Antimicrobial evaluation of Huilliche plant medicine used to treat wounds

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Journal of Ethnopharmacology 138 (2011) 219– 227

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

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ntimicrobial evaluation of Huilliche plant medicine used to treat wounds

er Mølgaarda, Jes Gitz Hollera, Betül Asarb, Iwona Libernaa, Lise Bakkestrøm Rosenbæka,hristina Ploug Jebjerga, Lene Jørgensena, Jeanette Lauritzena, Alfonso Guzmana, Anne Adsersena,enrik Toft Simonsenc,∗

Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, DenmarkDepartment of System Biology, Technical University of Denmark, Søltofts Plads, DK-2800 Kgs. Lyngby, DenmarkDepartment of Plant Biology and Biotechnology, VKR Research Centre Pro-active plants, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg,enmark

r t i c l e i n f o

rticle history:eceived 31 May 2011eceived in revised form 10 August 2011ccepted 6 September 2011vailable online 12 September 2011

eywords:uillichentimicrobialhile

a b s t r a c t

Ethnopharmacological relevance: The traditional use of 40 plant species used for treatment of wounds andassociated infections by the Huilliche people of Chile was evaluated against bacterial and fungal humanpathogens, especially including wound pathogens.Materials and methods: The extracts were tested against the fungi Penicillium expansum, Candida albicansand the bacteria Bacillus subtilis, Pseudomonas aeruginosa (four different strains), Staphylococcus aureus,Escherichia coli (four different strains), Streptococcus pneumoniae (four different strains with one beingresistance to streptomycin).Results: Thirteen of the plant species have interesting antimicrobial activities, with that of Acaena argentea,Aristotelia chilensis, Blechnum chilense, Francoa appendiculta, Gevuina avellana and Laureliopsis philippiana

ound infection being the most noteworthy.Conclusions: The findings in the manuscript support the traditional use by the Huilliche people, and allowenhanced economical benefit and use by the locals. The results obtained on Acaena argentea, Aristoteliachilensis, Blechnum chilense, Francoa appendiculta, Gevuina avellana and Laureliopsis philippiana are ofspecific scientific interest, and further studies is needed in order to establish the active constituents ofthe species.

. Introduction

The present project was initiated in order to evaluate the effectf plant medicine used by Huilliche people in Chile to treat woundsnd associated infections. The plants have been tested for theirntimicrobial activity in order to establish the use as wound healingnd anti-infection agents and to evaluate the need for further inves-igation. This evaluation is an opportunity for the Huilliche peopleo make use of the plants for production of herbal medicine andventually as raw material to modern medicinal products, as newiologically active metabolites can be discovered through in-depthxaminations.

Chile has an extraordinary variety of plants and animals, thanks

o the latitudinal extent of the country and its great altitudinalange. Chile is a domicile for the Chilean Winter Rainfall-Valdivianorest, which is one of the world’s biodiversity hotspots. It encom-

∗ Corresponding author. Tel.: +45 353 33328; fax: +45 353 33302.E-mail addresses: [email protected] (P. Mølgaard), [email protected] (J.G. Holler),

[email protected] (H.T. Simonsen).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.09.006

© 2011 Elsevier Ireland Ltd. All rights reserved.

passes app. 40% of Chiles land area and harbours both endemicflora and fauna. About 50% of the 4000 vascular plant taxa found inthis area are endemic; some of the more notable are the Araucariatree (Araucaria araucana), known as the monkey puzzle tree, andLapageria rosea, Chile’s national flower (Brooks et al., 2011).

