ARTICLE IN PRESS

Phytomedicine 13 (2006) 272–276

www.elsevier.de/phymed

Antibacterial properties of tropical plants from Puerto Rico

P.A. Meléndeza,b,, V.A. Caprilesa,{
a
Department of Biology, Inter American University of Puerto Rico, San Germán Campus, San Germán, Puerto Rico
b
Hewlett-Packard Caribe Ltd., ISB-Americas R&D, Aguadilla, Puerto Rico 00603

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 (Euphorbia-
ceae) 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 compiled and described the most frequently used species

to treat infections (Liogier, 1990; Nuñez-Meléndez,
Exploring the healing power of plants is an ancient 1989; Morton, 1981). However, scientific evidence
concept. For many centuries people have been trying to confirming that these traditional remedies possess
alleviate and treat diseases with different plant extracts antibiotic properties is lacking.
and formulations (Cowan, 1999). It is estimated, Over the years there have been several studies
however, that of the 250,000–500,000 species found on documenting the antibacterial properties of plants from
Earth, only 1% have been studied for their pharmaceu- other regions of the Caribbean and South America
tical potential. In Puerto Rico, the use of plants for (Chariandy et al., 1999; Anesini and Perez, 1993;
medicinal purposes is a common practice, especially Martı́nez et al., 1996; Cáceres et al., 1993; Rojas-
among adults living in the countryside. Many plants of Hernández et al., 1978a; Rojas-Hernández et al., 1978b).
our flora are known to possess antimicrobial properties The Traditional Medicine in the Islands (TRAMIL)
and have been used by the local population in many research project series started in 1982 in Haiti, and has
instances to treat colds, coughs, bronchitis, diarrhea, documented a great deal of information concerning the
respiratory infections, urinary disorders and skin lesions medicinal use of plants in the Caribbean basin
(Hernández et al., 1984). In the past, other groups have (Robineau and Soejarto, 1996). In Puerto Rico,
published important ethnobotanical bibliographies that Guerrero and Robledo (1993) studied the effects of
some endemic plants; and more recently (Frame et al.,
Corresponding author. Hewlett-Packard Caribe Ltd., ISB-Amer-
1999) examined the anti-Mycobacterium tuberculosis
effects of 50 local plants.
icas R&D, Aguadilla, 00603 Puerto Rico. Tel.: +1 787 819 7644; fax:
+1 787 819 6270.
In Puerto Rico, as in many other countries, we are
E-mail address: [email protected] (P.A. Meléndez). unfortunately at risk of losing the ancestral knowledge
{
Deceased. of the healing properties of plants. In an effort to

0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2004.11.009
 ARTICLE IN PRESS
P.A. Meléndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276 273

corroborate, document, and expand the traditional use variable bacteria), Mycobacterium phlei, Mycobacterium
of medicinal plants with antibacterial properties in rodochrus, and Mycobacterium smegmatis] were supplied
Puerto Rico, we collected and evaluated the antibacter- by BD-IAU.
ial effects of a vast number of species against different
types of Gram-positive and Gram-negative bacteria.
Antibacterial tests

Antibacterial activity was tested using a modification

Materials and methods of the disc diffusion method originally described by
Bauer et al. (1966). A loop of bacteria from the agar-
Plant collection slant stock was cultured in nutrient broth overnight and
spread with a sterile cotton swap into petriplates
Between March 1983 and March 1985, 172 species of containing 10 ml of Mueller Hinton Agar. Sterile filter
tropical plants, from 73 different families, were collected paper discs (9 mm in diameter) impregnated with the
from their natural habitats in the north-western and plant extract were placed on the cultured plates and
western regions of Puerto Rico, specifically in the incubated at 25 or 37 1C, depending on the bacteria. The
municipalities of Isabela, Aguadilla, Quebradillas, San solvent without extracts served as negative control.
Germán and Sábana Grande. A specimen of each Standard antibiotics of chloramphenicol 30 mg, strepto-
species was dry-mounted, photographed and preserved mycin 10 mg, tetracycline 30 mg, erythromycin 15 mg,
for future reference in the herbarium of the Biology neomycin 30 mg, novobiocin 30 mg, kanamycin 30 mg,
Department of the Inter American University (BD- and penicillium G 10 units were used as positive
IAU), San Germán, Puerto Rico. The identity of each controls. After 24 h of incubation, the diameter in mm
plant was confirmed by Professor M. Vives, a plant of the inhibitory or clear zones around the disks was
taxonomist based in Quebradillas, Puerto Rico. recorded.

