Journal of Ethnopharmacology 95 (2004) 155–161

Free radical scavengers and antioxidants fromBaccharis grisebachii

Alejandro Tapiaa,c, Jaime Rodriguezb, Cristina Theodulozb, Susana Lopezd,Gabriela Egly Feresinc,e, Guillermo Schmeda-Hirschmanna,∗

a Laboratorio de Qu´ımica de Productos Naturales, Instituto de Qu´ımica de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chileb Departamento de Ciencias B´asicas Biom´edicas, Facultad de Ciencias de la Salud, Universidad de Talca, Casilla 747, Talca, Chilec Instituto de Ciencias B´asicas, Universidad Nacional de San Juan, Avda. Ignacio de La Roza 230 Oeste, 5400 San Juan, Argentina

d Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Espa˜nae Instituto de Biotecnolog´ıa, Universidad Nacional de San Juan, Av. Libertador General San Mart´ın 1109 (O), 5400 San Juan, Argentina

Received 23 October 2003; received in revised form 16 June 2004; accepted 16 June 2004Available online 27 September 2004

Abstract

andt s. Assay-g activityt tedhar ity wasf ndtd

ndt er©

K

1

(iciP

g(

d/orn

stro--al

e ofning

in-forms

0d

The exudate and seriated extracts from the aerial parts ofBaccharis grisebachii(Asteraceae) which is recommended as a digestiveo relieve gastric ulcers in Argentina, showed activity as free radical scavengers and inhibited lipoperoxidation in erythrocyteuided isolation led to sevenp-coumaric acid derivatives and six flavonoids as the main active constituents of the crude drug. The

owards the superoxide anion was mainly due to the flavonoid constituents. 5,7,4′-Trihydroxy-6-methoxyflavone and quercetin presenigh activity (64 and 79%) even at 12.5�g/ml. The xanthine oxidase inhibitory effect of the extracts can be related with thep-coumariccid derivatives drupanin, 4-acetyl-3,5-diprenylcinnamic acid andtrans-ferulic acidO-hexan-3-onyl-ether which showed IC50 values in theange 28–40�g/ml. Bothp-coumaric acid derivatives and flavonoids inhibited lipoperoxidation in erythrocytes. The highest activound for thep-coumaric acid derivatives 4-acetyl-3-prenyl-ethoxycinnamate, 3-prenyl-4-(4′-hydroxydihydrocinnamoyloxy)-cinnamate arans-ferulic acidO-hexan-3-onyl-ether (69–82%) and the flavonoids 5,7,4′-trihydroxy-6-methoxyflavone, quercetin, 5,7,4′-trihydroxy-6,3′-imethoxyflavone and 5,7,4′-trihydroxy-6,8-dimethoxyflavone (64–84%) at 100�g/ml.The most active free radical scavengers measured by the DPPH decoloration assay were thep-coumaric acid derivatives drupanin a

rans-ferulic acidO-hexan-3-onyl-ether (27–35% at 10�g/ml) and the flavonoid quercetin (97 and 23% at 10 and 1�g/ml, respectively). Thesults support the use ofBaccharis grisebachiiin Argentinian traditional medicine.

2004 Elsevier Ireland Ltd. All rights reserved.

eywords:Baccharis grisebachii; Asteraceae;p-Coumaric acid derivatives; Flavonoids; Labdane diterpenes; Antioxidants

. Introduction

The aerial parts of the shrubBaccharis grisebachiiHieronAsteraceae), known as ‘quilchamalı’ in the andean provincesn Argentina, are recommended in infusion to treat gastric ul-ers, as a digestive, local antiseptic and cicatrizant. The plants a bush, which grows in sandy soils in northern Argentina,eru, Uruguay and southern Brazil.

∗ Corresponding author. Tel.: +56 71 200288; fax: +56 71 200448.E-mail addresses:jrodrig@utalca.cl (J. Rodriguez),

feresin@unsj.edu.ar (G.E. Feresin), schmeda@utalca.clG. Schmeda-Hirschmann).

Decoctions from the aerial parts of someBaccharisspecies such asBaccharis articulata, B. crispa, B. trimeraandB. genistelloideshave been reported as digestives anantiseptic for external use (Gupta, 1995). Recently, it has beereported that a crude extract ofBaccharis illinita, used for itsprotective action to relieve stomach ulcers, showed gaprotective effects in rats (Baggio et al., 2003). Previous studies onBaccharis grisebachiipointed out to the antimicrobieffects of their constituents (Feresin et al., 2003).

Oxidative stress, the consequence of an imbalancprooxidants and antioxidants in the organism, is gairecognition as a key phenomenon in chronic illness likeflammatory and heart diseases, hypertension and some

378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2004.06.035

156 A. Tapia et al. / Journal of Ethnopharmacology 95 (2004) 155–161

of cancer. Several gastrointestinal tract diseases seem to beinduced by oxidative stress (Oh et al., 2001). On the otherhand, the traditional use of manyBaccharisspecies are ashepatoprotective and digestive crude drugs.

