- Email: [email protected]
Antibacterial properties of tropical plants from Puerto Rico
ARTICLE IN PRESS
Phytomedicine 13 (2006) 272–276 www.elsevier.de/phymed
Antibacterial properties of tropical plants from Puerto Rico P.A. Mele´ndeza,b,, V.A. Caprilesa,{ a
Department of Biology, Inter American University of Puerto Rico, San Germa´n Campus, San Germa´n, Puerto Rico Hewlett-Packard Caribe Ltd., ISB-Americas R&D, Aguadilla, Puerto Rico 00603
b
Abstract In an effort to document the antibacterial properties of plants commonly used by the people of Puerto Rico, we studied the effects of 172 plant species, utilizing the disc diffusion method, against Escherichia coli and Staphylococcus aureus. The methanolic extracts of 14 species showed antibacterial activities during this preliminary screen. These positive plant extracts were tested successively over 15 additional species. The results showed that extracts from Citrus aurantifolia (Rutaceae), Citrus aurantium (Rutaceae), Punica granatum (Punicaceae), Phyllanthus acidus (Euphorbiaceae) and Tamarindus indica (Caesalpiniaceae) possess strong in vitro antibacterial activity against the bacteria tested. r 2005 Elsevier GmbH. All rights reserved. Keywords: Plant antibacterials; Antibacterial plants from; Puerto Rico; Tropical plants; Antimicrobials
Introduction Exploring the healing power of plants is an ancient concept. For many centuries people have been trying to alleviate and treat diseases with different plant extracts and formulations (Cowan, 1999). It is estimated, however, that of the 250,000–500,000 species found on Earth, only 1% have been studied for their pharmaceutical potential. In Puerto Rico, the use of plants for medicinal purposes is a common practice, especially among adults living in the countryside. Many plants of our flora are known to possess antimicrobial properties and have been used by the local population in many instances to treat colds, coughs, bronchitis, diarrhea, respiratory infections, urinary disorders and skin lesions (Herna´ndez et al., 1984). In the past, other groups have published important ethnobotanical bibliographies that Corresponding author. Hewlett-Packard Caribe Ltd., ISB-Americas R&D, Aguadilla, 00603 Puerto Rico. Tel.: +1 787 819 7644; fax: +1 787 819 6270. E-mail address: [email protected] (P.A. Mele´ndez). { Deceased.
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.11.009
compiled and described the most frequently used species to treat infections (Liogier, 1990; Nun˜ez-Mele´ndez, 1989; Morton, 1981). However, scientific evidence confirming that these traditional remedies possess antibiotic properties is lacking. Over the years there have been several studies documenting the antibacterial properties of plants from other regions of the Caribbean and South America (Chariandy et al., 1999; Anesini and Perez, 1993; Martı´ nez et al., 1996; Ca´ceres et al., 1993; RojasHerna´ndez et al., 1978a; Rojas-Herna´ndez et al., 1978b). The Traditional Medicine in the Islands (TRAMIL) research project series started in 1982 in Haiti, and has documented a great deal of information concerning the medicinal use of plants in the Caribbean basin (Robineau and Soejarto, 1996). In Puerto Rico, Guerrero and Robledo (1993) studied the effects of some endemic plants; and more recently (Frame et al., 1999) examined the anti-Mycobacterium tuberculosis effects of 50 local plants. In Puerto Rico, as in many other countries, we are unfortunately at risk of losing the ancestral knowledge of the healing properties of plants. In an effort to
ARTICLE IN PRESS P.A. Mele´ndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276
corroborate, document, and expand the traditional use of medicinal plants with antibacterial properties in Puerto Rico, we collected and evaluated the antibacterial effects of a vast number of species against different types of Gram-positive and Gram-negative bacteria.
Materials and methods Plant collection Between March 1983 and March 1985, 172 species of tropical plants, from 73 different families, were collected from their natural habitats in the north-western and western regions of Puerto Rico, specifically in the municipalities of Isabela, Aguadilla, Quebradillas, San Germa´n and Sa´bana Grande. A specimen of each species was dry-mounted, photographed and preserved for future reference in the herbarium of the Biology Department of the Inter American University (BDIAU), San Germa´n, Puerto Rico. The identity of each plant was confirmed by Professor M. Vives, a plant taxonomist based in Quebradillas, Puerto Rico.
273
variable bacteria), Mycobacterium phlei, Mycobacterium rodochrus, and Mycobacterium smegmatis] were supplied by BD-IAU.
Antibacterial tests Antibacterial activity was tested using a modification of the disc diffusion method originally described by Bauer et al. (1966). A loop of bacteria from the agarslant stock was cultured in nutrient broth overnight and spread with a sterile cotton swap into petriplates containing 10 ml of Mueller Hinton Agar. Sterile filter paper discs (9 mm in diameter) impregnated with the plant extract were placed on the cultured plates and incubated at 25 or 37 1C, depending on the bacteria. The solvent without extracts served as negative control. Standard antibiotics of chloramphenicol 30 mg, streptomycin 10 mg, tetracycline 30 mg, erythromycin 15 mg, neomycin 30 mg, novobiocin 30 mg, kanamycin 30 mg, and penicillium G 10 units were used as positive controls. After 24 h of incubation, the diameter in mm of the inhibitory or clear zones around the disks was recorded.
Preparation of the crude extracts The extraction procedures were kept as simple as possible. Plant material (usually leaves and sometimes fruits) was oven-dried in a finger bowl at 65 1C for 2 days, then hand-ground and stored in plastic bags for further use. A 50-g sample of each specimen was extracted using 100 ml methanol in an electric blender left running for 15 min. The resultant suspension was filtered twice, first through cloth (50% cotton/50% polyester) and then through filter paper (Whatman No. 2). In some cases (for the densest extracts), the material was vacuum filtered using a Bu¨chner funnel. Each final filtrate was concentrated to dryness in a Rotovapor rotary-evaporation unit (Bu¨chi Labortechnik; Flawil, Switzerland) and then stored at 20 1C until further use. Before tests, concentrates were dissolved in 50 ml of double distilled water for a final concentration of 1000 mg/ml of dry plant powder. Aqueous dilutions were store in close containers at 4 1C for a maximum of 14 days.