Preparation of the crude extracts

Results and discussion
The extraction procedures were kept as simple as
possible. Plant material (usually leaves and sometimes After evaluating the effects of 172 different plant
fruits) was oven-dried in a finger bowl at 65 1C for 2 extracts against Gram-negative E. coli and Gram-
days, then hand-ground and stored in plastic bags for positive S. aureus, we found 14 species with antibacterial
further use. A 50-g sample of each specimen was properties (Table 1). Five extracts inhibited E. coli while
extracted using 100 ml methanol in an electric blender 14 inhibited S. aureus. It has been documented
left running for 15 min. The resultant suspension was (Grosvenor et al., 1995; Martı́nez et al., 1996; Chariandy
filtered twice, first through cloth (50% cotton/50% et al., 1999; Stickler and King, 1992) that S. aureus is
polyester) and then through filter paper (Whatman No. one of the bacteria most susceptible to plant extracts.
2). In some cases (for the densest extracts), the material Citrus aurantifolia and Tamarindus indica were the most
was vacuum filtered using a Büchner funnel. Each final effective extracts against E. coli. The inhibitory zones
filtrate was concentrated to dryness in a Rotovapor reached 20 and 17-mm diameter, respectively. Two
rotary-evaporation unit (Büchi Labortechnik; Flawil, members of the Rutaceae family, Citrus aurantifolia (30-
Switzerland) and then stored at 20 1C until further use. mm diam.) and Citrus aurantium (27-mm diam.) were
Before tests, concentrates were dissolved in 50 ml of the most effective against S. aureus, followed by Punica
double distilled water for a final concentration of granatum (22-mm diam.), Phyllanthus acidus (20-mm
1000 mg/ml of dry plant powder. Aqueous dilutions diam.) and Crescentia cujete (18-mm diam.). The
were store in close containers at 4 1C for a maximum of inhibitory activities of the extracts were compared to
14 days. known antibiotics, which served as positive controls
(Table 2). Novobiocin and Penicillin G inhibited S.
Microorganisms aureus and the rest inhibited both E. coli and S. aureus.
In every case, the negative controls showed no
The test organisms used [Escherichia coli, Enterobac- antibacterial activity. The antibacterial properties of
ter cloacae, Pseudomonas fluorescens, Proteus vulgaris, members of the Citrus genera have been documented by
Alcaligenes faecalis, Serratia marcescens, Enterobacter others (Anesini and Perez, 1993; Reagor et al., 2002).
aerogenes (Gram-negative bacteria), Staphylococcus Likewise, there is evidence of the antibacterial properties
aureus, Arthrobacter globiformis, Micrococcus luteus, of P. granatum (Prashanth et al., 2001; Anesini and
Bacillus cereus, Bacillus subtilis (Gram-positive bacter- Perez, 1993; Rojas-Hernández et al., 1978b; Navarro et
ia), Bacillus coagulans, Micrococcus roseus (Gram- al., 1996), Crescentia cujete (Bignoniaceae) (Binutu and
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274 P.A. Meléndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276