The selection of plants examined in this report is based on thetraditional use in Region de Los Lagos in southern Chile, which isan area part of the Winter Rainfall-Valdivian Forest. The deforesta-tion threatens this area and the evaluation might help to conservethe area with its natural richness. Furthermore the evaluationalso contributes to the preservation of the Huilliche culture anda sustainable production and/or collection of plants may create aneconomic foundation as an alternative to the deforestation. Previ-ous publications have described the traditional use of plants by theMapuche people, being a tribe using much of the same medicineas the Huilliche. These publications give a broad insight to thetraditional use by the Mapuche and can be related to that of the

Huilliche peoples. The most frequently observed traditional usesby the Mapuche have therapeutic purposes such as wound healing,and treatment of digestive ailments/gastro-intestinal disorders,as analgesics, as anti-inflammatory, and against fevers (Estomba

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Table 1Overview of the plants examined. With voucher number, Latin names, local name and use. Other used are also given, and reference added. Collected parts, L = leaf, S = stem,R = root, AP = arial part, F = flower, Fr = fruit, and B = bark.

Family Voucher number Latin plant name Collected part Common name Local Use

Araliaceae PM01-44 Pseudopanax laetevirens(Gay.) Baill.

L, S Sauco Leaves, fruit and bark are used for woundhealing, as anti-inflammatory, laxative and asdiuretic.a

Asteraceae JGH003 Acrisione denticulata (Hook.& Arn.) B. Nord.

L Pal-Pal Traditionally used (Aal et al., 1988)

Asteraceae PM07-03 Baccharis elaeoides Rémy L Vautro, Chilco Leaves are used for wound healing, asanti-pyretic and analgesic.a

Asteraceae PM05-33 Baccharis magellanica Lam.(Pers.)

L Pichi Leaves are used as antibiotics.a

Asteraceae JGH005 Baccharis sphaerocephalaHook & Arn.

L Collected due to phylogenetic relationship.

Atherospermataceae PM01-31, JGH016 Laurelia sempervirens (R. &P.) Tul.

L, S Laurel Leaves, flowers and bark are used asexpectorant, and against colds.a

Atherospermataceae PM01-21, JGH017 Laureliopsis philippiana(Looser) Schodde

L, S Tepa Leaves, flowers and bark are used asexpectorant, anti-inflammatory, and againstcolds.a

Berberidaceae JGH006 Berberis buxifolia Lam. AP, R Leaves and bark are used towards infections(Anesini and Perez, 1993).

Blechnaceae PM01-18 Blechnum chilense (Kaulf.)Mett.

L, S, R Costilla de vaca The whole plant is used towards gonorrhoeaand wound and eye infections.a

Buddlejaceae PM01-23 Buddleja globosa Hope L, S Matico Leaves and flowers are used towards intestinediseases and for wound healing.a

Campanulaceae JGH018 Lobelia tupa L. L Tupa The whole plant is known to be toxic.a

Coriariaceae PM03-15, JGH007 Coriaria ruscifolia L. L, S, Fr Deu The whole plant is used for wound healing.a

Cunoniaceae JGH024 Weinmannia trichospermaCav.

L Teníu, Madén Tea and poultice of bark is used to controldiarrhea and, externally, wounds (Houghtonand Manby, 1985).

Durvillaeaceae PM01-54 Durvillaea antarctica(Cham.) Hariot

S Cochayuyo The whole plant is used as anti-inflammation,against ulcers and intestine diseases, laxativeand blood pressure regulation.a

Elaeocarpaceae PM07-02, JGH001 Aristotelia chilensis (Mol.)Stuntz.

L Quëlón Leaves are used towards diarrhea, woundhealing, and anti-pyretic.a

Elaeocarpaceae PM07-04 Crinodendron hookerianumGay

L Chaquihue Leaves are used towards diabetes and forwound healing.a

Gesneriaceae JGH019 Mitraria coccinea Cav. L Leaves are used to treat wounds.a

Gleicheniaceae JGH015 Gleichenia quadripartida(Poiret) T. Moore

L Leaves are used to treat wounds and rashes.a

Grossulariaceae PM01-07, JGH023 Ribes magellanicum Poir. L Parrilla Leaves are used to treat liver and intestineailments (Eyssartier et al., 2009).