Microorganisms The test organisms used [Escherichia coli, Enterobacter cloacae, Pseudomonas fluorescens, Proteus vulgaris, Alcaligenes faecalis, Serratia marcescens, Enterobacter aerogenes (Gram-negative bacteria), Staphylococcus aureus, Arthrobacter globiformis, Micrococcus luteus, Bacillus cereus, Bacillus subtilis (Gram-positive bacteria), Bacillus coagulans, Micrococcus roseus (Gram-
Results and discussion After evaluating the effects of 172 different plant extracts against Gram-negative E. coli and Grampositive S. aureus, we found 14 species with antibacterial properties (Table 1). Five extracts inhibited E. coli while 14 inhibited S. aureus. It has been documented (Grosvenor et al., 1995; Martı´ nez et al., 1996; Chariandy et al., 1999; Stickler and King, 1992) that S. aureus is one of the bacteria most susceptible to plant extracts. Citrus aurantifolia and Tamarindus indica were the most effective extracts against E. coli. The inhibitory zones reached 20 and 17-mm diameter, respectively. Two members of the Rutaceae family, Citrus aurantifolia (30mm diam.) and Citrus aurantium (27-mm diam.) were the most effective against S. aureus, followed by Punica granatum (22-mm diam.), Phyllanthus acidus (20-mm diam.) and Crescentia cujete (18-mm diam.). The inhibitory activities of the extracts were compared to known antibiotics, which served as positive controls (Table 2). Novobiocin and Penicillin G inhibited S. aureus and the rest inhibited both E. coli and S. aureus. In every case, the negative controls showed no antibacterial activity. The antibacterial properties of members of the Citrus genera have been documented by others (Anesini and Perez, 1993; Reagor et al., 2002). Likewise, there is evidence of the antibacterial properties of P. granatum (Prashanth et al., 2001; Anesini and Perez, 1993; Rojas-Herna´ndez et al., 1978b; Navarro et al., 1996), Crescentia cujete (Bignoniaceae) (Binutu and
ARTICLE IN PRESS P.A. Mele´ndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276
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Table 1.
Antibacterial properties of tropical plants from Puerto Rico against Gram-negative and Gram-positive bacteria
Scientific name
Family
Crescentia cujete L. Tamarindus indica L. Terminalia catappa L. Phyllanthus acidus (L.) Skeels Rosmarinus officinalis L. Lagerstroemia speciosa (L.) Pers. Urena lobata L. ssp. Lobata Clidemia hirta (L.) D. Don Punica granatum L. Psychotria nervosa Sw. Citrus aurantifolia (Chrism.) Swingle Citrus aurantium L. Melicoccus bijugatus Jacq. Petitia domingensis Jacq. a
Bignoniaceae Caesalpiniaceae Combretaceae Euphorbiaceae Lamiaceae Lythraceae Malvaceae Melastomataceae Punicaceae Rubiaceae Rutaceae Rutaceae Sapindaceae Verbenaceae
Part testeda
L L L F L L L L F L F F L L
Antimicrobial activityb E. coli
S. aureus
0 17 0 11 0 0 0 0 12 0 20 12 0 0
18 13 13 20 15 11 9 12 22 12 30 27 17 11
Plant part tested: L., leaves; F., fruits. Measured by the diameter of zone of inhibition in mm.
b
Table 2. Antibiotic controls against Gram-negative and Gram-positive bacteria Antibiotics
Chloramphenicol Streptomycin Tetracycline Erythromycin Neomycin Novobiocin Kanamycin Penicillium G
Concentration
30 mg 10 mg 30 mg 15 mg 30 mg 30 mg 30 mg 10 units
Inhibitory zone (mm) E. coli
S. aureus
31 33 26 16 22 0 26 0
30 26 38 35 26 38 38 43
Lajubutu, 1994), Rosmarinus officinalis (Lamiaceae) (Takenaka et al., 1997), and Urena lobata (Malvaceae) (Mazumder et al., 2001). Not much is known, however, about the antibacterial compounds present in the leaves of T. indica. Plants showing no activity – The following 158 plants did not show any activity against E. coli and S. aureus: Odontonema strictum (Nees) Kuntz, Furcraea tuberosa (Miller) Ait. f, Achyranthes indica (L.) Miller, Amaranthus viridis L., Gomphrena dispersa Standl., Allium cepa L., Allium sativum L., Anethum graveolens L., Apium petroselinum L., Foeniculum vulgare Miller, Comocladia dodonaea (L.) Urban, Mangifera indica L., Spondias purpurea L., Annona muricata L., Annona reticulata L., Catharanthus roseus (L.) G. Don, Plumeria rubra L., Rauvolfia nitida Jacq., Caladium colocasia (L.) W. F. Wight, Epipremnun aureum (Linden & Andre´) Bunt, Asclepias curassavica L., Calotropis procera (Ait.)