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

Scientific name Family Part testeda Antimicrobial activityb

E. coli S. aureus

Crescentia cujete L. Bignoniaceae L 0 18

Tamarindus indica L. Caesalpiniaceae L 17 13
Terminalia catappa L. Combretaceae L 0 13
Phyllanthus acidus (L.) Skeels Euphorbiaceae F 11 20
Rosmarinus officinalis L. Lamiaceae L 0 15
Lagerstroemia speciosa (L.) Pers. Lythraceae L 0 11
Urena lobata L. ssp. Lobata Malvaceae L 0 9
Clidemia hirta (L.) D. Don Melastomataceae L 0 12
Punica granatum L. Punicaceae F 12 22
Psychotria nervosa Sw. Rubiaceae L 0 12
Citrus aurantifolia (Chrism.) Swingle Rutaceae F 20 30
Citrus aurantium L. Rutaceae F 12 27
Melicoccus bijugatus Jacq. Sapindaceae L 0 17
Petitia domingensis Jacq. Verbenaceae L 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 Ait.f., Anthemis nobilis L., Bidens pilosa L., Calendula
Gram-positive bacteria officinalis L., Cichorium intybus L., Eupatoriun odoratum
L., Leptilon pusillum (Nutt.) Britton, Parthenuim hyster-
Antibiotics Concentration Inhibitory zone (mm)
ophorus L., Senecio aizoides Sch. Bip, Wedelia reticulata
E. coli S. aureus DC., Wedelia trilobata (L.) A. S. Hitchcock, Anredera
leptostachys (moq.) v. Steenis, Spathodea campanulata
Chloramphenicol 30 mg 31 30 Beauv., Bixa orellana L., Tournefortia hirsutissima L.,
Streptomycin 10 mg 33 26 Brassica hirta Moench, Brassica oleracea var. capitata
Tetracycline 30 mg 26 38
L., Lepidium virginicum L., Ananas comosus (L.) Merill,
Erythromycin 15 mg 16 35
Neomycin 30 mg 22 26 Bromelia pinguin L., Nidularium billbergioides L. B. Sm.,
Novobiocin 30 mg 0 38 Bursera simaruba (L.) Sarg., Bauhinia monandra Kurz,
Kanamycin 30 mg 26 38 Ditremexa occidentalis (L.) Britton & Rose, Hymenaea
Penicillium G 10 units 0 43 courbaril L., Isotoma longiflora (L.) Presl., Sambucus
simpsonii Rehder, Carica papaya L., Chenopodium
ambrosioides L, Commelina elegans HBK, Rhoeo spatha-
cea (Sw.) Stearn, Ambrosia peruviana Willd., Cuscuta
Lajubutu, 1994), Rosmarinus officinalis (Lamiaceae) americana L., Ipomoea batatas (L.) Lam., Ipomoea
(Takenaka et al., 1997), and Urena lobata (Malvaceae) carnea Jacq., Ipomoea pes-caprae (L.), Bryophylium
(Mazumder et al., 2001). Not much is known, however, pinnatum (Lam.) Oken, Kalanchoë daigremontiana Ha-
about the antibacterial compounds present in the leaves met & Perrier, Momordica charantia L., Pepo moschata
of T. indica. (Duch.) Britton, Cyperus alternifolius L., Cyperus rotun-
Plants showing no activity – The following 158 plants dus L., Cnidoscolus aconitifolius (Miller) I. M. Johnst.,
did not show any activity against E. coli and S. aureus: Croton rigidus (Muell. Arg.) Britton, Curcas curcas (L.)
Odontonema strictum (Nees) Kuntz, Furcraea tuberosa Britton & Millsp., Jatropha gossypifolia L., Jatropa
(Miller) Ait. f, Achyranthes indica (L.) Miller, Amar- multifida L., Manihot esculenta Crantz, Ricinus commu-
anthus viridis L., Gomphrena dispersa Standl., Allium nis L., Andira inermis (W. Wright) HBK, Crotalaria
cepa L., Allium sativum L., Anethum graveolens L., pallida Ait., Indigoferra spp., Sabinea florida (Vahl) DC.,
Apium petroselinum L., Foeniculum vulgare Miller, Casearia bicolor Urban, Andropogon leucostachyus
Comocladia dodonaea (L.) Urban, Mangifera indica HBK, Bambusa vulgaris Schrad. Ex Wendl., Cymbopo-
L., Spondias purpurea L., Annona muricata L., Annona gon citratus (DC.) Stapf, Saccharum officinarum
reticulata L., Catharanthus roseus (L.) G. Don, Plumeria L., Clusia rosea Jacq., Mammea americana L., Coleus
rubra L., Rauvolfia nitida Jacq., Caladium colocasia (L.) amboinicus Lour., Leonotis nepetifolia (L.) Ait.f. in Ait.,
W. F. Wight, Epipremnun aureum (Linden & André) Melissa officinalis L., Mentha nemorosa Willd., Menthax
Bunt, Asclepias curassavica L., Calotropis procera (Ait.) piperita L. var.citrata (J.F. Ehrh.) Briq., Salvia splendens
 ARTICLE IN PRESS
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F. Sellow ex Roem & Schult, Persea americana Miller, camara L., Lippia dulcis Trev., Lippia helleri Britton,
Aloe vera (L.) Burm. f., Sansevieria hyacinthoides (L.) Lippia stochadifolia (L.) HBK, Valerianoides jamaicense
Druce, Lycopodium cernuum L., Malpighia emarginata (L.) Kuntze, Cissus sicyoides L., Alpinia exaltata (L. f.)
Sessé & Moc, Malachra capitata (L.) L., Heterotrichum R. & S., Curcuma longa L., Zingiber officinale Rosc.,
cymosum (Wendl.) Urban, Trichilia hirta L., Acasia and Zingiber zerumbet (L.) J. E. Smith.
macracantha Humb. & Bonpl., Albizia lebbeck (L.) The initial screening helped us identify plants that will
Benth., Inga quaternata Poepp. & Endl, Artocarpus be used in future studies. We studied the inhibitory
communis Forst., Cecropia peltata L., Musa ssp., effects of these plants against 15 other bacteria; six
Callistemon citrinus (Curtis) Stapf, Pimenta racemosa Gram-negative, six Gram-positive, and three Gram-
Millar, Psidium guajava L., Argemone mexicana L., variable bacteria (Table 3). After evaluating the effects
Cajanus cajan (L.) Huth, Passiflora edulis Sims, Sesa- on the Gram-negative bacteria we found that extracts of
mum indicum L., Petiveria alliacea L., Peperomia ten plants inhibited P. fluorescens, nine inhibited
pellucida L. HBK, Piper aduncum L., Piper marginatum A. faecalis, eight inhibited P.vulgaris, five inhibited
Jacq., Pothomorphe peltata (L.) Miq., Plantago major L., E. cloacae and S. marcescens, and three E. aerogenes.
Plumbago indica L., Coccoloba uvifera (L.) L., Adiantum Among the Gram-positive bacteria, ten plants inhibited
cristatum L., Nephrolepis spp., Polypodium heterophyl- M. luteus and A. globiformis, nine B. cereus, B.
lum L., Portulaca oleracea L., Reynosia uncinata Urban, coagulans and B. subtilis, and six M. rodochrus.
Prunus occidentalis Sw., Chiococca alba (L.) A. S. Overall, the plants that showed the highest antibac-
Hitchcock, Coffea arabiga L., Genipa americana L., terial activity among the different bacterial strains as
Hamelia patens Jacq., Psychotria microdon (DC.) judged by the average diameter of their inhibitory zones
Urban, Ruta chalepensis L., Paullinia pinnata L., were Citrus aurantifolia, Citrus aurantium, Punica
Chrysophyllum cainito L., Manilkara zapota (L.) v. granatum, Phyllanthus acidus and Tamarindus indica.
Royen, Capsicum frutescens L., Cestrum diurnum L., Of these, C. aurantium was the only plant that inhibited
Datura metel L., Datura mollis Saff., Lycopersicum all 17 (100%) bacteria tested. C. aurantifolia and P.
esculentum Miller, Nicotiana tabacum L., Physalis acidus showed activity against 16 (94%) bacteria, P.
angulata L., Solanum americanum var. nodiflorum (Jacq.) granatum was effective against 15 (88%) of the bacteria
Edm., Solanum torvum Sw., Sterculia apetala (Jacq.) tested, and T. indica against over 13 (81%) bacteria.
Karst., Typha domingensis Pers., Trema lamarckianum Interestingly, in Puerto Rico it is a very common
(R. & S.) Blume, Urera baccifera (L.) Wedd., Citharex- practice to use these plant extracts as remedies for colds
ylum caudatum L., Citharexylum fruticosum L., Lantana and bacterial infections. These results provide evidence