Following our ethnopharmacological studies on SouthAmerican crude drugs, we now report the isolation and iden-tification of free radical scavengers and antioxidants from theexudate and seriated extracts from the aerial parts ofBaccha-ris grisebachii.

2. Materials and methods

2.1. Plant material

The plant was collected from Bauchazeta district, San JuanProvince, Argentina, during the flowering time in Decem-ber 2002. The plant was identified by Dr. L. Ariza-Espinar,IMBIV-CONICET, Universidad Nacional de Cordoba, Ar-gentina and a voucher specimen has been deposited atthe herbarium of the Escuela de Quımica y Farmacia,Universidad de Chile (SQF 21011), Santiago de Chile,Chile.

TP y extra olic exB inhibitio xicity oft

Ec

totoxic�M

l)

EEHDMEEA

C0 (232

00 (2744 (67)8 (1776 (98)9 (1504 (42)6 (47)

00 (>344) – 18± 3 4± 2 0 0 0

C

H

2.2. Isolation

The resinous exudate of the aerial parts was obtained bydipping the fresh plant (1 kg) in cold CH2Cl2 (10 l) at roomtemperature (36◦C) for 40 seconds. The CH2Cl2 solution wasfiltered and taken to dryness under reduced pressure to givea semisolid yellow-orange residue (120 g).

The exudate (40 g) was chromatographed over silica gel ina column (60 cm length, 10 cm i.d.) eluting with a petroleumether (PE)—PE: ethyl acetate (EtOAc)—EtOAc gradientyielding 33 fractions of 700 ml each. After TLC compari-son, fractions with a similar TLC pattern were pooled into9 groups: I: fractions 1–11; II: fractions 12–14; III: frac-tions 15–19; IV: fractions 20–22; V: fraction 23; VI: fractions24–25; VII: fractions 26–27; VIII: fractions 28–29; IX: frac-tions 30–33. Compounds1–11, 13and14were isolated fromthe exudate by extensive chromatography. While compounds1–10 were previously isolated fromBaccharis grisebachiiby Feresin et al. (2003)or byGianello and Giordano (1987),the flavonoids11, 13and14were obtained from the pooledfraction VI as described below (Table 1).

The pooled fraction VI (1.54 g) was permeated onSephadex LH 20 (45 cm length, 8 cm i.d.) with PE:

able 1ercentage of activity relative to the corresponding control induced baccharis grisebachiion superoxide anion scavenging, XO inhibition,

he compounds1–12and14

xtracts andompound

Superoxide anion XO inhib. CyIC50

(�g/m

50�g/ml

25�g/ml

12.5�g/ml

50�g/ml

xtractsxudate DCM 47± 3 – – 0 –

0 – – 0 –CM 0 – – 0 –eOH 43± 3 – – 0 –thyl ether 66± 2 – – 53±3 –tOAc 74± 3 – – 41±5 –queous 57± 4 – – 0 –

ompounds1 37± 2 – – 81 (IC50 28) >1002 36± 2 – – 0 >103 52± 1 – – 37±4 224 34± 5 – – 57±3 585 48± 3 – – 88 (IC50 31) 286 21± 2 – – 58±2 647 38± 3 – – 65 (IC50 40) 148 31± 3 – – 4±3 159 39± 2 – – 0 >10

10 29± 4 – – 0 176 (63)11 84± 4 71± 5 64± 4 30±3 826 (24812 95± 5 86± 3 79± 5 37±4 322 (65)13 – – – –14 60± 3 – – 28±3 215 (71)

atechin 72± 5 – – – –

, hexane extract; DCM, dichloromethane extract; MeOH, methanol extract;

cts and compounds isolated from the resinous exudate and methantract ofn of lipoperoxidation in erythrocytes, DPPH decoloration and cytoto

ity Inhibition oflipoperoxidationin erythrocytes

DPPH decoloration

500�g/ml

100�g/ml

100�g/ml

50�g/ml

10�g/ml

1�g/ml

32± 3 – 47± 3 27± 3 8± 4 –38± 4 – 44± 4 23± 4 12± 2 –47± 4 – 45± 3 36± 2 5± 2 –40± 5 – 73± 4 54± 4 23± 4 –94± 4 – 81± 4 65± 5 27± 3 –72± 3 – 91± 5 77± 3 37± 2 –58± 4 – 61± 3 35± 4 8± 2 –

) 78± 4 43± 3 81± 4 65± 3 27± 2 5± 2) 85± 5 47± 2 0 0 0 0

69± 4 4± 2 0 0 0) 82± 3 17± 4 27± 3 16± 4 6± 3 0

– 20± 2 14± 3 9± 3 2± 1 0) – 82± 3 – – – –

– 76± 4 83± 5 71± 3 35± 2 8± 3– 0 0 0 0 0

– 28± 2 0 0 0 0) – 74± 2 56± 4 27± 4 0 0

– 84± 4 97± 5 97± 5 97± 4 23± 2– 71± 4 – – – –– 64± 3 25± 3 9± 4 0 0

– 73± 3 98± 3 97± 2 96± 2 36± 2

EtOAc, ethyl acetate extract; –, not done.

