Journal of Ethnopharmacology 97 (2005) 293299

Antibacterial activity of crude extracts from Mexican medicinal plants and puried coumarins and xanthones
Kakuko Yasunakaa, , Fumiko Abeb , Ariaki Nagayamaa , Hikaru Okabeb , Lucio Lozada-P rezc , Edith L pez-Villafrancod , Elizabeth Estrada Mu ize , e o n Abigail Aguilarf , Ricardo Reyes-Chilpae
a

Department of Microbiology and Immunology, School of Medicine, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan b 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, Mexico d 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, Mexico f 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 2004 Available online 12 January 2005

Abstract Thirty-two extracts from 22 Mexican medicinal plants of 15 different families were assayed to determine their antibacterial activity against Escherichia coli and Staphylococcus aureus. Seventeen plants showed antibacterial activity, while ve plants showed no activity against both bacteria. All of the extracts showed higher activity against Staphylococcus aureus (methicillin-sensitive and methicillin-resistant) than against Escherichia coli, except one. Among the plants examined, Bursera simaruba (L.) Sarg. (Burseraceae), Haematoxylum brasiletto H. Karst. (Fabaceae), Calophyllum brasiliense Cambess. (Clusiaceae), and Mammea americana L. (Clusiaceae) were highly active against Staphylococcus aureus. Coumarins (mammea A/BA and mammea A/AA) and xanthones, namely jacareubin and 1,3,5,6-tetrahydroxy-2-(3,3dimethylallyl) xanthone, were isolated as the principle compounds from the last two plants. 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antibacterial activity; Mexican medicinal plants; Coumarins; Xanthones; Escherichia coli; Staphylococcus aureus

1. Introduction Many plants or their components are used as folk medicines in many parts of the world to cure various infectious diseases such as urinary tract infections, bronchitis, diarrhea, cutaneous abscesses and parasitic diseases. On the other hand, antibiotics have dramatically reduced the incidence of many infectious diseases. Nevertheless, many problems remain unresolved due to occasional serious side effects and the appearance of antibiotic-resistant mutant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA), for 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: kakuko@fukuoka-u.ac.jp (K. Yasunaka).

nosocomial infections in hospitals worldwide (Ichiyama et al., 1991; Yasunaka and Kono, 1999; Takeda et al., 2000). In recent years, the emergence of MRSA has become a serious problem because of its resistance against numerous antibiotics. Although many studies on antimicrobial activity of medicinal plants have been made over the past 30 years (Khan et al., 1980; Samy et al., 1998; Essawi and Srour, 2000), a large number of plants still have not been studied. In this context, we examined the antibacterial properties of medicinal plants from Mexico. It has been estimated that nearly 3103 higher plant species are used in Mexican traditional medicine, of which nearly one-third (1024) are used to treat diseases of the digestive tract (Argueta-Villamar et al., 1994). The survey by the IMSSM herbarium also indicates that Mexican medicinal

0378-8741/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.014

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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 study on antibacterial activity of crude extracts of 22 Mexican medicinal plants against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. These plants are used in Mexican traditional medicine for the treatment of diseases presumably caused by bacteria. We also examined the effects of pure compounds (two coumarins, three xanthones, and a triterpene) isolated from the active plants.

been deposited in the IMSSM herbarium of the Mexican Institute for Social Security, and IZTA and MEXU herbaria of the National University of Mexico. The ethnobotanical literature of the States of Puebla and Veracruz in particular (L pez-Villafranco, 1988; Martnez-Alfaro et al., 1995; o Cano-Asseleith, 1997; Mendoza-M rquez, 2000) and other a references on Mexican medicinal plants (Aguilar et al., 1994; Soto-Nu ez and Sousa, 1995; Reyes-Chilpa et al., 2003) were n also consulted. Investigated plant materials and their applications in Mexican traditional medicine are listed in Table 1. 2.2. Preparation of plant extracts