Ait.f., Anthemis nobilis L., Bidens pilosa L., Calendula officinalis L., Cichorium intybus L., Eupatoriun odoratum L., Leptilon pusillum (Nutt.) Britton, Parthenuim hysterophorus L., Senecio aizoides Sch. Bip, Wedelia reticulata DC., Wedelia trilobata (L.) A. S. Hitchcock, Anredera leptostachys (moq.) v. Steenis, Spathodea campanulata Beauv., Bixa orellana L., Tournefortia hirsutissima L., Brassica hirta Moench, Brassica oleracea var. capitata L., Lepidium virginicum L., Ananas comosus (L.) Merill, Bromelia pinguin L., Nidularium billbergioides L. B. Sm., Bursera simaruba (L.) Sarg., Bauhinia monandra Kurz, Ditremexa occidentalis (L.) Britton & Rose, Hymenaea courbaril L., Isotoma longiflora (L.) Presl., Sambucus simpsonii Rehder, Carica papaya L., Chenopodium ambrosioides L, Commelina elegans HBK, Rhoeo spathacea (Sw.) Stearn, Ambrosia peruviana Willd., Cuscuta americana L., Ipomoea batatas (L.) Lam., Ipomoea carnea Jacq., Ipomoea pes-caprae (L.), Bryophylium pinnatum (Lam.) Oken, Kalanchoe¨ daigremontiana Hamet & Perrier, Momordica charantia L., Pepo moschata (Duch.) Britton, Cyperus alternifolius L., Cyperus rotundus L., Cnidoscolus aconitifolius (Miller) I. M. Johnst., Croton rigidus (Muell. Arg.) Britton, Curcas curcas (L.) Britton & Millsp., Jatropha gossypifolia L., Jatropa multifida L., Manihot esculenta Crantz, Ricinus communis L., Andira inermis (W. Wright) HBK, Crotalaria pallida Ait., Indigoferra spp., Sabinea florida (Vahl) DC., Casearia bicolor Urban, Andropogon leucostachyus HBK, Bambusa vulgaris Schrad. Ex Wendl., Cymbopogon citratus (DC.) Stapf, Saccharum officinarum L., Clusia rosea Jacq., Mammea americana L., Coleus amboinicus Lour., Leonotis nepetifolia (L.) Ait.f. in Ait., Melissa officinalis L., Mentha nemorosa Willd., Menthax piperita L. var.citrata (J.F. Ehrh.) Briq., Salvia splendens
ARTICLE IN PRESS P.A. Mele´ndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276
F. Sellow ex Roem & Schult, Persea americana Miller, Aloe vera (L.) Burm. f., Sansevieria hyacinthoides (L.) Druce, Lycopodium cernuum L., Malpighia emarginata Sesse´ & Moc, Malachra capitata (L.) L., Heterotrichum cymosum (Wendl.) Urban, Trichilia hirta L., Acasia macracantha Humb. & Bonpl., Albizia lebbeck (L.) Benth., Inga quaternata Poepp. & Endl, Artocarpus communis Forst., Cecropia peltata L., Musa ssp., Callistemon citrinus (Curtis) Stapf, Pimenta racemosa Millar, Psidium guajava L., Argemone mexicana L., Cajanus cajan (L.) Huth, Passiflora edulis Sims, Sesamum indicum L., Petiveria alliacea L., Peperomia pellucida L. HBK, Piper aduncum L., Piper marginatum Jacq., Pothomorphe peltata (L.) Miq., Plantago major L., Plumbago indica L., Coccoloba uvifera (L.) L., Adiantum cristatum L., Nephrolepis spp., Polypodium heterophyllum L., Portulaca oleracea L., Reynosia uncinata Urban, Prunus occidentalis Sw., Chiococca alba (L.) A. S. Hitchcock, Coffea arabiga L., Genipa americana L., Hamelia patens Jacq., Psychotria microdon (DC.) Urban, Ruta chalepensis L., Paullinia pinnata L., Chrysophyllum cainito L., Manilkara zapota (L.) v. Royen, Capsicum frutescens L., Cestrum diurnum L., Datura metel L., Datura mollis Saff., Lycopersicum esculentum Miller, Nicotiana tabacum L., Physalis angulata L., Solanum americanum var. nodiflorum (Jacq.) Edm., Solanum torvum Sw., Sterculia apetala (Jacq.) Karst., Typha domingensis Pers., Trema lamarckianum (R. & S.) Blume, Urera baccifera (L.) Wedd., Citharexylum caudatum L., Citharexylum fruticosum L., Lantana Table 3.
