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Antibacterial properties of tropical plants from Puerto Rico.


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.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Plant antibacterials; Antibacterial plants from; Puerto Rico; Tropical plants; Antimicrobials


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 (Hernandez et al., 1984). In the past, other groups have published important ethnobotanical bibliographies that compiled and described the most frequently used species to treat infections (Liogier, 1990; Nunez-Melendez, 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; Martinez et al., 1996; Caceres et al., 1993; Rojas-Hernandez et al., 1978a; Rojas-Hernandez 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 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 German and Sabana 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 (BD-IAU), San German, Puerto Rico. The identity of each plant was confirmed by Professor M. Vives, a plant taxonomist based in Quebradillas, Puerto Rico.

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 [degrees]C 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 Buchner funnel. Each final filtrate was concentrated to dryness in a Rotovapor rotary-evaporation unit (Buchi Labortechnik; Flawil, Switzerland) and then stored at 20 [degrees]C 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 [degrees]C for a maximum of 14 days.


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-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 agar-slant 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 [degrees]C, depending on the bacteria. The solvent without extracts served as negative control. Standard antibiotics of chloramphenicol 30 [micro]g, streptomycin 10 [micro]g, tetracycline 30 [micro]g, erythromycin 15 [micro]g, neomycin 30 [micro]g, novobiocin 30 [micro]g, kanamycin 30 [micro]g, 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.

Results and discussion

After evaluating the effects of 172 different plant extracts against Gram-negative E. coli and Gram-positive 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; Martinez 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 (30-mm 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-Hernandez et al., 1978b; Navarro et al., 1996), Crescentia cujete (Bignoniaceae) (Binutu and 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, Amar-anthus 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 hyster-ophorus 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 Ha-met & 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 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 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 Gram-variable 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 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.


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 Negron from the Biology Department, Inter American University in San German, Puerto Rico for providing us with the bacterial stains. We are very grateful to Mr. Pedro J. Melendez, Mrs. Carmen H. Lopez, 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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P.A. Melendez (a,b,*), V.A. Capriles (a,*)

(a) Department of Biology, Inter American University of Puerto Rico, San German Campus, San German, Puerto Rico

(b) Hewlett-Packard Caribe Ltd., ISB-Americas R & D, Aguadilla, Puerto Rico 00603

*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: (P.A. Melendez).

Table 1. Antibacterial properties of tropical plants from Puerto Rico
against Gram-negative and Gram-positive bacteria

Scientific name Family Part tested (a)

Crescentia cujete L. Bignoniaceae L
Tamarindus indica L. Caesalpiniaceae L
Terminalia catappa L. Combretaceae L
Phyllanthus acidus (L.) Skeels Euphorbiaceae F
Rosmarinus officinalis L. Lamiaceae L
Lagerstroemia speciosa (L.) Pers. Lythraceae L
Urena lobata L. ssp. Lobata Malvaceae L
Clidemia hirta (L.) D. Don Melastomataceae L
Punica granatum L. Punicaceae F
Psychotria nervosa Sw. Rubiaceae L
Citrus aurantifolia (Chrism.) Swingle Rutaceae F
Citrus aurantium L. Rutaceae F
Melicoccus bijugatus Jacq. Sapindaceae L
Petitia domingensis Jacq. Verbenaceae L

 Antimicrobial activity (b)
Scientific name E. coli S. aureus

Crescentia cujete L. 0 18
Tamarindus indica L. 17 13
Terminalia catappa L. 0 13
Phyllanthus acidus (L.) Skeels 11 20
Rosmarinus officinalis L. 0 15
Lagerstroemia speciosa (L.) Pers. 0 11
Urena lobata L. ssp. Lobata 0 9
Clidemia hirta (L.) D. Don 0 12
Punica granatum L. 12 22
Psychotria nervosa Sw. 0 12
Citrus aurantifolia (Chrism.) Swingle 20 30
Citrus aurantium L. 12 27
Melicoccus bijugatus Jacq. 0 17
Petitia domingensis Jacq. 0 11

(a) Plant part tested: L., leaves; F., fruits.
(b) Measured by the diameter of zone of inhibition in mm.

Table 2. Antibiotic controls against Gram-negative and Gram-positive

 Inhibitory zone (mm)
Antibiotics Concentration E. coli S. aureus

Chloramphenicol 30 [micro]g 31 30
Streptomycin 10 [micro]g 33 26
Tetracycline 30 [micro]g 26 38
Erythromycin 15 [micro]g 16 35
Neomycin 30 [micro]g 22 26
Novobiocin 30 [micro]g 0 38
Kanamycin 30 [micro]g 26 38
Penicillium G 10 units 0 43

Table 3. Antibacterial properties of tropical plants from Puerto Rico
against multiple bacteria

 Gram (a) - - - -
 activity (b)
Scientific name Family tested (c) Af (d) Ea Ec Pf

Crescentia cujete Bignoniaceae L 16 0 0 19
Tamarindus indica Caesalpiniaceae L 9 0 0 0
Terminalia catappa Combretaceae L 0 9 0 14
Phyllanthus acidus Euphorbiaceae F 12 0 21 15
Rosmarinus officinalis Lamiaceae L 12 0 0 18
Lagerstroemia speciosa Lythraceae L 0 0 0 0
Urena lobata Malvaceae L 0 0 0 0
Clidemia hirta Melastomataceae L 0 0 11 17
Punica granatum Punicaceae F 17 0 11 12
Psychotria nervosa Rubiaceae L 0 0 0 12
Citrus aurantifolia Rutaceae F 25 16 45 43
Citrus aurantium Rutaceae F 16 11 26 20
Melicoccus bijugatus Sapindaceae L 9 0 0 0
Petitia domingensis Verbenaceae L 9 0 0 18

 - - + + + + + + v v v
 activity (b)
Scientific name Pv Sm Ag Bc Bco Bs Ml Mr Mp Mro Ms

Crescentia cujete 0 0 20 12 16 14 0 14 13 12 12
Tamarindus indica 0 9 19 11 10 8 10 22 7 14 19
Terminalia catappa 12 0 0 0 17 0 23 0 0 0 9
Phyllanthus acidus 17 16 20 15 11 10 25 29 13 20 14
Rosmarinus officinalis 0 0 15 0 12 0 14 18 12 14 12
Lagerstroemia speciosa 0 0 0 0 0 12 0 0 0 0 0
Urena lobata 0 0 0 0 0 0 0 7 0 0 7
Clidemia hirta 14 0 0 0 0 0 18 0 10 9 15
Punica granatum 20 12 26 10 16 12 31 30 15 15 0
Psychotria nervosa 0 0 12 9 0 0 9 0 10 10 9
Citrus aurantifolia 16 17 35 23 24 20 37 0 24 25 21
Citrus aurantium 26 11 25 21 20 17 30 44 20 22 14
Melicoccus bijugatus 14 0 12 11 0 13 14 0 11 0 0
Petitia domingensis 11 0 12 11 13 11 0 0 10 13 0

(a) Gram test results; -: negative, +: positive, v: variable.
(b) 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.
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Author:Melendez, P.A.; Capriles, V.A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2006
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