2. Materials and methods 2.1. Plant materials Plant materials were collected during the period between 2000 and 2003 in Cuetzal n del Progreso in the State of a Puebla, and in several localities of the State of Veracruz. Calophyllum brasiliense Cambess. (Clusiaceae) was collected in Santa Marta and Los Tuxtlas of the State of Veracruz. Plant materials were also purchased at Sonora Medicinal Plant Market in Mexico City, or from surplus herbarium specimens. Taxonomic identication was performed and conrmed by three of us (A. Aguilar, E. L pez-Villafranco, o and L. Lozada-P rez) based on voucher specimens that had e
Table 1 Plants examined and their ethnomedical applicationsa Family Annonaceae Aristolochiaceae Asteraceae Bixaceae Burseraceae Chenopodiaceae Clusiaceae Scientic name Annona cherimola Mill. Annona muricata L. Aristolochia taliscana Hook. et Arn. Piqueria trinervia Cav. Bixa orellana L. Bursera simaruba (L.) Sarg. Chenopodium graveolens Willd. Chenopodium ambrosioides L. Calophyllum brasiliense Cambess. Clusia salvinii Donn. Sm. Mammea americana L. Muntingia calabura L. Croton draco Schltdl. Gliricidia sepium (Jacq.) Kunth ex Walp. Haematoxylum brasiletto H. Karst. Zornia thymifolia Kunth Malvaviscus arboreus Cav. Piper amalago L. Piper auritum Kunth Phlebodium aureum (L.) J. Sm. Hamelia patens Jacq. Aloysia triphylla Royle

Plant extracts were prepared according to the procedures of Abe et al. (2002). Each dried plant part was extracted with organic solvents (mainly with methanol). Fresh plant parts and other solvents used in this study are shown in Table 2. The solvent of each extract was dried in vacuo and the residue was then dissolved in dimethyl sulfoxide (DMSO) to produce a nal concentration of 100 mg/ml. 2.3. Preparation of coumarins, xanthones and a triterpene Coumarin mammea A/BA (1) and triterpene friedelin (6) were extracted from the leaves of Calophyllum brasiliense, collected in Santa Marta of the State of Veracruz (Reyes-

Local name Chirimoya Guan bana a Guaco Hierba de San Nicol s a Achiote Palo mulato Epazote de zorrillo Epazote morado Bari Oreja de coyote Zapote Domingo P an, Capuln rojo u Sangre de grado Cocuite Palo de Brasil Hierba de la Vbora Azocopacle, manzanita Cordoncillo Acuyo Lengua de ciervo Balletilla T cedr n e o

Ethnomedical applicationb Diarrhea, fever, anthelmintic, cough, emetic Diarrhea, dysentery, leprae Cutaneous disease Dysentery, fever Dysentery, measles, mouth pimples Diarrhea, fever, gingivitis, cough, measles Diarrhea, amebiasis, stomach ache, cough Diarrhea, stomach ache, cramps Diarrhea, intestinal worms Gonorrhea, kidney ache Fever, cutaneous disease Measles, mouth pimples, stomach ache Dysentery, pimples, sores Antipyretic, measles Fever Fever Dysentery, stomach ache, sores, cough Fever Fever, skin infections Fever, kidney diseases, ulcer Pimples, malaria, sores Diarrhea, stomach ache

Elaeocarpaceae Euphorbiaceae Fabaceae

Malvaceae Piperaceae Polypodiaceae Rubiaceae Verbenaceae

a b

See Table 2 for voucher herbarium specimens. L pez-Villafranco (1988), Aguilar et al. (1994); Martnez-Alfaro et al. (1995), Soto-Nu ez and Sousa (1995), Cano-Asseleith (1997), Mendoza-M rquez, o n a (2000), and Reyes-Chilpa et al. (2003).