275
camara L., Lippia dulcis Trev., Lippia helleri Britton, Lippia stochadifolia (L.) HBK, Valerianoides jamaicense (L.) Kuntze, Cissus sicyoides L., Alpinia exaltata (L. f.) R. & S., Curcuma longa L., Zingiber officinale Rosc., and Zingiber zerumbet (L.) J. E. Smith. The initial screening helped us identify plants that will be used in future studies. We studied the inhibitory effects of these plants against 15 other bacteria; six Gram-negative, six Gram-positive, and three Gramvariable bacteria (Table 3). After evaluating the effects on the Gram-negative bacteria we found that extracts of ten plants inhibited P. fluorescens, nine inhibited A. faecalis, eight inhibited P.vulgaris, five inhibited E. cloacae and S. marcescens, and three E. aerogenes. Among the Gram-positive bacteria, ten plants inhibited M. luteus and A. globiformis, nine B. cereus, B. coagulans and B. subtilis, and six M. rodochrus. Overall, the plants that showed the highest antibacterial activity among the different bacterial strains as judged by the average diameter of their inhibitory zones were Citrus aurantifolia, Citrus aurantium, Punica granatum, Phyllanthus acidus and Tamarindus indica. Of these, C. aurantium was the only plant that inhibited all 17 (100%) bacteria tested. C. aurantifolia and P. acidus showed activity against 16 (94%) bacteria, P. granatum was effective against 15 (88%) of the bacteria tested, and T. indica against over 13 (81%) bacteria. Interestingly, in Puerto Rico it is a very common practice to use these plant extracts as remedies for colds and bacterial infections. These results provide evidence
Antibacterial properties of tropical plants from Puerto Rico against multiple bacteria
Scientific name
Family
Grama
+
+ +
+ +
+
v
v
v
Antimicrobial activityb Part testedc Afd Ea Ec Pf Pv Sm Ag Bc Bco Bs Ml Mr Mp Mro Ms Crescentia cujete Tamarindus indica Terminalia catappa Phyllanthus acidus Rosmarinus officinalis Lagerstroemia speciosa Urena lobata Clidemia hirta Punica granatum Psychotria nervosa Citrus aurantifolia Citrus aurantium Melicoccus bijugatus Petitia domingensis a
Bignoniaceae Caesalpiniaceae Combretaceae Euphorbiaceae Lamiaceae Lythraceae Malvaceae Melastomataceae Punicaceae Rubiaceae Rutaceae Rutaceae Sapindaceae Verbenaceae
L L L F L L L L F L F F L L
16 9 0 12 12 0 0 0 17 0 25 16 9 9
0 0 9 0 0 0 0 0 0 0 16 11 0 0
0 0 0 21 0 0 0 11 11 0 45 26 0 0
19 0 14 15 18 0 0 17 12 12 43 20 0 18
0 0 12 17 0 0 0 14 20 0 16 26 14 11
0 9 0 16 0 0 0 0 12 0 17 11 0 0
20 19 0 20 15 0 0 0 26 12 35 25 12 12
12 11 0 15 0 0 0 0 10 9 23 21 11 11
16 10 17 11 12 0 0 0 16 0 24 20 0 13
14 8 0 10 0 12 0 0 12 0 20 17 13 11
0 10 23 25 14 0 0 18 31 9 37 30 14 0
14 22 0 29 18 0 7 0 30 0 0 44 0 0
13 7 0 13 12 0 0 10 15 10 24 20 11 10
12 14 0 20 14 0 0 9 15 10 25 22 0 13
12 19 9 14 12 0 7 15 0 9 21 14 0 0
Gram test results; : negative, +: positive, v: variable. Measured by the diameter of zone of inhibition in mm. c Plant part tested: L., leaves; F., fruits. d Microorganisms: Af, A. faecalis; Ea, E. aerogenes; Ec, E. cloacae; Pf, P. fluorescens; Pv, P. vulgaris; Sm, S. marcescens; Ag, A. globiformis; Bc, B. cereus; Bco, B. coagulans; Bs, B. subtilis; Ml, M. luteus; Mr, M. roseus; Mp, M. phlei; Mro, M. rodochrus; Ms, M. smegmatis. b
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P.A. Mele´ndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276
for the presence of antimicrobial compounds in the crude methanolic extracts of these plants. This study validates and documents, in a systematic way, the antibacterial properties of a large number of plants used for many years by the people of Puerto Rico. It also provides valuable information for further phytochemical isolation and characterization studies of active compounds, necessary for the development of new drugs.
Acknowledgements This manuscript is dedicated to the memory of my first science mentor, Victor A. Capriles (‘‘El Doctor’’) who was an inspiration for many students in Puerto Rico. We acknowledge Irvin Toro and Isidro Negro´n from the Biology Department, Inter American University in San Germa´n, Puerto Rico for providing us with the bacterial stains. We are very grateful to Mr. Pedro J. Mele´ndez, Mrs. Carmen H. Lo´pez, and Mrs. Carmen M. Alvarez for their invaluable help collecting the plant species from their natural habitats. We also thank Prof. Miguel Vives, plant taxonomist, for his help authenticating every specimen.
References Anesini, C., Perez, C., 1993. Screening of plants used in Argentine folk medicine for antimicrobial activity. J. Ethnopharmacol. 39, 119–128. Bauer, A.W., Kirby, M.D.K., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by standard single disc diffusion method. Am. J. Clin. Pathol. 45, 493–496. Binutu, O.A., Lajubutu, B.A., 1994. Antimicrobial potentials of some plants species of the Bignoniaceae family. African J. Med. Med. Sci. 23, 269–273. Ca´ceres, A., Figueroa, L., Taracera, A.M., Samoya, B., 1993. Plants used in Guatemala for the treatment of respiratory diseases. 2. Evaluation of activity of 16 plants against Gram-positive bacteria. J. Ethnopharmacol. 39, 77–92. Chariandy, C.M., Seaforth, C.E., Phelps, R.H., Pollard, G.V., Khambay, B.P.S., 1999. Screening of medicinal plants from Trinidad and Tobago for antimicrobial and insecticidal properties. J. Ethnopharmacol. 64, 265–270. Cowan, M.M., 1999. Plants products as antimicrobial agents. Clin. Microbiol. Rev. 14, 564–584. Frame, A.D., Rı´ os-Olivares, E., De Jesu´s, L., Ortiz, D., Paga´n, J., Me´ndez, S., 1999. Puerto Rico Health Sci. J. 17, 243–252. Grosvenor, P.W., Supriono, A., Gray, D.O., 1995. Medicinal plants from the Riau Providence, Sumatra, Indonesia. Part