A. Tapia et al. / Journal of Ethnopharmacology 95 (2004) 155–161 157

CHCl3:MeOH (2:1:1). Some 78 fractions of 200 ml eachwere collected and pooled as follows: 1: 1; 2: 2–17, 3: 18;4: 19–27; 5: 28–30; 6: 31–41; 7: 42–57; 8: 58–63; 9: 64–68;10: 69–72; 11: 73–76; 12: 77–78. Fraction 7 afforded 30 mgof the flavonoid14, while fraction 8 contained 45 mg of thecompound13and fraction 10 yielded 18 mg of the compound11.

A second plant sample (1.125 kg) was extracted suc-cessively with hexane (H), dichloromethane (DCM) andmethanol (MeOH) at room temperature to afford an H, DCMand MeOH extract, respectively. The w/w yields in terms ofdry starting material for the H, DCM and MeOH extract were1.98% (22.29 g), 11.39% (128.20 g) and 9.50% (106.97 g),respectively. Each extract was tested for free radical scav-enging effect by the DPPH decoloration test as well as in thesuperoxide anion scavenging and lipid peroxidation assay.

The active MeOH extract (50 g) was resuspended in water(1 l) and partitioned with diethylether (Et2O, 3× 500 ml) andethyl acetate (EtOAc, 3× 500 ml) to afford an Et2O (15 g)and an EtOAc extract (2 g). The remaining aqueous phase waslyophilized (40 g). The isolation of the free radical scavengingcompounds was monitored by the DPPH autographic assay.

The Et2O-soluble extract (14 g) was dissolved in MeOHand applied to a Sephadex LH-20 column (column length33 cm, diameter 5.5 cm) equilibrated with MeOH. Some 30f oleda tica frac-t PLC(2 entf ithf )( g ofq den-t ract.T di gm

23

443

43

TL5

5

5,7,4′-Trihydroxy-6-methoxyflavone11: 15 mg/kg.1H-NMR (MeOH-d4): � 7.89 d (8.8) (H-2′, H-6′); 6.95 d(8.8) (H-3′, H-5′); 6.62 s (H-3); 6.27 s (H-8); OMe:3.90 s.

3,5,7,3′,4′-Pentahydroxyflavone (Quercetin)12: 80 mg/kg.5,7,4′-Trihydroxy-6,3′-dimethoxyflavone13: 45 mg/kg.

1H-NMR (MeOH-d4): � 7.56 brd (8.5) (H-6′); 7.55 br s(H-2′), 6.95 d (8.5) (H-5′); 6.65 s (H-3); 6.26 s (H-8);OMe: 3.96 s, 3.93 s.

5,7,4′-Trihydroxy-6,8-dimethoxyflavone14: 30 mg/kg. Forthe1H-NMR data seeGianello and Giordano (1987). Forthe1H and13C-NMR data of compounds11–14seeAgrawal (1989). For the UV spectra of the flavonoids seeMabry et al. (1970).

2.3. Assays

2.3.1. DPPH decoloration assayThe free radical scavenging effect of the samples

was assessed by the decoloration of a methanolic so-lution of 1,1,-diphenyl-2-picrylhydrazyl radical (DPPH,Aldrich) as previously reported (Schmeda-Hirschmannet al., 2003). Crude extracts were assayed at 100, 50 and10�g/ml, while at 100, 50, 10 and 1�g/ml for the pure com-pounds. Values are presented as mean± standard deviationo

undsw t thea eta asu ol andt Thed ginge refer-e ecol-o -H

2O

i llu-l eac-t BT),l n at5 ed ofg ureds ,1 al-ud

2n-

t sedf O

ractions of 75 ml each were obtained. Fractions were poccording to their TLC profiles (cellulose; glacial acecid:water 20% as the mobile phase). The combined

ions 11–12 (187 mg) were submitted to preparative Hcolumn: Lichrospher RP-18, 250 mm× 25 mm, 7�m; �:80 nm; flow rate: 5 ml/min) using a 40 min linear gradi

rom 50 to 80% acetonitrile in water adjusted to pH 2.5 wormic acid to yield 100 mg of compound9 (NevandensinRt: 33 min). The combined fractions 22–23 afford 30 muercetin12. The same compounds were isolated and i

ified as the main compounds from the whole MeOH exthe NMR spectral data of the compounds1–10 is presente

n Feresin et al. (2003). The w/w yields in terms of dry startinaterial is presented below.

.2.1. Isolated compounds-Prenyl-4-hydroxycinnamic acid (3-Prenyl-p-coumaricacid = drupanin)1: 2.40 g/kg.

-Acetyl-3-prenyl-p-coumaric acid2: 370 mg/kg.-Acetyl-3-prenyl-ethoxycinnamate3: 70 mg/kg.,5-Diprenyl-4-hydroxycinnamic acid(3,5-Diprenyl-p-coumaric acid)4: 5.00 g/kg.