Table 3. 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 Bignoniaceae L 16 0 0 19 0 0 20 12 16 14 0 14 13 12 12

Tamarindus indica Caesalpiniaceae L 9 0 0 0 0 9 19 11 10 8 10 22 7 14 19
Terminalia catappa Combretaceae L 0 9 0 14 12 0 0 0 17 0 23 0 0 0 9
Phyllanthus acidus Euphorbiaceae F 12 0 21 15 17 16 20 15 11 10 25 29 13 20 14
Rosmarinus officinalis Lamiaceae L 12 0 0 18 0 0 15 0 12 0 14 18 12 14 12
Lagerstroemia speciosa Lythraceae L 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0
Urena lobata Malvaceae L 0 0 0 0 0 0 0 0 0 0 0 7 0 0 7
Clidemia hirta Melastomataceae L 0 0 11 17 14 0 0 0 0 0 18 0 10 9 15
Punica granatum Punicaceae F 17 0 11 12 20 12 26 10 16 12 31 30 15 15 0
Psychotria nervosa Rubiaceae L 0 0 0 12 0 0 12 9 0 0 9 0 10 10 9
Citrus aurantifolia Rutaceae F 25 16 45 43 16 17 35 23 24 20 37 0 24 25 21
Citrus aurantium Rutaceae F 16 11 26 20 26 11 25 21 20 17 30 44 20 22 14
Melicoccus bijugatus Sapindaceae L 9 0 0 0 14 0 12 11 0 13 14 0 11 0 0
Petitia domingensis Verbenaceae L 9 0 0 18 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. smegmatis.
 ARTICLE IN PRESS
276 P.A. Meléndez, V.A. Capriles / Phytomedicine 13 (2006) 272–276

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