2005 antibacterial activity of crude extracts from mexican medicinal

Journal of Ethnopharmacology 97 (2005) 293–299

Antibacterial activity of crude extracts from Mexican medicinalplants and purified coumarins and xanthones

Kakuko Yasunakaa,∗, Fumiko Abeb, Ariaki Nagayamaa, Hikaru Okabeb,Lucio Lozada-Perezc, Edith Lopez-Villafrancod, Elizabeth Estrada Munize,

Abigail Aguilarf, Ricardo Reyes-Chilpae

a Department of Microbiology and Immunology, School of Medicine, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japanb Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

c Department of Biology, Faculty of Science, National University of Mexico, Ciudad Universitaria, 04510 Mexico, Mexicod Herbarium IZTA, National University of Mexico, Campus Iztacala, Tlalnepantla, Estado de Mexico 54090, Mexico

e Institute of Chemistry, National University of Mexico, Ciudad Universitaria, 04510 Mexico, Mexicof Herbarium IMSSM, Mexican Institute for Social Security, National Medical Center S. XXI, Cuahtemoc 330, 06725 Mexico, D.F. Mexico

Received 30 November 2003; received in revised form 1 November 2004; accepted 15 November 2004Available online 12 January 2005

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Thirty-two extracts from 22 Mexican medicinal plants of 15 different families were assayed to determine their antibacterial activitscherichia coliandStaphylococcus aureus. Seventeen plants showed antibacterial activity, while five plants showed no activity aoth bacteria. All of the extracts showed higher activity againstStaphylococcus aureus(methicillin-sensitive and methicillin-resistant) thgainstEscherichia coli, except one. Among the plants examined,Bursera simaruba(L.) Sarg. (Burseraceae),Haematoxylum brasilett. Karst. (Fabaceae),Calophyllum brasilienseCambess. (Clusiaceae), andMammea americanaL. (Clusiaceae) were highly active agaitaphylococcus aureus. Coumarins (mammea A/BA and mammea A/AA) and xanthones, namely jacareubin and 1,3,5,6-tetrahydroximethylallyl) xanthone, were isolated as the principle compounds from the last two plants.2004 Elsevier Ireland Ltd. All rights reserved.

eywords:Antibacterial activity; Mexican medicinal plants; Coumarins; Xanthones;Escherichia coli; Staphylococcus aureus

. Introduction

Many plants or their components are used as folkedicines in many parts of the world to cure various in-

ectious diseases such as urinary tract infections, bronchi-is, diarrhea, cutaneous abscesses and parasitic diseases. Onhe other hand, antibiotics have dramatically reduced thencidence of many infectious diseases. Nevertheless, manyroblems remain unresolved due to occasional serious sideffects and the appearance of antibiotic-resistant mutant bac-

eria. Methicillin-resistantStaphylococcus aureus(MRSA),or example, is one of the main species of bacteria that cause

∗ Corresponding author. Tel.: +81 92 801 1011; fax: +81 92 801 9390.E-mail address:[email protected] (K. Yasunaka).

nosocomial infections in hospitals worldwide (Ichiyama eal., 1991; Yasunaka and Kono, 1999; Takeda et al., 20).In recent years, the emergence of MRSA has become aous problem because of its resistance against numerotibiotics. Although many studies on antimicrobial activitymedicinal plants have been made over the past 30 yearsKhanet al., 1980; Samy et al., 1998; Essawi and Srour, 200), alarge number of plants still have not been studied. In thistext, we examined the antibacterial properties of medicplants from Mexico.

It has been estimated that nearly 3103 higher plant spare used in Mexican traditional medicine, of which neone-third (1024) are used to treat diseases of the dtive tract (Argueta-Villamar et al., 1994). The survey by thIMSSM herbarium also indicates that Mexican medic

378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2004.11.014

294 K. Yasunaka et al. / Journal of Ethnopharmacology 97 (2005) 293–299

plants are used primarily to treat digestive (473 species),respiratory (247 species) and skin (277 species) diseases(Aguilar et al., 1994).

In the present paper, we describe the results of the studyon antibacterial activity of crude extracts of 22 Mexicanmedicinal plants against Gram-negativeEscherichia coliandGram-positiveStaphylococcus aureus. These plants are usedin Mexican traditional medicine for the treatment of diseasespresumably caused by bacteria. We also examined the effectsof pure compounds (two coumarins, three xanthones, and atriterpene) isolated from the active plants.