-Acetyl-3,5-diprenylcinnamic acid5: 1.30 g/kg.-Prenyl-4-(4′-hydroxydihydrocinnamoyloxy)-cinnamate6:30 mg/kg.rans-Ferulic acidO-hexan-3-onyl-ether7: 50 mg/kg.abda-7,13E-dien-2�,15-diol8: 410 mg/kg.,7-Dihydroxy-6,8,4′-trimethoxyflavone (Nevadensin)9:12.60 g/kg.

-Hydroxy-6,7,8,4′-tetramethoxyflavone(5-Desmethyltangeretin)10: 100 mg/kg.

f three determinations.The quenching of free radicals by extracts and compo

as evaluated spectrophotometrically at 517 nm againsbsorbance of the DPPH radical (Schmeda-Hirschmannl., 2003). A freshly prepared DPPH solution (20 mg/l) wsed for the assays. Samples were dissolved in methan

he methanolic solution of DPPH served as a control.egree of decoloration indicates the free radical scavenfficiency of the substances. Catechin was used as ance free radical scavenger. The percentage of DPPH dration was calculated as previously described (Schmedairschmann et al., 2003).

.3.2. Superoxide anionThe enzyme xanthine oxidase is able to generate

•−2

n vivo by oxidation of reduced products from intracear ATP metabolism. The superoxide generated in this rion sequence reduces the nitro blue tetrazolium dye (Neading to a chromophore with a maximum of absorptio60 nm. Superoxide anion scavengers reduce the speeneration of the chromophore. The activity was measpectrophotometrically as reported previously (Paya et al.992; Masaki et al., 1995). Extracts and products were evated at 50�g/ml. Values are presented as mean± standardeviation of three determinations.

.3.3. Xanthine oxidase activityXanthine oxidase (XO) derived from cow’s milk, xa

hine and the standard inhibitor allopurinol were purcharom Sigma Chemical Co. (St. Louis, MO, USA). The X

158 A. Tapia et al. / Journal of Ethnopharmacology 95 (2004) 155–161

activities with xanthine as substrate were measured spec-trophotometrically as previously reported using a Thermo-spectronic Genesys 10 UV-scanning equipment (Schmeda-Hirschmann et al., 2003). Extracts and products were evalu-ated at 50�g/ml. Results are presented as mean± standarddeviation of three determinations.

2.3.4. Lipoperoxidation in erythrocytesStudies on erythrocyte lipid peroxidation were carried out

as described byDe Azevedo et al. (2000)with slight modifica-tions. Human red blood cells obtained from healthy donorswere washed three times in cold phosphate buffered saline(PBS) by centrifugation at 3500 rpm. After the last wash-ing cells were suspended in PBS and its density adjusted to1 mM haemoglobin in each reaction tube. The final cell sus-pensions were incubated with different concentrations of thetest compounds dissolved in DMSO and PBS during 10 minat 37◦C. The final concentration of DMSO in the samplesand controls was 1%. After incubation cells were exposed totert-butylhydroperoxide (1 mM) during 15 min at 37◦C un-der vigorous shaking. After treatment lipid peroxidation wasdetermined indirectly by the TBARs formation as describedpreviouslyDe Azevedo et al. (2000). Results are expressedas percentage of inhibition compared to controls. Each deter-mination was repeated four times.

2cell

v l lined 1).M ume Ml ntedw /mlp di da -fl iumc from8 edi ofD ellsw outt wast paratee take(H retw es.

3

-b and

six flavonoids were isolated and assessed as free radicalscavengers/antioxidants in selected assays. Most of the com-pounds were previously reported in an article on the antimi-crobial effect of the crude drug (Feresin et al., 2003). In ad-dition, four further flavonoids were isolated from the activeextract by permeation on Sephadex LH-20 (Fig. 1).

The crude exudate showed a moderate effect towards thesuperoxide anion, with nearly 50% inhibition at 50�g/ml. At100�g/ml, the activity of the same extract was about 50%against the free radical DPPH. The unpolar extracts of theplant (hexane and DCM-soluble) were devoid of activity inthe superoxide and XO assays with a moderate effect as aninhibitor of lipoperoxidation in erythrocytes. Most of the an-tioxidant/free radical scavenging effect was found in the ethylether and EtOAc extracts obtained by partition of the defattedmethanol extract with Et2O and EtOAc, respectively.

The activity towards the superoxide anion was mainlydue to the flavonoid constituents of the crude drug. Com-pounds11and12presented high activity (64 and 79%) evenat 12.5�g/ml. The xanthine oxidase inhibitory effect of theextracts can be related with thep-coumaric acid derivatives1,5and7, which showed IC50 values in the range 28–40�g/ml.