Gunneraceae PM01-09 Gunnera chilensis Lam. L, S Nalca Stem and root are used against uterus pains,and as haemostatic and anti-inflammatory.a

Lauraceae JGH020 Persea lingue Nees L Linge, Lingue Bark is employed in traditional medicine forthe treatment of dysentery, leucorrhoea,endometritis and some kinds of tumours(Sepulveda-Boza et al., 1990).

Loganiaceae JGH009 Desfontainia spinosa Ruizand Pav.

L Leaves are narcotic and analgesic (Houghtonand Manby, 1985).

Loranthaceae PM01-30 Tristerix tetrandrus (R. & P.)Martius

L, S, R, F Quintral Leaves are used in wound healing, ashaemostatic, against ulcers, towards epilepsyand as aliment against worms.a

Malvaceae PM01-10 Corynabutilon vitifolium(Cav.) Kearney

L, S Huella Bark, stem and leaves are used for liverdiseases and uterus contractions.a

Myrtaceae PM01-17 Amomyrtus meli (Phil.) D.Legrand & Kausel

L, S Meli Leaves are used to decrease blood pressure andcholesterol levels, to treat liver diseases and asdiureticum.a

Myrtaceae PM01-16 Ugni molinae Turcz. L, S Murta The fruit is stimulating and refreshing.a

Onagraceae PM01-19, JGH013 Fuchsia magellanica Lam. L, S Leaves are used as anti-pyretic, blood pressureregulator, diuretic and wound healing.a

Poaceae PM01-22 Chusquea quila Kunth L, S Quila Root and stem are used towards diabetes andwound and eye infections.a

Podocarpaceae JGH021 Podocarpus nubigenus Lindl. L The species is used for wood and as anornamental, but have shown antimicrobialactivity (Abdillahi et al., 2010).

Polypodiaceae PM01-34 Polypodium feuillei Bertero L, S, R Calahuala The root is used as expectorant, against ulcers,bronchial diseases and syphilis.a

Proteaceae JGH011 Embothrium coccineumForst.

L, B The plant is used to wash open wounds (teaand to dress wounds (leaves).a

Proteaceae PM01-36, JGH014 Gevuina avellana Mol. L, S, Fr Avellana The essential oil, bark, fruit and leaves are usedagainst ulcers, diarrhea, intestinal diseases,and gonorrhoea.a

Proteaceae PM01-12 Lomatia hirsuta (Lam.)Diels. ex Macbr.

L, S Radal, Raral Leaves and bark are used as laxative,expectorant and as anti-inflammatory.a

Rosaceae PM07-01, JGH002 Acaena argentea Ruiz & Pav. L, S, R Cadillo Leaves used against diarrhea, for woundhealing, antivenereal, and ulcers.a

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Table 1 (Continued)

Family Voucher number Latin plant name Collected part Common name Local Use

Saxifragaceae JGH012 Francoa appendiculata Cav. L, R Tea of the root is used for wound healing.a

Solanaceae PM05-35 Cestrum parqui L‘Hér L, S Palqui Leaves are used to relief fevers, and towardsskin diseases.a

Solanaceae PM01-08 Latua pubiflora Baill L, S Leaves, bark and fruit are hallucinogenic, andare used as anti-inflammatory agent.a

Verbenaceae JGH022 Rhaphithamnus spinosus(Juss.) Moldenke

L Bark is used as emetic. Fruit as treatment forgastro-intestinal infections (Houghton andManby, 1985).

Vitaceae PM01-49 Cissus striata Ruiz & Pav. L, S Voqui Colorado Leaves and stem are used for wound healing,and wound infections.a

Winteraceae PM07-05, JGH010 Drimys winteri J.R. & G.Forster

L, B Leaves are used as anti-pyretic, in woundhealing, as diurectic anti-inflammatory agent,

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a New information on local names and local use has been collected by Dr. Alfonso

t al., 2006; Houghton and Manby, 1985; Molares and Ladio, 2009).n order to fully understand the therapeutic efficacy of differentlants, the cultural background, in which they are used, needs to benown, since this will influence the compliance of the users (Torri,010).