2: antibacterial and antifungal activity. J. Ethnopharmacol. 45, 97–111. Guerrero, R.O., Robledo, I., 1993. Endemic plants of Puerto Rico: brine shrimp lethality and antibacterial activity. Puerto Rico Health Sci. J. 12, 259–262. Herna´ndez, L., Mun˜oz, R.A., Miro´, G., Martı´ nez, M., SilviaParra, J., Chavez, P., 1984. Use of medicinal plants by ambulatory patients in Puerto Rico. Am. J. Hosp. Pharm. 41, 2060–2064. Liogier, H.A., 1990. Plantas Medicinales de Puerto Rico y del Caribe. Iberoamericana Ediciones, Inc., San Juan, Puerto Rico. Martı´ nez, M.J., Betancourt, J., Alonso-Gonza´lez, N., Jauregui, A., 1996. Screening of some Cuban medicinal plants for antimicrobial activity. J. Ethnopharmacol. 52, 171–174. Mazumder, U.K., Gupta, M., Manikandan, L., Bhattacharya, S., 2001. Antibacterial activity of Urena lobata root. Fitoterapia 72, 927–929. Morton, J.F., 1981. Atlas of Medicinal Plants of Middle America. Charles C. Thomas Publishers, Springfield, IL, USA. Navarro, V., Villareal, M.L., Rojas, G., Lozoya, X., 1996. Antimicrobial evaluation of some plants used in Mexican traditional medicine for the treatment of infectious diseases. J. Ethnopharmacol. 53, 143–147. Nun˜ez-Mele´ndez, E., 1989. Plantas Medicinales de Puerto Rico: folklore y fundamentos. Editorial de la Universidad de Puerto Rico, San Juan, Puerto Rico Prashanth, D., Asha, M.K., Amit, A., 2001. Antibacterial activity of Punica granatum. Fitoterapia 72, 171–173. Reagor, L., Gusman, L., McCoy, L., Carino, E., Heggers, J.P., 2002. The effectiveness of processed grapefruit-seed extract as an antibacterial agent: I. An in vitro agar assay. J. Altern. Comp. Med. 8, 325–332. Robineau, L., Soejarto, D.D., 1996. Tramil: a research project on the medicinal plant resources of the Caribbean. In: Balick, J., Elizabetsly, E., Laird, S.A. (Eds.), Medicinal Resources of the Tropical Forest. Columbia University Press, New York. Rojas-Herna´ndez, N.M., Lo´pez-Abraham, A.M., JimenezMisas, C.A., 1978a. Plantas con propiedades antimicrobianas: primera parte. Rev. Cubana Med.Trop. 30, 109–118. Rojas-Herna´ndez, N.M., Lo´pez-Abraham, A.M., JimenezMisas, C.A., 1978b. Plantas con propiedades antimicrobianas: Segunda parte. Rev. Cubana Med. Trop. 30, 119–127. Stickler, D.J., King, J.B., 1992. Bacterial sensitivity and resistance. Intrinsic resistance. In: Russell, A.D., Hugo, W.B., Ayliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilization. Blackwell Scientific Publications, Oxford. Takenaka, M., Watanabe, T., Sugahara, K., Harada, Y., Yoshida, S., Sugawara, F., 1997. New antimicrobial substances against Streptomyces scabies from Rosemary (Rosmarinus officinalis L.). Biosci. Biotech. Biochem. 61, 1440–1444.
Phytomedicine 13 (2006) 272–276 www.elsevier.de/phymed
Antibacterial properties of tropical plants from Puerto Rico P.A. Mele´ndeza,b,, V.A. Caprilesa,{ a
Department of Biology, Inter American University of Puerto Rico, San Germa´n Campus, San Germa´n, Puerto Rico Hewlett-Packard Caribe Ltd., ISB-Americas R&D, Aguadilla, Puerto Rico 00603
b
Abstract In an effort to document the antibacterial properties of plants commonly used by the people of Puerto Rico, we studied the effects of 172 plant species, utilizing the disc diffusion method, against Escherichia coli and Staphylococcus aureus. The methanolic extracts of 14 species showed antibacterial activities during this preliminary screen. These positive plant extracts were tested successively over 15 additional species. The results showed that extracts from Citrus aurantifolia (Rutaceae), Citrus aurantium (Rutaceae), Punica granatum (Punicaceae), Phyllanthus acidus (Euphorbiaceae) and Tamarindus indica (Caesalpiniaceae) possess strong in vitro antibacterial activity against the bacteria tested. r 2005 Elsevier GmbH. All rights reserved. Keywords: Plant antibacterials; Antibacterial plants from; Puerto Rico; Tropical plants; Antimicrobials
Introduction Exploring the healing power of plants is an ancient concept. For many centuries people have been trying to alleviate and treat diseases with different plant extracts and formulations (Cowan, 1999). It is estimated, however, that of the 250,000–500,000 species found on Earth, only 1% have been studied for their pharmaceutical potential. In Puerto Rico, the use of plants for medicinal purposes is a common practice, especially among adults living in the countryside. Many plants of our flora are known to possess antimicrobial properties and have been used by the local population in many instances to treat colds, coughs, bronchitis, diarrhea, respiratory infections, urinary disorders and skin lesions (Herna´ndez et al., 1984). In the past, other groups have published important ethnobotanical bibliographies that Corresponding author. Hewlett-Packard Caribe Ltd., ISB-Americas R&D, Aguadilla, 00603 Puerto Rico. Tel.: +1 787 819 7644; fax: +1 787 819 6270. E-mail address: [email protected] (P.A. Mele´ndez). { Deceased.
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.11.009
compiled and described the most frequently used species to treat infections (Liogier, 1990; Nun˜ez-Mele´ndez, 1989; Morton, 1981). However, scientific evidence confirming that these traditional remedies possess antibiotic properties is lacking. Over the years there have been several studies documenting the antibacterial properties of plants from other regions of the Caribbean and South America (Chariandy et al., 1999; Anesini and Perez, 1993; Martı´ nez et al., 1996; Ca´ceres et al., 1993; RojasHerna´ndez et al., 1978a; Rojas-Herna´ndez et al., 1978b). The Traditional Medicine in the Islands (TRAMIL) research project series started in 1982 in Haiti, and has documented a great deal of information concerning the medicinal use of plants in the Caribbean basin (Robineau and Soejarto, 1996). In Puerto Rico, Guerrero and Robledo (1993) studied the effects of some endemic plants; and more recently (Frame et al., 1999) examined the anti-Mycobacterium tuberculosis effects of 50 local plants. In Puerto Rico, as in many other countries, we are unfortunately at risk of losing the ancestral knowledge of the healing properties of plants. In an effort to
ARTICLE IN PRESS P.A. Mele´ndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276
corroborate, document, and expand the traditional use of medicinal plants with antibacterial properties in Puerto Rico, we collected and evaluated the antibacterial effects of a vast number of species against different types of Gram-positive and Gram-negative bacteria.