K. Yasunaka et al. / Journal of Ethnopharmacology 97 (2005) 293299 Table 2 Plants examined and their antibacterial activities Family Scientic name Herbarium and register number Parta (solvent)b MICc ( g/ml) Escherichia coli C600 Seeds (M) Seeds, S (M) R (M) G (M) Seeds (M) L, T (M) fresh S (M) fresh G (M) G (M) L (H)e L (A)e L (last M)e L (M) H (M + A) L (M + C) P (H) P (AcOEt insol) P (AcOEt) P (A) Seeds (M) F (M) fresh L (M) fresh L (M) fresh L (M) S (M) L (M) L (M) fresh L (M) fresh L (M) Whole (M) L, T (M) L (M) fresh >1024 >1024 >1024 >1024 >1024 >1024 >1024 512 >1024 512 >1024 1024 >1024 128 1024 >1024 >1024 512 512 256 1024 512 >1024 >1024 128 >1024 >1024 512 >1024 >1024 >1024 1024

295

Staphylococcus aureus 209P 512 >1024 1024 1024 128 8 64 256 1024 32 2 32 16 8 128 8 256 16 4 2 256 128 64 64 16 128 256 1024 1024 512 256 64

Annonaceae Aristolochiaceae Asteraceae Bixaceae Burseraceae Chenopodiaceae Clusiaceae

Annona cherimola Annona muricata Aristolochia taliscana Piqueria trinervia Bixa orellana Bursera simaruba Bursera simaruba Chenopodium graveolens Chenopodium ambrosioides Calophyllum brasiliensed Calophyllum brasiliensed Calophyllum brasiliensed Calophyllum brasiliensef Calophyllum brasiliensed Clusia salvinii Mammea americana Mammea americana Mammea americana Mammea americana Mammea americana Muntingia calabura Muntingia calabura Croton draco Gliricidia sepium Haematoxylum brasiletto Zornia thymifolia Malvaviscus arboreus Piper amalago Piper auritum Phlebodium aureum Hamelia patens Aloysia triphylla

IZTA 879 IMSSM 14760 IMSSM 14718 IZTA 880 IMSSM 14759 IZTA 868 IZTA 868 IMSSM 14751 IZTA 881 IMSSM 14439 IMSSM 14439 IMSSM 14439 IMSSM 14425 IMSSM 14754 IMSSM 14434 IMSSM 14420 IMSSM 14420 IMSSM 14420 IMSSM 14420 IMSSM 14420 IZTA 870 IZTA 870 IZTA 871 IZTA 25146 IMSSM 14726 IMSSM 14725 IMSSM 14726 FECME 85564 IZTA 872 IMSSM 14727 IZTA 28222 IMSSM 14754

Elaeocarpaceae Euphorbiaceae Fabaceae

Malvaceae Piperaceae Polypodiaceae Rubiaceae Verbenaceae

a b c d e f

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

Chilpa et al., 2004). Coumarin mammea A/AA (2) was extracted with hexane at room temperature from the fruit peels (421 g) of Mammea americana L. (Clusiaceae). The extract spontaneously yielded yellow crystals, which were recrystallized from hexane/CH2 Cl2 (6 g). Xanthones jacareubin (3), 1,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl) xanthone (4) and 6-deoxyjacareubin (5) were extracted from the heartwood of Calophyllum brasiliense (Reyes-Chilpa et al., 1997). In the isolation process, each of these extracts was subjected to various chromatographies to yield pure product. The structural determination of pure compounds was carried out using 1 H and 13 C nuclear magnetic resonance, infrared and ultraviolet spectroscopies, and mass spectrometry. For bioassay, each

pure compound was dissolved in DMSO to produce a nal concentration of 20 mg/ml. 2.4. Bacteria and media Antibacterial evaluations were performed against Escherichia coli K12 strain C600, methicillin-sensitive Staphylococcus aureus (MSSA) strain FDA 209P and two methicillin-resistant Staphylococcus aureus (MRSA) strains: no. 3208 and no. 80401. These two MRSA strains were clinically isolated in Fukuoka City, Japan, and clearly determined to be MRSA (Yasunaka and Kono, 1999). Strain no. 3208 did not produce any -lactamase but no. 80401