Since ancient times, people have used plants and preparationshereof to accelerate the wound healing process (Reuter et al.,009). Often their use is merely based on tradition, without anycientific evidence of efficacy, and only little knowledge aboututative active compounds or their mode of action. Plants, andspecially water and alcohol extracts of plants, are often usedor treatment of chronic wounds, e.g. wounds which have a highacterial infection that often delays or hinder the healing (Dowt al., 1999). The use of topical antibiotics and antiseptics is oftenestricted due to problems with multi resistant bacteria; there isherefore a continuous need for new drugs in this area, and also aeed for the Huilliche people to have an evaluation of the plantssed against different bacterial infections.

Skin and wound infections are common and may be causedy bacteria, fungi or viruses. Bacterial wound infections are oftenaused by staphylococci and streptococcal, both causing severenfections. Candidal infections occur in moist areas, such as theulva, mouth, skinfolds and diaper area. Wounds with such con-itions are prone to candidal infection and are often also seen inatients with low immune responses (O’Dell, 1998). In chronicounds infections are caused by many different bacteria and fungi,here Staphylococcus aureus and Pseudomonas aeruginosa are the

est described and most predominant (Edwards and Harding,004).

Huilliche plants have not previously been examined for antimi-robial activity, but from the closely related Mapuche peoples manylants have been evaluated, and some of the reports also include

solation of active constituents. This is the case with Lomatia hir-uta (Simonsen et al., 2006), two Baccharis species (Simonsen et al.,009), four Pseudognaphalium speices (Mendoza et al., 1997), andine Haplopappus species (Urzúa et al., 2006, 1995). Buddleja glo-osa was recently reported to have a slight effect on proliferation ofbroblasts, but it was not significant, and at higher concentrations iteemed to be cytotoxic (Houghton et al., 2005; Mensah et al., 2001).n a recent report the results of several Mapuche plants testedn a blood coagulation assay established that even though somelants are used against wound healing, the effect on blood coag-lation might be of interest with respect to anti-platelet activityFalkenberg et al., 2012).

The aim of this study is to evaluate the local knowledge and usef Huilliche plants against a range of fungi and bacterial strains.

ikewise, the uses of two different traditional methods of tea prepa-ation have been evaluated for efficacy and toxicity. Additionally,he plants have been tested for their cholinesterase inhibition inrder to enhance their local use and knowledge.

and against ulcers.a

án and is reported here

2. Material and methods

2.1. Plant material

All the collected plants are used as traditional medicine by theHuilliche Indians, and the collection took place during the summers(January–February) in the period from 2001 to 2009 by MSc andPhD students from the Faculty of Pharmaceutical Sciences underguidance by the botanist, Professor Alfonso Gúzman.

All the plants have been collected in Region de Los Lagos, whichis located in region X, part of the Chilean Winter Rainfall-ValdivianForest. The different plant parts available were collected, e.g. leaves,stems, flowers and roots (see Table 1). After collection the plantswere dried in a ventilated room and transported to Denmark. Aftersuccessful return, the material was subjected further to a dryingprocess in heating cabinet for 72 h at 40 ◦C.

Voucher specimens are deposited in the herbarium of the Botan-ical Garden and Museum, University of Copenhagen (C), see Table 1for voucher specimen number.

2.2. Extraction and sample preparation

Dry and grinded plant material was subjected to extrac-tion using different solvents by the following procedure: 5 mlDCM:MeOH 1:1 was added to 0.5 g dry plant material and ultrasoni-cated for 30 min. After sonication the solvent and plant mixture wasfiltered through an air suction filter. The solvent was saved for laterevaporation, and once again 5 ml DCM:MeOH 1:1 was added to theplant material followed by 30 min of ultrasonication; the combinedextracts were dried by evaporation and stored at −20 ◦C. The sameprocedure was used also for extraction with 96% EtOH and MeOH.Water-extraction was performed by adding 50 ml of water to 0.5 gof plant powder and boiled for 1 h under reflux. Extraction mixturewas filtrated and hereafter lyophilised.