Materials and methods Plant collection Between March 1983 and March 1985, 172 species of tropical plants, from 73 different families, were collected from their natural habitats in the north-western and western regions of Puerto Rico, specifically in the municipalities of Isabela, Aguadilla, Quebradillas, San Germa´n and Sa´bana Grande. A specimen of each species was dry-mounted, photographed and preserved for future reference in the herbarium of the Biology Department of the Inter American University (BDIAU), San Germa´n, Puerto Rico. The identity of each plant was confirmed by Professor M. Vives, a plant taxonomist based in Quebradillas, Puerto Rico.
273
variable bacteria), Mycobacterium phlei, Mycobacterium rodochrus, and Mycobacterium smegmatis] were supplied by BD-IAU.
Antibacterial tests Antibacterial activity was tested using a modification of the disc diffusion method originally described by Bauer et al. (1966). A loop of bacteria from the agarslant stock was cultured in nutrient broth overnight and spread with a sterile cotton swap into petriplates containing 10 ml of Mueller Hinton Agar. Sterile filter paper discs (9 mm in diameter) impregnated with the plant extract were placed on the cultured plates and incubated at 25 or 37 1C, depending on the bacteria. The solvent without extracts served as negative control. Standard antibiotics of chloramphenicol 30 mg, streptomycin 10 mg, tetracycline 30 mg, erythromycin 15 mg, neomycin 30 mg, novobiocin 30 mg, kanamycin 30 mg, and penicillium G 10 units were used as positive controls. After 24 h of incubation, the diameter in mm of the inhibitory or clear zones around the disks was recorded.
Preparation of the crude extracts The extraction procedures were kept as simple as possible. Plant material (usually leaves and sometimes fruits) was oven-dried in a finger bowl at 65 1C for 2 days, then hand-ground and stored in plastic bags for further use. A 50-g sample of each specimen was extracted using 100 ml methanol in an electric blender left running for 15 min. The resultant suspension was filtered twice, first through cloth (50% cotton/50% polyester) and then through filter paper (Whatman No. 2). In some cases (for the densest extracts), the material was vacuum filtered using a Bu¨chner funnel. Each final filtrate was concentrated to dryness in a Rotovapor rotary-evaporation unit (Bu¨chi Labortechnik; Flawil, Switzerland) and then stored at 20 1C until further use. Before tests, concentrates were dissolved in 50 ml of double distilled water for a final concentration of 1000 mg/ml of dry plant powder. Aqueous dilutions were store in close containers at 4 1C for a maximum of 14 days.
Microorganisms The test organisms used [Escherichia coli, Enterobacter cloacae, Pseudomonas fluorescens, Proteus vulgaris, Alcaligenes faecalis, Serratia marcescens, Enterobacter aerogenes (Gram-negative bacteria), Staphylococcus aureus, Arthrobacter globiformis, Micrococcus luteus, Bacillus cereus, Bacillus subtilis (Gram-positive bacteria), Bacillus coagulans, Micrococcus roseus (Gram-
Results and discussion After evaluating the effects of 172 different plant extracts against Gram-negative E. coli and Grampositive S. aureus, we found 14 species with antibacterial properties (Table 1). Five extracts inhibited E. coli while 14 inhibited S. aureus. It has been documented (Grosvenor et al., 1995; Martı´ nez et al., 1996; Chariandy et al., 1999; Stickler and King, 1992) that S. aureus is one of the bacteria most susceptible to plant extracts. Citrus aurantifolia and Tamarindus indica were the most effective extracts against E. coli. The inhibitory zones reached 20 and 17-mm diameter, respectively. Two members of the Rutaceae family, Citrus aurantifolia (30mm diam.) and Citrus aurantium (27-mm diam.) were the most effective against S. aureus, followed by Punica granatum (22-mm diam.), Phyllanthus acidus (20-mm diam.) and Crescentia cujete (18-mm diam.). The inhibitory activities of the extracts were compared to known antibiotics, which served as positive controls (Table 2). Novobiocin and Penicillin G inhibited S. aureus and the rest inhibited both E. coli and S. aureus. In every case, the negative controls showed no antibacterial activity. The antibacterial properties of members of the Citrus genera have been documented by others (Anesini and Perez, 1993; Reagor et al., 2002). Likewise, there is evidence of the antibacterial properties of P. granatum (Prashanth et al., 2001; Anesini and Perez, 1993; Rojas-Herna´ndez et al., 1978b; Navarro et al., 1996), Crescentia cujete (Bignoniaceae) (Binutu and
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Table 1.