2. Materials and methods

2.1. Plant materials

Plant materials were collected during the period between2000 and 2003 in Cuetzalan del Progreso in the State ofPuebla, and in several localities of the State of Veracruz.Calo-phyllum brasilienseCambess. (Clusiaceae) was collected inSanta Marta and Los Tuxtlas of the State of Veracruz. Plantmaterials were also purchased at “Sonora Medicinal PlantMarket” in Mexico City, or from surplus herbarium spec-imens. Taxonomic identification was performed and con-fi ,a had

been deposited in the IMSSM herbarium of the Mexican In-stitute for Social Security, and IZTA and MEXU herbariaof the National University of Mexico. The ethnobotanicalliterature of the States of Puebla and Veracruz in particu-lar (Lopez-Villafranco, 1988; Martınez-Alfaro et al., 1995;Cano-Asseleith, 1997; Mendoza-Marquez, 2000) and otherreferences on Mexican medicinal plants (Aguilar et al., 1994;Soto-Nunez and Sousa, 1995; Reyes-Chilpa et al., 2003) werealso consulted. Investigated plant materials and their appli-cations in Mexican traditional medicine are listed inTable 1.

2.2. Preparation of plant extracts

Plant extracts were prepared according to the proceduresof Abe et al. (2002). Each dried plant part was extracted withorganic solvents (mainly with methanol). Fresh plant partsand other solvents used in this study are shown inTable 2.The solvent of each extract was dried in vacuo and the residuewas then dissolved in dimethyl sulfoxide (DMSO) to producea final concentration of 100 mg/ml.

2.3. Preparation of coumarins, xanthones and atriterpene

Coumarin mammea A/BA (1) and triterpene friedelin (6)wc -

TP

F Lo

A Chi ticGu

A GuA HieB AchB Pa les

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rmed by three of us (A. Aguilar, E. Lopez-Villafrancond L. Lozada-Perez) based on voucher specimens that

able 1lants examined and their ethnomedical applicationsa

amily Scientific name

nnonaceae Annona cherimolaMill.Annona muricataL.

ristolochiaceae Aristolochia taliscanaHook. et Arn.steraceae Piqueria trinerviaCav.ixaceae Bixa orellanaL.urseraceae Bursera simaruba(L.) Sarg.

henopodiaceae Chenopodium graveolensWilld.Chenopodium ambrosioidesL.

lusiaceae Calophyllum brasilienseCambess.Clusia salviniiDonn. Sm.Mammea americanaL.

laeocarpaceae Muntingia calaburaL.uphorbiaceae Croton dracoSchltdl.

abaceae Gliricidia sepium(Jacq.) Kunth ex Walp.Haematoxylum brasilettoH. Karst.Zornia thymifoliaKunth

alvaceae Malvaviscus arboreusCav.iperaceae Piper amalagoL.

Piper auritumKunth

olypodiaceae Phlebodium aureum(L.) J. Sm.ubiaceae Hamelia patensJacq.
erbenaceae Aloysia triphyllaRoyle Te
a SeeTable 2for voucher herbarium specimens.b Lopez-Villafranco (1988), Aguilar et al. (1994); Martınez-Alfaro et al. (1995,

2000), andReyes-Chilpa et al. (2003).

ere extracted from the leaves ofCalophyllum brasiliense,ollected in Santa Marta of the State of Veracruz (Reyes

cal name Ethnomedical applicationb

rimoya Diarrhea, fever, anthelmintic, cough, emeanabana Diarrhea, dysentery, leprae

aco Cutaneous diseaserba de San Nicolas Dysentery, feveriote Dysentery, measles, mouth pimpleslo mulato Diarrhea, fever, gingivitis, cough, meas

zote de zorrillo Diarrhea, amebiasis, stomach ache, cozote morado Diarrhea, stomach ache, cramps

ari Diarrhea, intestinal wormseja de coyote Gonorrhea, kidney acheote Domingo Fever, cutaneous disease

n, Capulın rojo Measles, mouth pimples, stomach achengre de grado Dysentery, pimples, sores

ocuite Antipyretic, measleslo de Brasil Feverrba de la Vıbora Fever

ocopacle, manzanita Dysentery, stomach ache, sores, cougrdoncillo Feveruyo Fever, skin infections

ngua de ciervo Fever, kidney diseases, ulcerlletilla Pimples, malaria, sores
cedron Diarrhea, stomach ache
)Soto-Nunez and Sousa (1995), Cano-Asseleith (1997), Mendoza-Marquez,