Bothp-coumaric acid derivatives and flavonoids inhibitedlipoperoxidation in erythrocytes. The highest activity wasfound for thep-coumaric acid derivatives3,6and7 (69–82%)ac c-tt c-i

y theD -ta

iiwfla bout0 com-p nI

l-4-h asf sh rdsT eap -iAv moralaa leica

teepl sa d by

.3.5. Cytotoxicity assayThe cytotoxic effect of the compounds, expressed as

iability, was assessed on a permanent fibroblast celerived from human lung (MRC-5) (ATCC Nr CCL-17RC-5 fibroblasts were grown as monolayers in minimssential Eagle medium (MEM), with Earle’s salts, 2 m-glutamine and 1.5 g/l sodium bicarbonate, supplemeith 10% heat-inactivated fetal calf serum (FCS), 100 IUenicillin and 100�g/ml streptomycin in a humidifie

ncubator with 5% CO2 in air at 37◦C. Cells were platet a density of 2.5× 103 per well in 96-well plates. Conuent cultures of MRC-5 cells were treated with medontaining the compounds at concentrations ranging0 up to 1000�M. The substance was firstly dissolv

n DMSO and then in MEM. The final concentrationMSO in the test medium and controls was 1%. Cere exposed for 24 hours to test medium with or with

he compound (control). Each drug concentrationested in quadruplicate, and repeated three times in sexperiments. At the end of incubation, the neutral red upNRU) assay was carried out as described byRodrıguez andaun (1999). To calculate the IC50 values the results we

ransformed to percentage of controls and the IC50 valuesere graphically obtained from the dose-response curv

. Results and discussion

From the exudate and polar extracts ofBaccharis griseachii, sevenp-coumaric acid derivatives, a diterpene

nd the flavonoids11–14 (64–84%) at 100�g/ml. For thep-oumaric acid derivatives, compound6displayed the best aivity (82%) with low cytotoxicity (IC50 150�g/ml). Amonghe flavonoids, compound11 showed the better cytotoxity:activity relationship with an IC50 value of 248�g/ml.

The most active free radical scavengers measured bPPH decoloration assay were thep-coumaric acid deriva

ives1 and7 (27–35% at 10�g/ml) and the flavonoid12 (97nd 23% at 10 and 1�g/ml, respectively).

The main compounds isolated fromBaccharis grisebachere thep-coumaric acid derivatives1, 4 and 5 and theavonoid9. It can be estimated that the content ofp-coumariccid derivatives and flavonoids in the crude drug are a.9–1 and 1.3%, respectively. However, from these threeounds only the derivative1showed low cytotoxicity with a

C50 value up to 232�g/ml.Several biological activities were reported for 3-preny

ydroxycinnamic acid1, (also known as drupanin) as wellor 3,5-diprenyl-4-hydroxycinnamic acid4. The compoundave been shown to display tripanocidal activity towarypanosoma cruziand induced a relaxant effect in guinig isolated trachea (Marcucci et al., 2001). Drupanin inhib

ted cell growth in human tumor cells (Akao et al., 2003).rtepillin C (3,5-diprenyl-4-hydroxycinnamic acid4) acti-ates the immune system, and possesses direct antituctivity (Kimoto et al., 1998, Kimoto et al., 2001) as well asntioxidant effect measured by the peroxidation of linocid in a micelle solution (Aga et al., 1994).

In a study on the antioxidant compounds from corn siquor,Niwa et al. (2001)isolatedp-coumaric acid derivatives the active products. The activity was mainly affecte

A. Tapia et al. / Journal of Ethnopharmacology 95 (2004) 155–161 159

Fig. 1. Structure of the compounds isolated from the resinous exudate and methanolic extract ofBaccharis grisebachii.

their functional groups at the 3-position and less by the conju-gated side chain. Thep-coumaric acid derivatives were moreactive than tocopherols and ascorbic acid on peroxynitrite-mediated lipoprotein nitration suggesting thatp-coumaricacid derivatives might play a beneficial role against oxida-tive damage.

The aromaticortho-dihydroxy groups of the cinnamicacid derivatives combined with a lipophilic residue seemsto be a prerequisite for an optimal binding within the ac-tive site of the human neutrophil elastase (Siedle et al.,2003). In a study on the structure-hepatoprotective effect of3,4-dihydroxycinnamic acid (caffeic acid) derivatives,Perez-Alvarez et al. (2001)reported that the double bond in the C-3moiety of the acids does not account for the liver pharmaco-logical properties of caffeic acid derivatives. The 4-hydroxysubstituent, however, proved to be very important for hepato-protective activity because the 4-hydroxy analogue improved

almost every hepatic injury marker altered by carbon tetra-chloride in a better way than caffeic acid.

According toLaranjinha et al. (1996), naturally occur-ring phenolic acids can prevent ferrylmyoglobin-dependentLDL oxidation and the effect was dependent on the substi-tution pattern on the phenol ring. The authors found thato-dihydroxy derivatives of cinnamic and benzoic acids (caffeic,chlorogenic, and protocatechuic acids), in a molar ratio of 1to metmyoglobin, efficiently blocked LDL oxidation initiatedby ferrylmyoglobin. Furthermore, the replacement of one OHgroup from catecholic structure with an H (p-coumaric acid)or methoxy group (ferulic acid) decreased the antioxidantactivity.