Antibacterial properties of tropical plants from Puerto Rico against Gram-negative and Gram-positive bacteria
Scientific name
Family
Crescentia cujete L. Tamarindus indica L. Terminalia catappa L. Phyllanthus acidus (L.) Skeels Rosmarinus officinalis L. Lagerstroemia speciosa (L.) Pers. Urena lobata L. ssp. Lobata Clidemia hirta (L.) D. Don Punica granatum L. Psychotria nervosa Sw. Citrus aurantifolia (Chrism.) Swingle Citrus aurantium L. Melicoccus bijugatus Jacq. Petitia domingensis Jacq. a
Bignoniaceae Caesalpiniaceae Combretaceae Euphorbiaceae Lamiaceae Lythraceae Malvaceae Melastomataceae Punicaceae Rubiaceae Rutaceae Rutaceae Sapindaceae Verbenaceae
Part testeda
L L L F L L L L F L F F L L
Antimicrobial activityb E. coli
S. aureus
0 17 0 11 0 0 0 0 12 0 20 12 0 0
18 13 13 20 15 11 9 12 22 12 30 27 17 11
Plant part tested: L., leaves; F., fruits. Measured by the diameter of zone of inhibition in mm.
b
Table 2. Antibiotic controls against Gram-negative and Gram-positive bacteria Antibiotics
Chloramphenicol Streptomycin Tetracycline Erythromycin Neomycin Novobiocin Kanamycin Penicillium G
Concentration
30 mg 10 mg 30 mg 15 mg 30 mg 30 mg 30 mg 10 units
Inhibitory zone (mm) E. coli
S. aureus
31 33 26 16 22 0 26 0
30 26 38 35 26 38 38 43
Lajubutu, 1994), Rosmarinus officinalis (Lamiaceae) (Takenaka et al., 1997), and Urena lobata (Malvaceae) (Mazumder et al., 2001). Not much is known, however, about the antibacterial compounds present in the leaves of T. indica. Plants showing no activity – The following 158 plants did not show any activity against E. coli and S. aureus: Odontonema strictum (Nees) Kuntz, Furcraea tuberosa (Miller) Ait. f, Achyranthes indica (L.) Miller, Amaranthus viridis L., Gomphrena dispersa Standl., Allium cepa L., Allium sativum L., Anethum graveolens L., Apium petroselinum L., Foeniculum vulgare Miller, Comocladia dodonaea (L.) Urban, Mangifera indica L., Spondias purpurea L., Annona muricata L., Annona reticulata L., Catharanthus roseus (L.) G. Don, Plumeria rubra L., Rauvolfia nitida Jacq., Caladium colocasia (L.) W. F. Wight, Epipremnun aureum (Linden & Andre´) Bunt, Asclepias curassavica L., Calotropis procera (Ait.)
Ait.f., Anthemis nobilis L., Bidens pilosa L., Calendula officinalis L., Cichorium intybus L., Eupatoriun odoratum L., Leptilon pusillum (Nutt.) Britton, Parthenuim hysterophorus L., Senecio aizoides Sch. Bip, Wedelia reticulata DC., Wedelia trilobata (L.) A. S. Hitchcock, Anredera leptostachys (moq.) v. Steenis, Spathodea campanulata Beauv., Bixa orellana L., Tournefortia hirsutissima L., Brassica hirta Moench, Brassica oleracea var. capitata L., Lepidium virginicum L., Ananas comosus (L.) Merill, Bromelia pinguin L., Nidularium billbergioides L. B. Sm., Bursera simaruba (L.) Sarg., Bauhinia monandra Kurz, Ditremexa occidentalis (L.) Britton & Rose, Hymenaea courbaril L., Isotoma longiflora (L.) Presl., Sambucus simpsonii Rehder, Carica papaya L., Chenopodium ambrosioides L, Commelina elegans HBK, Rhoeo spathacea (Sw.) Stearn, Ambrosia peruviana Willd., Cuscuta americana L., Ipomoea batatas (L.) Lam., Ipomoea carnea Jacq., Ipomoea pes-caprae (L.), Bryophylium pinnatum (Lam.) Oken, Kalanchoe¨ daigremontiana Hamet & Perrier, Momordica charantia L., Pepo moschata (Duch.) Britton, Cyperus alternifolius L., Cyperus rotundus L., Cnidoscolus aconitifolius (Miller) I. M. Johnst., Croton rigidus (Muell. Arg.) Britton, Curcas curcas (L.) Britton & Millsp., Jatropha gossypifolia L., Jatropa multifida L., Manihot esculenta Crantz, Ricinus communis L., Andira inermis (W. Wright) HBK, Crotalaria pallida Ait., Indigoferra spp., Sabinea florida (Vahl) DC., Casearia bicolor Urban, Andropogon leucostachyus HBK, Bambusa vulgaris Schrad. Ex Wendl., Cymbopogon citratus (DC.) Stapf, Saccharum officinarum L., Clusia rosea Jacq., Mammea americana L., Coleus amboinicus Lour., Leonotis nepetifolia (L.) Ait.f. in Ait., Melissa officinalis L., Mentha nemorosa Willd., Menthax piperita L. var.citrata (J.F. Ehrh.) Briq., Salvia splendens
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F. Sellow ex Roem & Schult, Persea americana Miller, Aloe vera (L.) Burm. f., Sansevieria hyacinthoides (L.) Druce, Lycopodium cernuum L., Malpighia emarginata Sesse´ & Moc, Malachra capitata (L.) L., Heterotrichum cymosum (Wendl.) Urban, Trichilia hirta L., Acasia macracantha Humb. & Bonpl., Albizia lebbeck (L.) Benth., Inga quaternata Poepp. & Endl, Artocarpus communis Forst., Cecropia peltata L., Musa ssp., Callistemon citrinus (Curtis) Stapf, Pimenta racemosa Millar, Psidium guajava L., Argemone mexicana L., Cajanus cajan (L.) Huth, Passiflora edulis Sims, Sesamum indicum L., Petiveria alliacea L., Peperomia pellucida L. HBK, Piper aduncum L., Piper marginatum Jacq., Pothomorphe peltata (L.) Miq., Plantago major L., Plumbago indica L., Coccoloba uvifera (L.) L., Adiantum cristatum L., Nephrolepis spp., Polypodium heterophyllum L., Portulaca oleracea L., Reynosia uncinata Urban, Prunus occidentalis Sw., Chiococca alba (L.) A. S. Hitchcock, Coffea arabiga L., Genipa americana L., Hamelia patens Jacq., Psychotria microdon (DC.) Urban, Ruta chalepensis L., Paullinia pinnata L., Chrysophyllum cainito L., Manilkara zapota (L.) v. Royen, Capsicum frutescens L., Cestrum diurnum L., Datura metel L., Datura mollis Saff., Lycopersicum esculentum Miller, Nicotiana tabacum L., Physalis angulata L., Solanum americanum var. nodiflorum (Jacq.) Edm., Solanum torvum Sw., Sterculia apetala (Jacq.) Karst., Typha domingensis Pers., Trema lamarckianum (R. & S.) Blume, Urera baccifera (L.) Wedd., Citharexylum caudatum L., Citharexylum fruticosum L., Lantana Table 3.