K. Yasunaka et al. / Journal of Ethnopharmacology 97 (2005) 293–299 295

Table 2Plants examined and their antibacterial activities

Family Scientific name Herbarium andregister number

Parta (solvent)b MICc (�g/ml)

Escherichiacoli C600

Staphylococcusaureus209P

Annonaceae Annona cherimola IZTA 879 Seeds (M) >1024 512Annona muricata IMSSM 14760 Seeds, S (M) >1024 >1024

Aristolochiaceae Aristolochia taliscana IMSSM 14718 R (M) >1024 1024Asteraceae Piqueria trinervia IZTA 880 G (M) >1024 1024Bixaceae Bixa orellana IMSSM 14759 Seeds (M) >1024 128

Burseraceae Bursera simaruba IZTA 868 L, T (M) fresh >1024 8Bursera simaruba IZTA 868 S (M) fresh >1024 64

Chenopodiaceae Chenopodium graveolens IMSSM 14751 G (M) 512 256Chenopodium ambrosioides IZTA 881 G (M) >1024 1024

Clusiaceae Calophyllum brasiliensed IMSSM 14439 L (H)e 512 32Calophyllum brasiliensed IMSSM 14439 L (A)e >1024 2Calophyllum brasiliensed IMSSM 14439 L (last M)e 1024 32Calophyllum brasiliensef IMSSM 14425 L (M) >1024 16Calophyllum brasiliensed IMSSM 14754 H (M + A) 128 8Clusia salvinii IMSSM 14434 L (M + C) 1024 128Mammea americana IMSSM 14420 P (H) >1024 8Mammea americana IMSSM 14420 P (AcOEt insol) >1024 256Mammea americana IMSSM 14420 P (AcOEt) 512 16Mammea americana IMSSM 14420 P (A) 512 4Mammea americana IMSSM 14420 Seeds (M) 256 2

Elaeocarpaceae Muntingia calabura IZTA 870 F (M) fresh 1024 256Muntingia calabura IZTA 870 L (M) fresh 512 128

Euphorbiaceae Croton draco IZTA 871 L (M) fresh >1024 64Fabaceae Gliricidia sepium IZTA 25146 L (M) >1024 64

Haematoxylum brasiletto IMSSM 14726 S (M) 128 16Zornia thymifolia IMSSM 14725 L (M) >1024 128

Malvaceae Malvaviscus arboreus IMSSM 14726 L (M) fresh >1024 256

Piperaceae Piper amalago FECME 85564 L (M) fresh 512 1024Piper auritum IZTA 872 L (M) >1024 1024

Polypodiaceae Phlebodium aureum IMSSM 14727 Whole (M) >1024 512Rubiaceae Hamelia patens IZTA 28222 L, T (M) >1024 256Verbenaceae Aloysia triphylla IMSSM 14754 L (M) fresh 1024 64

a F, fruits; G, ground parts; H, heartwoods; L, leaves; P, fruit peels; R, roots; S, stems; T, twigs; fresh, fresh plant parts.b A, acetone; C, dichloromethane; AcOEt, ethyl acetate; H, hexane; M, methanol; insol, insoluble fraction.c Minimum inhibitory concentration.d Corrected in Santa Marta, State of Veracruz.e Extracted with firstly hexane, secondly acetone, lastly methanol.f Corrected in Los Tuxtlas, State of Veracruz.

Chilpa et al., 2004). Coumarin mammea A/AA (2) was ex-tracted with hexane at room temperature from the fruit peels(421 g) ofMammea americanaL. (Clusiaceae). The extractspontaneously yielded yellow crystals, which were recrystal-lized from hexane/CH2Cl2 (6 g). Xanthones jacareubin (3),1,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl) xanthone (4) and6-deoxyjacareubin (5) were extracted from the heartwood ofCalophyllum brasiliense(Reyes-Chilpa et al., 1997). In theisolation process, each of these extracts was subjected to var-ious chromatographies to yield pure product. The structuraldetermination of pure compounds was carried out using1Hand13C nuclear magnetic resonance, infrared and ultravioletspectroscopies, and mass spectrometry. For bioassay, each

pure compound was dissolved in DMSO to produce a finalconcentration of 20 mg/ml.