Hydroxycinnamic acid derivatives have been shown to dis-play antioxidant activity measured by the decoloration of theDPPH radical and correlated with the methyl linoleate assay(Kikuzaki et al., 2002). Ferulic acid was the most effective

160 A. Tapia et al. / Journal of Ethnopharmacology 95 (2004) 155–161

phenolic acid and activity increases after esterification. Theresults indicated that lipophilicity of the antioxidants mightbe an important feature in their activity. Hydroxycinnamicacids such as ferulic acid, sinapic acid, andp-coumaric acidlinked to the plant cell wall polymers can be released by in-testinal esterases (Andreasen et al., 2001).

In an in vitro investigation of the inhibitory effect ofphenylpropanoids on copper-induced protein oxidative mod-ification of mice brain homogenate,Toda (2002)found thatphenolic carboxylic acids with 3,4-dihydroxy or 4-hydroxy-3-methoxy substituents and benzo-alpha-pyrons with 6,7-dihydroxy or 7-hydroxy-6-methoxy substituents in phenyl-propanoid metabolites inhibited metal-induced protein ox-idative modification of the brain.

The main flavonoid of Baccharis grisebachiiwasnevadensin. This compound has been reported as anti-inflammatory (Reddy et al., 1990) and hypotensive (Liu etal., 1991). Quercetin is a well-known free radical scavengerand antioxidant (Middleton et al., 2000).

Several biological activities have been reported so far forflavonoids. Their role in oxidative stress is well known and re-lated to their antioxidant and antiproliferative effects. More-over, they also have an inhibitory action on inflammatoryprocesses (Middleton et al., 2000) and several diseases ofthe gastrointestinal tract seem to be induced by oxidatives

f theo romp n thatf magec l (e im-p ronici rteryd 0;M

4

cant sti-n aH hlow,1 ec utedc tivesi ectso o thea om-p e re-pa -P

-f the

crushed leaves and flowers are recommended as an antisep-tic and wound healing agent. The plant exudate as well assome of its main compounds have been shown to display an-timicrobial effect towards dermatophytic fungi and bacteria(Feresin et al., 2003).

Flavonoids and phenolics of higher plants are known tobe excellent antioxidants in vitro and numerous studies sug-gest that dietary intake of plant polyphenol antioxidants mayhave positive effects in oxidative-stress related pathologies(Urquiaga and Leighton, 2000). Several diseases of the gas-trointestinal tract seem to be induced by oxidative stress (Ohet al., 2001).

The free radical scavenging and antioxidant activity foundin Baccharis grisebachiicould be associated with their mainphenolic compounds, comprising both flavonoids andp-coumaric acid derivatives, giving support to its use as a medic-inal plant to treat gastrointestinal troubles in the traditionalmedicine of San Juan Province in Argentina.

It is well known that antioxidant activity is com-monly related with gastroprotective (El-Abhar et al., 2003;Kahraman et al., 2003), cytoprotective (Potapovich andKostyuk, 2003; Mahakunakorn et al., 2003) and hepatopro-tective effects (Ferreira et al., 2003; Hsiao et al., 2003; Sohnet al., 2003). Extracts and compounds fromB. grisebachii,including the aqueous extract exhibited strong antioxidant ac-t itros ns ofB rnA

A

ro-g up-p ant.WN icalw dd( uthA

R

A 94.lian46.

A anic

A K.,wthhu-26,

A .T.,rox-

tress.Supplementation of the antioxidant defense system o

rganism with natural antioxidant compounds derived flants may have beneficial effects since it has been show

ree radical scavengers prevent esophageal mucosal daaused by ischemia, anti-inflammatory drugs, or ethanoOht al., 2001). Furthermore, flavonoids can be useful torove other pathophysiological processes, including ch

nflammatory and allergic diseases, as well as coronary aisease and breast cancer (Urquiaga and Leighton, 200iddleton et al., 2000).

. Conclusions

Baccharisspecies are widely used in South Ameriradicional medicine mainly for the treatment of gastrointeal disorders and hepatic alterations (Gupta, 1995; Schmedirschmann and Bordas, 1990; Soicke and Leng-Pesc987; Giberti, 1983). SomeBaccharis, known under thommon name of ‘carqueja’ are among the most reprude drugs for treating hepatic troubles and as digesn the southern part of the continent. The beneficial efff some species can be attributed, at least in part, tntioxidant/free radical scavenging effect of phenolic counds such as the dimethoxybenzyl-caffeate derivativorted byDe Oliveira et al. (2003)fromBaccharis articulatand the flavonoids fromBaccharis trimera(Soicke and Lengeschlow, 1987).The aerial parts ofBaccharis grisebachiiare used in in

usion to treat gastric ulcers and as a digestive, while

s

ivity and free radical scavenging effect in different in vystems. Our results support the traditional use of infusioaccharis grisebachiiby the Andean population in westergentina.

cknowledgements

We are grateful to the Universidad de Talca (Chile), Prama ‘Desarrollo de Productos Bioactivos’ for financial sort. A. Tapia thanks the Universidad de Talca for a gre also thank the Secretarıa de Investigacion, Universidadacional de San Juan, Argentina and the skilful technork of Sergio Reyes A. and Lily C. Munoz, Universidae Talca. This work is part of a CYTED-CAB CyT N◦ 1700K1-2002) project on bioactive compounds from the Somerican flora.