2.3. Antimicrobial screening

A direct bioautographic method was used to determine theminimum inhibiting amount (MIA) for activity against Penicilliumexpansum (IMI 285521) (Simonsen et al., 2002). A thin-layer chro-matographic agar overlay technique (Simonsen et al., 2004) wasused to determine the MIA against Candida albicans (IMI 349010),Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 9027,PAO1, m1 II AEA, S4 231, AEA S6-white), Staphylococcus aureus(ATCC 6538), and Escherichia coli (ATCC 11229, EDL 933 (ATCC700927), MG1655 (ATCC 700926), CFT073, Nissle 1917), Strepto-

coccus pneumoniae (1472-04 (serotype 14), 1327-05 (serotype 14),1205-05 (serotype 14) and 1322-05 (serotype 6B)). The extractswere dissolved in an appropriate solvent. A maximum of 1 mg ofeach extract was tested and the activity observed visually as total

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Table 2Antimicrobial activity. +: MIA ≈ 1 mg; ++: MIA < 1 mg; +++: MIA < 0.5 mg; ++++: MIA < 0.25 mg; +++++: MIA < 0.125 mg; ++++++: MIA < 0.0625 mg; –: no activity; space: no experimental data. MIA: minimum inhibiting amount.Plant parts L = leaf, B = bark, S = stem, R = root, F = flower, Fr = fruit, and AP = arial part; extracts D:M = DCM:MeOH. In bold are plants of interest.

Bacterial species Escherichia coli Pseudomonasaeruginosa

Staphylococcusaureus

Bacillus subtilis Streptococcus pneumoniae Candida albicans Penicillium expansum

Strain ATCC 11229 EDL 933 ATCC 9027 PAO1 ATCC 6538 ATCC 6633 1472-04 1327-05 1205-05 1322-05 IMI 349010 IMI 285521Plant species Part Extract

Acaena argentea L D:M 1:1 +++ + – – +++ –EtOH + –MeOH – – ++ + ++++ ++ ++ ++++ – ++++H2O – – – – ++ –

S MeOH + + +++ + – +R MeOH + + ++ + – +

Acrisione denticulata L D:M 1:1 – –EtOH – +H2O – –

Aristotelia chilensis L D:M 1:1 – + – +++ ++ – –EtOH + –H2O – – – ++ ++ – –

Amomyrtus meli L MeOH – – – – – –S MeOH – – – – – –

Baccharis elaeoides L D:M 1:1 – – +++ ++ – –H2O

Baccharis magellanica L D:M 1:1 – –EtOH – –MeOH ++++ ++++ ++++ ++++H2O – –

Baccharis spaerocephala L D:M 1:1 – –EtOH – –H2O – –

Berberis buxifolia AP D:M 1:1 + –EtOH – –H2O – –

R D:M 1:1 +EtOH – +H2O – –

Blechnum chilense L MeOH – – + + – –S MeOH + – + – – –R MeOH ++ ++ ++ + – +

Buddleja globosa L MeOH – – – – – –S MeOH – – – – – –

Cestrum parqui L MeOH + – – – – –S MeOH – – – – – –

Chusquea quila L MeOH – – – – – –S MeOH – – + – – –

Cissus striata L MeOH – – + – – –S MeOH ++ – – – – –

Coriaria ruscifolia L D:M 1:1 + –

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EtOH + +MeOH +++++ +++ ++++++ ++++ ++++ ++++ ++++ ++ – –H2O – –