2.4. Bacteria and media

Antibacterial evaluations were performed againstEs-cherichia coliK12 strain C600, methicillin-sensitiveStaphy-lococcus aureus(MSSA) strain FDA 209P and twomethicillin-resistantStaphylococcus aureus(MRSA) strains:no. 3208 and no. 80401. These two MRSA strains wereclinically isolated in Fukuoka City, Japan, and clearly de-termined to be MRSA (Yasunaka and Kono, 1999). Strainno. 3208 did not produce any�-lactamase but no. 80401


did produce�-lactamase. All the bacterial strains were keptat −80◦C until used. Brain heart infusion agar (BHI agar)and Mueller–Hinton broth (MH broth) were purchased fromthe Nippon Becton Dickinson Co., Tokyo, Japan. Oxacillinwas purchased from Sigma-Aldrich Co., Tokyo, Japan.�-Lactamase production was detected according to the manu-facturer’s instructions using cefinase disks (the Nippon Bec-ton Dickinson Co., Tokyo).

2.5. Antibacterial activity assay: evaluation of minimuminhibitory concentration (MIC)

MIC evaluations were performed by the micro-dilutionmethod. MH broth was used as the dilution and incuba-tion broth. Bacteria grown on BHI agar plates for 16 h weresuspended in MH broth. The bacterial cell numbers wereadjusted to approximately 3–6× 106 cfu (colony formingunit)/ml. Plant extracts (100 mg/ml of DMSO) or pure com-pounds (20 mg/ml of DMSO) were subjected to serial two-fold dilution. Each 80�l of the bacterial suspensions wasadded to 20�l of each serial two-fold dilution of the test ma-terial in a 96-well dish plate and then was mixed. The bacteriawere incubated at 37◦C for 24 h, and growth or no-growthwas assessed by the naked eye, then MIC was determined.DMSO solutions that did not contain any test material oro aysw ainstE to purec e fourb eacha h

activity, while 128–512 and≥1024�g/ml were consideredto show moderate and no antibacterial activity, respectively.

3. Results

3.1. Antibacterial activity of plant extracts againstEscherichia coli and Staphylococcus aureus

To evaluate the antibacterial activity againstEscherichiacoli C600 andStaphylococcus aureus209P, the MIC of eachplant extract was assayed and the results shown inTable 2.Nine extracts out of 32 showed moderate activity, while 23extracts did not show any antibacterial activity againstEs-cherichia coliC600. On the other hand, many extracts ex-amined were active againstStaphylococcus aureus209P (15extracts, high; 11 extracts, moderate). Both acetone extractfrom the leaves ofCalophyllum brasilienseand methanolextract from the seeds ofMammea americanashowedthe highest antibacterial activity (MIC 2�g/ml) of all 32extracts.

3.2. Anti-MRSA activity of plant extracts

9Pw 208ae -foldM hata byt that

TA

P MICa

S MSSA )

B 128B 8B 64C 32C 2C 32C 16C 8M 8M 16M 4M 2C 64H 16A 64

in-resisR, roo; M, m

nly contained oxacillin were used as controls. All assere performed three times for each plant extract agscherichia coliC600 andStaphylococcus aureus209P, bunly one time against each MRSA strains. The assays forompounds were performed three times against all thesacteria. MIC values were shown as a mean value ofssay. An MIC value at≤64�g/ml was judged to show hig

able 3ntibacterial activities of plant extracts

lant

cientific name Part usedc (solvent)d

ixa orellana Seeds (M)ursera simaruba L, T (M) freshursera simaruba S (M) freshalophyllum brasiliensee L (H)f

alophyllum brasiliensee L (A) f

alophyllum brasiliensee L (last M)f

alophyllum brasilienseg L (M)alophyllum brasiliensee H (M + A)ammea americana P (H)ammea americana P (AcOEt)ammea americana P (A)ammea americana Seeds (M)roton draco L (M) freshaematoxylum brasiletto S (M)loysia triphylla L (M) fresh

a Minimum inhibitory concentration.b MSSA, methicillin-sensitiveStaphylococcus aureus; MRSA, methicillc F, fruits; G, ground parts; H, heartwoods; L, leaves; P, fruit peels;d A, acetone; C, dichloromethane; AcOEt, ethyl acetate; H, hexanee Corrected in Santa Marta, State of Veracruz.f Extracted with firstly hexane, secondly acetone, lastly methanol.g Corrected in Los Tuxtlas, State of Veracruz.