eferences

ga, H., Shibuya, T., Sugimoto, T., Kurimoto, M., Nakajima, S., 19Isolation and identification of antimicrobial compounds in Brazipropolis. Bioscience Biotechnology and Biochemistry 58, 945–9

grawal, P.K., 1989. Carbon-13 NMR of Flavonoids. Studies in OrgChemistry 39, 564 (Elsevier).

kao, Y., Maruyama, H., Matsumoto, K., Ohguchi, K., Nishizawa,Sakamoto, T., Araki, Y., Mishima, S., Nozawa, Y., 2003. Cell groinhibitory effect of cinnamic acid derivatives from propolis onman tumor cell lines. Biological and Pharmaceutical Bulletin1057–1059.

ndreasen, M.F., Kroon, P.A., Williamson, G., Garcia-Conesa, M2001. Esterase activity able to hydrolyze dietary antioxidant hyd

A. Tapia et al. / Journal of Ethnopharmacology 95 (2004) 155–161 161

ycinnamates is distributed along the intestine of mammals. Journal ofAgricultural and Food Chemistry 49, 5679–5684.

Baggio, C.H., Freitas, C.S., Rieck, L., Marques, M.C., 2003. Gastropro-tective effects of a crude extract ofBaccharis illinita DC in rats.Pharmacological Research 47, 93–98.

De Azevedo, M.B.M., Alderete, J., Rodrıguez, J.A., Souza, A.O., Rettori,D., Torsoni, M.A., Faljoni-Alario, A., Haun, M., Duran, N., 2000.Biological activities of new antitumoral indole derivatives in an in-clusion complex with cyclodextrin. Journal of Inclusion Phenomenaand Macrocyclic Chemistry 37, 93–101.

De Oliveira, S.Q., Dal-Pizzol, F., Gosmann, G., Guillaume, D., Moreira,J.C., Schenkel, E.P., 2003. Antioxidant activity ofBaccharis articulataextracts: isolation of a new compound with antioxidant activity. FreeRadical Research 37, 555–559.

El-Abhar, H.S., Abdallah, D.M., Saleh, S., 2003. Gastroprotective activityof Nigella sativaoil and its constituent, thymoquinone, against gastricmucosal injury induced by ischaemia/reperfusion in rats. Journal ofEthnopharmacology 84, 251–258.

Feresin, G.E., Tapia, A., Gimenez, A., Gutierrez Ravelo, A., Zacchino, S.,Sortino, M., Schmeda-Hirschmann, G., 2003. Antimicrobial activityof the Argentinian medicinal plantBaccharis grisebachii(Asteraceae).Journal of Ethnopharmacology 89, 73–80.

Ferreira, M.A., Nunes, O., del, R., Leal, L.K., Pessoa, O.D., Lemos, T.L.,Viana, G.S., 2003. Antioxidant effects in the quinone fraction fromAuxemma oncocalyxTaub. Biological and Pharmaceutical Bulletin 26,595–599.

Gianello, J.C., Giordano, O.S., 1987. Constituents fromBaccharis grise-bachii. Anales de la Asociacion Quımica Argentina 75, 1–3.

Giberti, G.C., 1983. Herbal folk medicine in northwestern Argentina,Compositae. Journal of Ethnopharmacology 7, 321–341.

G . Pro-y

H in,ctived

K asap,cetin

K H.,nds.

K J.,morDe-

K ef,sisient

L ofxi-acol-

L .T.,

M den-

Mahakunakorn, P., Tohda, M., Murakami, Y., Matsumoto, K., Watan-abe, H., Vajaragupta, O., 2003. Cytoprotective and cytotoxic effectsof curcumin: dual action on H2O2-induced oxidative cell damagein NG108-15 cells. Biological and Pharmaceutical Bulletin 26, 725–728.

Marcucci, M.C., Ferreres, F., Garcia-Viguera, C., Bankova, V.S., De Cas-tro, S.L., Dantas, A.P., Valente, P.H., Paulino, N., 2001. Phenoliccompounds from Brazilian propolis with pharmacological activities.Journal of Ethnopharmacology 74, 105–112.

Masaki, H., Sakaki, S., Atsumi, T., Sakurai, H., 1995. Active oxygenscavenging activity of plant extracts. Biological and PharmaceuticalBulletin 18, 162–166.

Middleton, E., Kandaswami, K., Theoharides, T., 2000. The effects ofplant flavonoids on mammalian cells: implications for inflammation,heart disease, and cancer. Pharmacolological Reviews 52, 673–751.

Niwa, T., Doi, U., Kato, Y., Osawa, T., 2001. Antioxidative propertiesof phenolic antioxidants isolated from corn steep liquor. Journal ofAgricultural and Food Chemistry 49, 177–182.