S MeOH ++ – ++ ++ – –Fr MeOH ++ +++++ ++ ++ – –

Corynabutilon vitifolium L MeOH – – – – – –S MeOH – – – – – –

Crinodendronhookerianum

L D:M 1:1 +++ + – – ++ –

EtOH + +MeOH +++++ ++++++ ++++++ ++++++H2O – – – – ++ –

Desfontainia spinose L D:M 1:1 – –EtOH – –H2O – –

Drimys winterii L D:M 1:1 +++ – – – ++ +++EtOH – –MeOH ++++++ ++++++ ++++++ ++++++H2O ++ – – – + –

B D:M 1:1 – –EtOH – +MeOH ++++++ ++++++ +++++ ++++++H2O – –

Durvillea antarctica S MeOH – – – – – –

Embothrium coccineum L D:M 1:1 – –EtOH – –H2O – –

B D:M 1:1 – –EtOH – –H2O – –

Francoaappendiculata

L D:M 1:1 + –

EtOH – –H2O – –

R D:M 1:1 + –EtOH + +H2O + –

Fuchsia magellanica L D:M 1:1 +++ + +++ – +++ ++EtOH – –MeOH + + +++++ – +++++ +++++ +++++ +++++ – –H2O ++ + – – ++ –

S MeOH + – ++ – – –

Gevuina avellana L D:M 1:1 – –EtOH + +MeOH – ++ +++ +++ ++ ++ – – – –H2O + +

S MeOH – + ++ ++++ – – – – –Fr D:M 1:1 – –

EtOH + –

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Table 2 (Continued)

Bacterial species Escherichia coli Pseudomonasaeruginosa

Staphylococcusaureus

Bacillus subtilis Streptococcus pneumoniae Candidaalbicans

Penicilliumexpansum

Strain ATCC 11229 EDL 933 ATCC 9027 PAO1 ATCC 6538 ATCC 6633 1472-04 1327-05 1205-05 1322-05 IMI 349010 IMI 285521Plant species Part Extract

MeOH ++ ++ ++ + ++ +H2O – –

Gleicheniaquadripartida

L D:M 1:1 – –

EtOH – –H2O + –

Gunnera chilensis L MeOH + – – – –S MeOH + + + + – –

Latua pubiflora L MeOH – – – – –S MeOH – + – – –

Laurelia sempervirens L D:M 1:1 – –EtOH + +MeOH – – – – –H2O – –

S D:M 1:1 – –EtOH – –MeOH – – + – –H2O – –

Laureliopsisphilippiana

L D:M 1:1 – –

EtOH – –MeOH ++ +++ ++ ++ ++ ++ ++ ++ – ++++H2O – –

S MeOH – – – – –

Lobelia tupa L D:M 1:1 – –EtOH – –

Lomatia hirsuta L MeOH – – – ++ +++++S MeOH – – – – –

Mitraria coccinea L D:M 1:1 – –EtOH – –H2O – –

Persea lingue L D:M 1:1 – –EtOH – –H2O – –

Podocarpus nubigenus L D:M 1:1 – –EtOH – –H2O – –

Polypodium feuillei L MeOH – – – – –S + R MeOH – – – – –

Pseudopanax laetevirens L MeOH – – – – –S MeOH – – – – –

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rmacology 138 (2011) 219– 227 225

inhibition. A dilution range from 1 mg to 0.0625 mg was tested toyield approximate MIA values.

A microplate method as previously described (Simonsen et al.,2006) was used with slight modifications to determine minimuminhibitory concentration values (MIC) of active extracts againstEscherichia coli and Pseudomonas aeruginosa. The extracts were dis-solved in DMSO or water and diluted with Mueller–Hinton broth(MHB) to a final DMSO concentration of 2%. A dilution range of theextracts from 2048 �g/ml to 1 �g/ml was tested to yield approxi-mate MIC values.