MIC values of 15 extracts active against MSSA 20ere further estimated against two MRSA strains (no. 3nd no. 80401) and are shown inTable 3. All the extractsxamined showed nearly the same MIC values (1/2–2IC) against these two MRSA strains. It is obvious tntibacterial activity of these extracts is not influenced

he existence of�-lactamase in the bacteria, suggesting

(�g/ml)

209Pb MRSA (no. 3208) MRSA (no. 80401

128 1288 8

64 6432 328 8

32 3216 1616 864 64

8 162 48 4

128 6432 1664 64

tantStaphylococcus aureus.ts; S, stems; T, twigs; fresh, fresh plant parts.ethanol; insol, insoluble fraction.


Fig. 1. Chemical structures of coumarins, xanthones, and triterpene: (1) mammea A/BA; (2) mammea A/AA; (3) jacareubin; (4) 1,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl) xanthone; (5) 6-deoxyjacareubin; (6) friedelin.

the antibacterial principles in them do not contain any�-lactam rings. FromTables 2 and 3it was found thatBurserasimaruba(L.) Sarg. (Burseraceae),Haematoxylum brasilettoH. Karst. (Fabaceae),Calophyllum brasiliense, andMam-mea americanawere highly active against both MSSA andMRSA.

3.3. Antibacterial activity of coumarins, xanthones anda triterpene

To identify the principle compounds ofCalophyllumbrasilienseandMammea americana, we purified coumarins1and2, xanthones3–5and triterpene6(Fig. 1) as described in

Table 4Antibacterial activities of purified compounds

Compound Plant MICa (�g/ml)

Scientific name Partb Escherichiacoli C600

MSSAc

209PMRSA(no. 3208)

MRSA(no. 80401)

Mammea A/BA (1) Calophyllum brasiliense L >256 1 2 2Mammea A/AA (2) Mammea americana P >256 8 8 8Jacareubin (3) Calophyllum brasiliense H 128 4 4 41,3,5,6-Tetrahydroxy-2-(3,3-dimethylallyl)

xanthone (4)Calophyllum brasiliense H 128 1 2 4

6-Deoxyjacareubin (5) Calophyllum brasiliense H >512 256 >512 >512Friedelin (6) Calophyllum brasiliense L >512 >512 >512 >512Oxacillin <0.5 <0.5 >1024 512

a Minimum inhibitory concentration.b F, fruits; G, ground parts; H, heartwoods; L, leaves; P, fruit peels; R, roots; S, stems; T, twigs; fresh, fresh plant parts.c MSSA, methicillin-sensitiveStaphylococcus aureus; MRSA, methicillin-resistantStaphylococcus aureus.


Section2. MIC values of these pure compounds are shown inTable 4. Compounds1–4 are the compounds responsible forthe activity, since they showed higher activity againstStaphy-lococcus aureusthan that of the original extract. Xanthone5and triterpene6 showed no activity againstStaphylococcusaureus. Only xanthones3 and4 showed moderate activityagainstEscherichia coli.

Various studies have previously shown the biological ac-tivities of some coumarins and xanthones (Finnegan et al.,1972; Sundaram et al., 1983; Iinuma et al., 1996; Reyes-Chilpa et al., 1997; Ojala et al., 2000; Itoigawa et al., 2001;Ito et al., 2002). However, this is the first report of antibacte-rial activity of these six compounds againstEscherichia coliandStaphylococcus aureus(MSSA and MRSA).Calophyl-lum brasilienseandMammea americanacontain other typesof coumarins and xanthones (Crombie et al., 1967; Finneganet al., 1972; Finnegan et al., 1973; Ito et al., 2002), therefore,their antibacterial activity should be studied.

4. Discussion and conclusions

Twenty-two plants studied here are used in Mexican tra-ditional medicine against illness such as fever, diarrhea,malaria, dysentery, sores, and venereal diseases presumablyc 22m activ-i lysBb -iw d isw oul-t easesH alf redh l ap-p ases.H sa a,1 tt i-c era-p com-p in.

ortt allyu se oft

A

ra ke to

thank Dr. H. Akahane of Fukuoka University for his encour-agement in carrying out this study. This work was supportedin part by a grant (no. 001001) from the Central ResearchInstitute of Fukuoka University: 2000–2001.

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