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did produce -lactamase. All the bacterial strains were kept at 80 C until used. Brain heart infusion agar (BHI agar) and MuellerHinton broth (MH broth) were purchased from the Nippon Becton Dickinson Co., Tokyo, Japan. Oxacillin was purchased from Sigma-Aldrich Co., Tokyo, Japan. Lactamase production was detected according to the manufacturers instructions using cenase disks (the Nippon Becton Dickinson Co., Tokyo). 2.5. Antibacterial activity assay: evaluation of minimum inhibitory concentration (MIC) MIC evaluations were performed by the micro-dilution method. MH broth was used as the dilution and incubation broth. Bacteria grown on BHI agar plates for 16 h were suspended in MH broth. The bacterial cell numbers were adjusted to approximately 36 106 cfu (colony forming unit)/ml. Plant extracts (100 mg/ml of DMSO) or pure compounds (20 mg/ml of DMSO) were subjected to serial twofold dilution. Each 80 l of the bacterial suspensions was added to 20 l of each serial two-fold dilution of the test material in a 96-well dish plate and then was mixed. The bacteria were incubated at 37 C for 24 h, and growth or no-growth was assessed by the naked eye, then MIC was determined. DMSO solutions that did not contain any test material or only contained oxacillin were used as controls. All assays were performed three times for each plant extract against Escherichia coli C600 and Staphylococcus aureus 209P, but only one time against each MRSA strains. The assays for pure compounds were performed three times against all these four bacteria. MIC values were shown as a mean value of each assay. An MIC value at 64 g/ml was judged to show high
Table 3 Antibacterial activities of plant extracts Plant Scientic name Bixa orellana Bursera simaruba Bursera simaruba Calophyllum brasiliensee Calophyllum brasiliensee Calophyllum brasiliensee Calophyllum brasilienseg Calophyllum brasiliensee Mammea americana Mammea americana Mammea americana Mammea americana Croton draco Haematoxylum brasiletto Aloysia triphylla
a b c d e f g

activity, while 128512 and 1024 g/ml were considered to show moderate and no antibacterial activity, respectively.

3. Results 3.1. Antibacterial activity of plant extracts against Escherichia coli and Staphylococcus aureus To evaluate the antibacterial activity against Escherichia coli C600 and Staphylococcus aureus 209P, the MIC of each plant extract was assayed and the results shown in Table 2. Nine extracts out of 32 showed moderate activity, while 23 extracts did not show any antibacterial activity against Escherichia coli C600. On the other hand, many extracts examined were active against Staphylococcus aureus 209P (15 extracts, high; 11 extracts, moderate). Both acetone extract from the leaves of Calophyllum brasiliense and methanol extract from the seeds of Mammea americana showed the highest antibacterial activity (MIC 2 g/ml) of all 32 extracts. 3.2. Anti-MRSA activity of plant extracts MIC values of 15 extracts active against MSSA 209P were further estimated against two MRSA strains (no. 3208 and no. 80401) and are shown in Table 3. All the extracts examined showed nearly the same MIC values (1/22-fold MIC) against these two MRSA strains. It is obvious that antibacterial activity of these extracts is not inuenced by the existence of -lactamase in the bacteria, suggesting that

MICa ( g/ml) Part usedc (solvent)d Seeds (M) L, T (M) fresh S (M) fresh L (H)f L (A)f L (last M)f L (M) H (M + A) P (H) P (AcOEt) P (A) Seeds (M) L (M) fresh S (M) L (M) fresh MSSA 209Pb 128 8 64 32 2 32 16 8 8 16 4 2 64 16 64 MRSA (no. 3208) 128 8 64 32 8 32 16 16 64 8 2 8 128 32 64 MRSA (no. 80401) 128 8 64 32 8 32 16 8 64 16 4 4 64 16 64

Minimum inhibitory concentration. MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus. F, fruits; G, ground parts; H, heartwoods; L, leaves; P, fruit peels; R, roots; S, stems; T, twigs; fresh, fresh plant parts. A, acetone; C, dichloromethane; AcOEt, ethyl acetate; H, hexane; M, methanol; insol, insoluble fraction. Corrected in Santa Marta, State of Veracruz. Extracted with rstly hexane, secondly acetone, lastly methanol. Corrected in Los Tuxtlas, State of Veracruz.