The plates were incubated for 20 h at 35 ◦C and the growthof bacteria assessed using a Micur-Viewer, in which the individ-ual plate-cells were read. Amphotericin B was used as a positivecontrol for Candida albicans (MIA = 1.2 �g), nystatin for Penicil-lium expansum (MIA = 0.5 �g), and streptomycin for the bacteria(Pseudomonas aeruginosa (m1 II) MIC = 64 �g/ml; Escherichia coli(EDL 933) MIC = 8 �g/ml), and erythromycin for the streptococcus(MIC = 12 �g/ml except for 1322-05 where it is >250 �g/ml).

3. Results

3.1. Screening of antimicrobial activity

Forty plants were tested for their antimicrobial activity. Theactivity of extracts of these is given in Table 2 (MIA) and Table 3(MIC). As seen in Tables 2 and 3, some of the species but not allshowed antimicrobial activity. Of special interest (marked withbold in Tables 2 and 3) are the effects seen for: Acaena argentea,Aristotelia chilensis, Baccharis elaeoides, Blechnum chilense, Cori-aria ruscifolia, Crinodendron hookerianum, Drimys winterii, Francoaappendiculata, Fuchsia magellanica, Gevuina avellana, Laurelia sem-pervirens, Laureliopsis philippiana and Lomatia hirusta. Among theplants tested against the Streptococcus a correlation between theresistance to streptomycin (strain 1322-05 have a significant resis-tance to streptomycin) and the plant extracts anti-microbial effectcould not be observed, indicating different mechanism of action. Allthese 13 findings support the traditional use of these plants againstinfections.

4. Discussion

Thirteen plant species were found to be active in the antimi-crobial tests performed. The present results support the previouscomprehensive reports regarding the antimicrobial and/or cyto-toxic effects of Baccharis elaeoides (Simonsen et al., 2009),Crinodendron hookerianum (Miró, 1995), Drimys winterii (Munoz-Concha et al., 2007; Verdeguer et al., 2011; Zapata and Smagghe,2010), Fuchsia magellanica (Bussmann et al., 2010), Laurelia sem-pervirens (Bittner et al., 2008, 2009; Zapata and Smagghe, 2010),and Lomatia hirsuta (Simonsen et al., 2006). Extracts of leaves ofUgni molinae (obtained with 100% MeOH, 60% and 100% H2O) andof Aristotelia chilensis (obtained with 60% EtOH) showed inhibitionactivity on Pseudomonas aeruginosa (ATTC 27653), Staphylococcusaureus (ATTC 6538P), Enterobacter aerogenes (UC-1), and Candidaalbicans (UC-A) (Avello et al., 2009). The results here supportssome of the present findings, since the H2O extract of Aristoteliachilensis was observed to have a MIA < 1 mg against Staphylococ-cus aureusaureus, although a different strain was used than theone used in this study (Avello et al., 2009). The previous activ-ity reports on Ugni molinae could not be reproduced, but severalissues such as time and place of collection will have influence

on the results (Avello et al., 2009). When the extracts of Ugnimolinae and Aristotelia chilensis were added as preservatives andantioxidants in cosmetic formulations, these formulations was pre-served with respect to sensory characteristics and physicochemical

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Table 3Antimicrobial activity. +: minimum inhibiting concentration (MIC) values in �g/ml below 512 �g/ml is indicated. If no number is given no activity was observed. Plant partsL = leaf, B = Bark, S = stem, R = root, F = Flower, Fr = Fruit, AP = Arial Part. Extracts D:M = DCM:MeOH. In bold are plants of interest.

