the literature for authentic samples.

Fresh mature leaves of A. unedo (100 g) were broken into little pieces and infused for 18 h, on a rotary shaker, inEtOH:H2O (1:1) solution (1 L). The crude hydroalcoholic extract (6.4 g) chromatographed on Amberlite XAD-4 elut-ing with H2O first, and then with MeOH to obtain a crude aqueous (3.35 g) and a crude organic (1.13 g) fraction.

The methanolic extract was chromatographed on a Sephadex LH-20 filled column with water and methanol solu-tions as eluents, to collect 490 fractions of 20 mL volumes. Fractions from 22 to 29, eluted with H2O, contained purearbutin (2) (62.7 mg); fractions from 183 to 193, eluted with H2O:MeOH (3:1), consisted of ethyl gallate (1)(44.0 mg). Fractions from 287 to 304, eluted with H2O:MeOH (1:1), were rechromatographed on preparative TLCeluting with CHCl3:MeOH (4:1), to obtain pure p-hydroxybenzoyl arbutin (3) (2.5 mg) and galloylarbutin (4)(11.4 mg). Fractions 317e340, eluted with H2O:MeOH (1:1), were purified by preparative TLC eluting withCHCl3:MeOH (4:1), to obtain (þ)�gallocatechin (5) (35.5 mg) and catechin (6) (54.6 mg). Fractions 406e430,eluted with H2O:MeOH (1:1), were purified by preparative TLC eluting with CHCl3:MeOH:H2O (13:9:3), to obtainkaempferol 3-O-a-L-ramnopyranoside (7) (11.0 mg), quercetin 3-O-a-L-ramnopyranoside (8) (33.0 mg), myricetin3-O-a-L-ramnopyranoside (9) (18.0 mg). Finally, fractions 445e458, eluted with H2O:MeOH (1:3), were purifiedby TLC eluting with CHCl3:MeOH:H2O (13:9:3), to obtain kaempferol 3-O-b-D-arabinofuranoside (10) (1.3 mg),quercetin 3-O-b-D-arabinofuranoside (11) (3.1 mg) and myricetin 3-O-b-D-arabinofuranoside (12) (2.8 mg).

HOOH

HOO

O

1

OO

OH

OHHO

HORO

2

3

4

R = H

R =

R =

O

OHOH

OH

O

OH

O

OH

HO

OH

OHOH

R

R = OHR = H

O

OH

HO

OR3

OH

O

R1

R2

R1 = H, R2 =H, R3 = -L-rhamnopyranosylR1 = OH, R2 = H, R3 = -L-rhamnopyranosylR1 = OH, R2 = OH, R3 = -L-rhamnopyranosylR1 = H, R2 =H, R3 = -D-arabinofuranosylR1 = OH, R2 = H, R3 = -D-arabinofuranosylR1 = OH, R2 = OH, R3 = -D-arabinofuranosyl

5

6

7

8

9

10

11

12

4. Ecological significance

A. unedo is a plant known for its phytotoxicity (Del Moral and Cates, 1970), which might play a key role in theformation of monospecific woodlands of this species, as often found in Mediterranean areas. Moreover, the leaf ex-tract of Arbutus utilized for the present study showed a toxic effect on soil nitrification activity in laboratory tests,supported by the absence of nitrate production in the field where Arbutus leaves were sampled, thus giving evidenceof an implication of metabolites produced by Arbutus on the biogeochemical cycles of nitrogen.

The most abundant metabolite from the hydroalcoholic extract of A. unedo was ethyl gallate (1). This compoundhad already been isolated from A. unedo (Sosa, 1950) and from other plants of the genus Rhodiola (Wang et al., 2006).

811A. Fiorentino et al. / Biochemical Systematics and Ecology 35 (2007) 809e811

Arbutin (2) was isolated from A. unedo (Heinrich and Delmir, 1950), while their derivatives 3 and 4 were found fromleaves of Myrsine seguinii (Zhong et al., 1998).

The gallocatechin 5 was the most abundant metabolite after compound 1, and it has not been previously reportedfrom A. unedo. This compound was previously characterized from the roots of Leucaena leucocephala and identified asthe main nitrification inhibitor isolated from this species (Erickson et al., 2000). The experimental [a]D value (þ10.4�)and the CD spectrum of the gallocatechin isolated from A. unedo were in accordance with the (þ)�gallocatechinenantiomer (Roux et al., 1961). Catechin (6) was identified as a phytotoxin by Bais et al. (2002) in a study onCentaurea maculosa. The authors reported that although the plant root exudes (�)�catechin, the (�)�catechinenantiomer was phytotoxic, while (þ)�catechin had antibacterial activity against root-infesting pathogens. Otherstudies on this species showed the inhibition of native species growth and germination in field soils at natural concen-trations of (�)�catechin (Bais et al., 2003). The catechin of A. unedo was isolated with 35% enantiomeric excess. Theflavonoids 7, 8, 10 and 11 had already been reported as constituents of A. unedo (Dauguet and Foucher, 1982), whereasthe glycosides of myricetin, 9 and 12, are isolated from this species for the first time.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bse.2007.04.005.

References

Bais, P.H., Walker, T.S., Stermitz, F.R., Hufbauer, R.A., Vivanco, J.M., 2002. Plant Physiol. 128, 1173.

Bais, P.H., Vepachedu, R., Gilroy, S., Callaway, R.M., Vivanco, J.M., 2003. Science 301, 1377.

Davini, E., Esposito, P., Iavarone, C., Trogolo, C., 1981a. Phytochemistry 20, 1583.

Davini, E., Esposito, P., Lavarone, C., Sen, A., Trogolo, C., Villa 1st, S., 1981b. Int. Conf. Chem. Biotechnol. Biol. Act. Nat. Prod., [Proc.] 3, 326.

Del Moral, R., Cates, R.G., 1970. Ecology 52 (6), 1030.

Dauguet, J.C., Foucher, J.P., 1982. Plant. Med. Phytother. 16, 185.

Erickson, A., Ramsewak, R.S., Smucker, A.J., Nair, M.G.J., 2000. J. Agric. Food Chem. 48, 6174.

Heinrich, T., Delmir, S., 1950. Pharmazie 5, 553.

Karikas, G.A., Giannitsaros, A., 1990. Plant. Med. Phytother. 24 (1), 27.

Roux, D.G., Maihs, E.A., Paulus, E., 1961. Biochem. J. 78, 834.

Sosa, A., 1950. Bull. Soc. Chim. Biol. 32, 344.

USDA-NCRS Soil Survey Staff, 1998. Agriculture Handbook n. 436. U.S.D.A., Natural Resources Conservation Service, New York, 870 pp.

Wang, H., Li, Y., Ding, C., Zhao, X., You, X., You, J., Suo, Y., 2006. J. Liq. Chromat. Related Technol. 29 (6), 857.

Zhong, X.-N., Otsuka, H., Ide, T., Hirata, E., Takushi, A., Takeda, Y., 1998. Phytochemistry 49, 2149.