Oh, T.Y., Lee, J.S., Ahn, B.O., Cho, H., Kim, W.B., Surch, Y.J., Cho,S.W., Hahm, K.B., 2001. Oxidative damages are critical in pathogen-esis of reflux esophagitis implication of antioxidants in its treatment.Free Radical Biology and Medicine 30, 905–915.

Paya, M., Halliwell, B., Hoult, R.S., 1992. Interactions of a series ofcoumarins with reactive oxygen species. Biochemical Pharmacology44, 205–214.

Perez-Alvarez, V., Bobadilla, R.A., Muriel, P., 2001. Structure-hepatoprotective activity relationship of 3,4-dihydroxycinnamic acid(caffeic acid) derivatives. Journal of Applied Toxicology 21, 527–531.

Potapovich, A.I., Kostyuk, V.A., 2003. Comparative study of antioxi-dant properties and cytoprotective activity of flavonoids. Biochemistry

R an,tain

R ninato-

S l com-

S dillo,d an-h

S tureitorstions.

S ep-

S fromdica

T s onho-

183–

U xida-

upta, M.P. (ed) 1995. 270 Plantas medicinales iberoamericanasgrama Iberoamericano de Ciencia y Tecnologıa para el DesarrolloConvenio Andres Bello, 73–84.

siao, G., Shen, M.Y., Lin, K.H., Lan, M.H., Wu, L.Y., Chou, D.S., LC.H., Su, C.H., Sheu, J.R., 2003. Antioxidative and hepatoproteeffects of Antrodia camphorataextract. Journal of Agriculture anFood Chemistry 51, 3302–3308.

ahraman, A., Erkasap, N., Koken, T., Serteser, M., Aktepe, F., ErkS., 2003. The antioxidative and antihistaminic properties of querin ethanol-induced gastric lesions. Toxicology 183, 133–142.

ikuzaki, H., Hisamoto, M., Hirose, K., Akiyama, K., Taniguchi,2002. Antioxidant properties of ferulic acid and its related compouJournal of Agricultural and Food Chemistry 50, 2161–2168.

imoto, T., Arai, S., Kohguchi, M., Aga, M., Nomura, Y., Micallef, M.Kurimoto, M., Mito, K., 1998. Apoptosis and suppression of tugrowth by artepillin C extracted from Brazilian propolis. Cancertection and Prevention 22, 506–515.

imoto, T., Aga, M., Hino, K., Koya-Miyata, S., Yamamoto, Y., MicallM.J., Hanaya, T., Arai, S., Ikeda, M., Kurimoto, M., 2001. Apoptoof human leukemia cells induced by Artepillin C, an active ingredof Brazilian propolis. Anticancer Research 21, 221–228.

aranjinha, J., Vierira, O., Almeida, L., Madeira, V., 1996. Inhibitionmetmyoglobin/H2O2-dependent low density lipoprotein lipid perodation by naturally occurring phenolic acids. Biochemical Pharmogy 51, 395–402.

iu, M.C., Chen, Z.S., Chung, L.C., Yang, M.S., Ho, S.T., Chen, M1991. Studies on hypotensive constituents ofLimnophyla rugosa. Chi-nese Pharmaceutical Journal (Taipei) 43, 35–40.

abry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Itification of Flavonoids. Springer Verlag, New York.

(Moscow) 68, 514–519.eddy, G.B.S., Udupa, A.L., Shirwaikar, A., Aithal, K.S., Srinivas

K.K., 1990. Comparison of the anti-inflammatory activity of cermethoxyflavonoids. Fitoterapia 61, 460–461.

odrıguez, J.A., Haun, M., 1999. Cytotoxicity of trans-dehydrocrotofrom Croton cajucara(Euphorbiaceae) on V79 cells and rat hepcytes. Planta Medica 65, 522–526.

chmeda Hirschmann, G., Bordas, E., 1990. Paraguayan medicinapositae. Journal of Ethnopharmacology 28, 163–171.

chmeda-Hirschmann, G., Rodriguez, J.A., Theoduloz, C., AstuS.L., Feresin, G.E., Tapia, A., 2003. Free radical scavengers antioxidants fromPeumus boldusMol. (“Boldo”). Free Radical Researc37, 447–452.

iedle, B., Murillo, R., Hucke, O., Labahn, A., Merfort, I., 2003. Strucactivity relationship studies of cinnamic acid derivatives as inhibof human neutrophil elastase revealed by ligand docking calculaPharmazie 58, 337–339.

ohn, D.H., Kim, Y.C., Oh, S.H., Park, E.J., Li, X., Lee, B.H., 2003. Hatoprotective and free radical scavenging effects ofNelumbo nucifera.Phytomedicine 10, 165–169.

oicke, H., Leng-Peschlow, E., 1987. Characterisation of flavonoidsBaccharis trimeraand their antihepatotoxic properties. Planta Me53, 37–39.

oda, S., 2002. Inhibitory effects of phenylpropanoid metabolitecopper-induced protein oxidative modification of mice brainmogenate, in vitro. Biological Trace Element Research 85,188.

rquiaga, I., Leighton, F., 2000. Plant polyphenol antioxidant and otive stress. Biological Research 33, 55–64.