275
camara L., Lippia dulcis Trev., Lippia helleri Britton, Lippia stochadifolia (L.) HBK, Valerianoides jamaicense (L.) Kuntze, Cissus sicyoides L., Alpinia exaltata (L. f.) R. & S., Curcuma longa L., Zingiber officinale Rosc., and Zingiber zerumbet (L.) J. E. Smith. The initial screening helped us identify plants that will be used in future studies. We studied the inhibitory effects of these plants against 15 other bacteria; six Gram-negative, six Gram-positive, and three Gramvariable bacteria (Table 3). After evaluating the effects on the Gram-negative bacteria we found that extracts of ten plants inhibited P. fluorescens, nine inhibited A. faecalis, eight inhibited P.vulgaris, five inhibited E. cloacae and S. marcescens, and three E. aerogenes. Among the Gram-positive bacteria, ten plants inhibited M. luteus and A. globiformis, nine B. cereus, B. coagulans and B. subtilis, and six M. rodochrus. Overall, the plants that showed the highest antibacterial activity among the different bacterial strains as judged by the average diameter of their inhibitory zones were Citrus aurantifolia, Citrus aurantium, Punica granatum, Phyllanthus acidus and Tamarindus indica. Of these, C. aurantium was the only plant that inhibited all 17 (100%) bacteria tested. C. aurantifolia and P. acidus showed activity against 16 (94%) bacteria, P. granatum was effective against 15 (88%) of the bacteria tested, and T. indica against over 13 (81%) bacteria. Interestingly, in Puerto Rico it is a very common practice to use these plant extracts as remedies for colds and bacterial infections. These results provide evidence
Antibacterial properties of tropical plants from Puerto Rico against multiple bacteria
Scientific name
Family
Grama
+
+ +
+ +
+
v
v
v
Antimicrobial activityb Part testedc Afd Ea Ec Pf Pv Sm Ag Bc Bco Bs Ml Mr Mp Mro Ms Crescentia cujete Tamarindus indica Terminalia catappa Phyllanthus acidus Rosmarinus officinalis Lagerstroemia speciosa Urena lobata Clidemia hirta Punica granatum Psychotria nervosa Citrus aurantifolia Citrus aurantium Melicoccus bijugatus Petitia domingensis a
Bignoniaceae Caesalpiniaceae Combretaceae Euphorbiaceae Lamiaceae Lythraceae Malvaceae Melastomataceae Punicaceae Rubiaceae Rutaceae Rutaceae Sapindaceae Verbenaceae
L L L F L L L L F L F F L L
16 9 0 12 12 0 0 0 17 0 25 16 9 9
0 0 9 0 0 0 0 0 0 0 16 11 0 0
0 0 0 21 0 0 0 11 11 0 45 26 0 0
19 0 14 15 18 0 0 17 12 12 43 20 0 18
0 0 12 17 0 0 0 14 20 0 16 26 14 11
0 9 0 16 0 0 0 0 12 0 17 11 0 0
20 19 0 20 15 0 0 0 26 12 35 25 12 12
12 11 0 15 0 0 0 0 10 9 23 21 11 11
16 10 17 11 12 0 0 0 16 0 24 20 0 13
14 8 0 10 0 12 0 0 12 0 20 17 13 11
0 10 23 25 14 0 0 18 31 9 37 30 14 0
14 22 0 29 18 0 7 0 30 0 0 44 0 0
13 7 0 13 12 0 0 10 15 10 24 20 11 10
12 14 0 20 14 0 0 9 15 10 25 22 0 13
12 19 9 14 12 0 7 15 0 9 21 14 0 0
Gram test results; : negative, +: positive, v: variable. Measured by the diameter of zone of inhibition in mm. c Plant part tested: L., leaves; F., fruits. d Microorganisms: Af, A. faecalis; Ea, E. aerogenes; Ec, E. cloacae; Pf, P. fluorescens; Pv, P. vulgaris; Sm, S. marcescens; Ag, A. globiformis; Bc, B. cereus; Bco, B. coagulans; Bs, B. subtilis; Ml, M. luteus; Mr, M. roseus; Mp, M. phlei; Mro, M. rodochrus; Ms, M. smegmatis. b
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for the presence of antimicrobial compounds in the crude methanolic extracts of these plants. This study validates and documents, in a systematic way, the antibacterial properties of a large number of plants used for many years by the people of Puerto Rico. It also provides valuable information for further phytochemical isolation and characterization studies of active compounds, necessary for the development of new drugs.
Acknowledgements This manuscript is dedicated to the memory of my first science mentor, Victor A. Capriles (‘‘El Doctor’’) who was an inspiration for many students in Puerto Rico. We acknowledge Irvin Toro and Isidro Negro´n from the Biology Department, Inter American University in San Germa´n, Puerto Rico for providing us with the bacterial stains. We are very grateful to Mr. Pedro J. Mele´ndez, Mrs. Carmen H. Lo´pez, and Mrs. Carmen M. Alvarez for their invaluable help collecting the plant species from their natural habitats. We also thank Prof. Miguel Vives, plant taxonomist, for his help authenticating every specimen.
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