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297

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-(3,3dimethylallyl) xanthone; (5) 6-deoxyjacareubin; (6) friedelin.

the antibacterial principles in them do not contain any lactam rings. From Tables 2 and 3 it was found that Bursera simaruba (L.) Sarg. (Burseraceae), Haematoxylum brasiletto H. Karst. (Fabaceae), Calophyllum brasiliense, and Mammea americana were highly active against both MSSA and MRSA.
Table 4 Antibacterial activities of puried compounds Compound Plant Scientic name Mammea A/BA (1) Mammea A/AA (2) Jacareubin (3) 1,3,5,6-Tetrahydroxy-2-(3,3-dimethylallyl) xanthone (4) 6-Deoxyjacareubin (5) Friedelin (6) Oxacillin
a b c

3.3. Antibacterial activity of coumarins, xanthones and a triterpene To identify the principle compounds of Calophyllum brasiliense and Mammea americana, we puried coumarins 1 and 2, xanthones 35 and triterpene 6 (Fig. 1) as described in

MICa ( g/ml) Partb L P H H H L Escherichia coli C600 >256 >256 128 128 >512 >512 <0.5 MSSAc 209P 1 8 4 1 256 >512 <0.5 MRSA (no. 3208) 2 8 4 2 >512 >512 >1024 MRSA (no. 80401) 2 8 4 4 >512 >512 512

Calophyllum brasiliense Mammea americana Calophyllum brasiliense Calophyllum brasiliense Calophyllum brasiliense Calophyllum brasiliense

Minimum inhibitory concentration. F, fruits; G, ground parts; H, heartwoods; L, leaves; P, fruit peels; R, roots; S, stems; T, twigs; fresh, fresh plant parts. MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus.

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Section 2. MIC values of these pure compounds are shown in Table 4. Compounds 14 are the compounds responsible for the activity, since they showed higher activity against Staphylococcus aureus than that of the original extract. Xanthone 5 and triterpene 6 showed no activity against Staphylococcus aureus. Only xanthones 3 and 4 showed moderate activity against Escherichia coli. Various studies have previously shown the biological activities of some coumarins and xanthones (Finnegan et al., 1972; Sundaram et al., 1983; Iinuma et al., 1996; ReyesChilpa et al., 1997; Ojala et al., 2000; Itoigawa et al., 2001; Ito et al., 2002). However, this is the rst report of antibacterial activity of these six compounds against Escherichia coli and Staphylococcus aureus (MSSA and MRSA). Calophyllum brasiliense and Mammea americana contain other types of coumarins and xanthones (Crombie et al., 1967; Finnegan et al., 1972; Finnegan et al., 1973; Ito et al., 2002), therefore, their antibacterial activity should be studied.

thank Dr. H. Akahane of Fukuoka University for his encouragement in carrying out this study. This work was supported in part by a grant (no. 001001) from the Central Research Institute of Fukuoka University: 20002001.

References
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4. Discussion and conclusions Twenty-two plants studied here are used in Mexican traditional medicine against illness such as fever, diarrhea, malaria, dysentery, sores, and venereal diseases presumably caused mainly by bacteria or microorganisms. Of the 22 medicinal plants assayed, seven species showed high activity againstStaphylococcus aureus and nine moderate. Only six plants showed moderate activity against Escherichia coli. Bursera simaruba, Haematoxylon brasiletto, Calophyllum brasiliense, and Mammea americana showed high activity against Staphylococcus aureus. Bursera simaruba is a widely distributed tree in the tropical area in Mexico and is well known for its applications as water decoctions or poultices made from the leaves against bacteria related diseases. Haematoxylum brasiletto, a tree distributed in dry tropical forests, has red heartwood. This morphological feature red heartwood could probably be related to several medical applications, due to an association with blood or heart diseases. Haematoxylum brasiletto is also known in certain localities as a febrifuge (Aguilar et al., 1994; Soto-Nu ez and Sousa, n 1995). Antibacterial compounds (14) isolated from the last two species Calophyllum brasiliense and Mammea americana would serve as promising candidates for chemotherapeutic agents, especially against MRSA, because these compounds showed much higher activity than that of oxacillin. The results obtained in this work scientically support the effectiveness of medicinal plants locally and traditionally used to treat infection-related diseases in Mexico, because of their high antibacterial activity.

Acknowledgements The authors are grateful to Laura Cort s Z rraga for her e a assistance in collecting ethnobotanical data. We would like to

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