Species Part Extract Escherichia coli Pseudomonas aeruginosa

488 CFT 073 466 Nissle 317 Mn 1655 313 EDL 933 m1 II AEA Sa4 231 AEA S6 white PAO1

Acaena argentea L D:M 1:1 512 512 512 >512EtOH >512 >512 >512 >512

Acrisione denticulata L EtOH 128 >512 >512 >512

Aristotelia chilensis L D:M 1:1 >512 512 512 >512EtOH >512 >512 >512 >512

Berberis buxifolia R EtOH 512 >512 >512 >512AP D:M 1:1 >512 >512 >512 >512

Crinodendron hookerianum L D:M 1:1 512 512 512 512EtOH >512 >512 >512 >512 128 512 512 512

Coraria ruscifolia L D:M 1:1 >512 >512 256 512EtOH >512 >512 >512 256 256 384 >512

Drimys winterii B EtOH >512 >512 512 >512

Francoa appendiculata R D:M 1:1 512 512 512 >512EtOH >512 >512 >512 >512 32 >512 >512 >512H2O >512 >512 >512 >512

L D:M 1:1 >512 >512 512 >512

Fuchsia magellanica L D:M 1:1 512 512 512 >512H2O 512 512 >512 >512

Gevuina avellana Fr EtOH >512 >512 >512 >512AP EtOH >512 >512 512 >512 64 >512 >512 >512

H2O >512 >512 >512 >512 64 >512 >512 >512

Laurelia sempervirens L EtOH >512 >512 512 >512H2O >512 >512 >512 >512 64 512 >512 >512

Weinmannia trichosperma L D:M 1:1 >512 512 512 51251

pirt

wlascpfiiaect

fwsasjc

tnlatu

EtOH >512 >512

roperties (Avello et al., 2009; Céspedes et al., 2008). These resultsn combination with the results on toxicity with very high mortalityates highly support the traditional use, and the potential of thesewo species as natural additives for cosmetic preparations.

Two known sesquiterpene lactones, tutin and coriamyrtin, tohich the cytotoxicity of Coriaria ruscifolia is attributed, were iso-

ated from the fruit (Reyes et al., 1980). Also from fruits, ellagiccid 3,3′-dimethylether and corianin were obtained, but they onlyhowed scant antibacterial activity, and likewise, only moderateytotoxicity. These results were not conclusive with regard to theroperties attributed to the fruits (Valencia et al., 2001), but thendings of tutin and coriamyrtin (Reyes et al., 1980) in the fruits

ndicate that these two and probably other sesquiterpene lactonesre responsible for the activity observed (Perez et al., 2011). How-ver, the active constituents of leaves and stem still have to beonfirmed, since the sesquiterpene lactones are only reported fromhe fruits.

The essential oils of Laureliopsis philippiana have shownungistatic, repellent and toxic properties (Bittner et al., 2009),hich could explain the effect observed against Penicillium expan-

um, although Laureliopsis philippiana previously showed nontimicrobial activity, but this is clearly not the situation in thistudy (Anesini and Perez, 1993). Therefore this species will be sub-ected to further in-depth studies, in order to characterise the activeonstituents in the essential oil.

Regarding the very interesting and active species, Acaena argen-ea, Blechnum chilense, Francoa appendiculta, and Gevuina avellana,o previous reports on their antimicrobial activity has been pub-

ished. As shown in Tables 2 and 3, several extracts of these speciesre very potent against a range of bacteria. This clearly supportshe local use, but also asks for further investigation, which isnderway.

2 >512 512 512 >512 >512

5. Conclusion

The examinations of several extracts have revealed that the tra-ditional use is supported by the in vitro results published here. Ofthe 40 plants tested significant antimicrobial activity was observedfor Acaena argentea, Aristotelia chilensis, Baccharis elaeoides, Blech-num chilense, Coriaria ruscifolia, Crinodendron hookerianum, Drimyswinterii, Francoa appendiculata, Fuchsia magellanica, Gevuina avel-lana, Laurelia sempervirens, Laureliopsis philippiana and Lomatiahirusta. These findings support the traditional use by the Huillichepeople, and allow enhanced economical benefit and use by thelocals. The results obtained on Acaena argentea, Aristotelia chilensis,Blechnum chilense, Francoa appendiculta, Gevuina avellana and Lau-reliopsis philippiana are of specific scientific interest, and furtherexamination is ongoing in order to determine the active con-stituents.

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