Journal of Ethnopharmacology 64 (1999) 265 270

Short communication

Screening of medicinal plants from Trinidad and Tobago for antimicrobial and insecticidal properties
C.M. Chariandy a, C.E. Seaforth a,*, R.H. Phelps a, G.V. Pollard a, B.P.S. Khambay b
a

Departments of Life Sciences and Chemistry, Faculty of Agriculture and Natural Sciences, The Uni6ersity of the West Indies, St. Augustine, Trinidad, Trinidad and Tobago b IARC -Rothamsted, Harpenden, Herts., AL5 2JQ, England, UK Received 9 February 1998; received in revised form 21 June 1998; accepted 21 July 1998

Abstract Antibacterial activity in 51 extracts from 29 plant species currently used in traditional medicine in Trinidad and the neighbouring Caribbean islands was tested for by the agar dilution streak method using six bacteria: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis and Enterococ cus faecalis. The extracts from eight of the plants tested showed signicant activity against one or more micro-organisms and the most susceptible bacterium was Staphylococcus aureus. In the bioassays for toxicity towards the Aedes aegypti mosquito the most effective plant extracts were from Justicia pectoralis, Manihot utilissima and Stachytarpheta jamaicensis. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Medicinal plants; Antibacterial activity; Trinidad; Insecticidal activity; Aedes aegypti

1. Introduction There are hundreds of medicinal plants still used in Trinidad and Tobago and in the other Caribbean islands, to treat colds, coughs and respiratory ailments associated with microbial infections (Morton, 1981; Seaforth et al., 1985;
* Corresponding author.

Robineau and Soejarto, 1996). There is increasing popular and scientic interest in the higher plants as potential sources of agents against microbial and insect pests (Mitscher et al., 1987; Grainge and Ahmed, 1988; Recio et al., 1989). Using the methods of Meyer et al. (1982), we pre-screened the extracts from a number of Caribbean medicinal plants to indicate general bio-activity through their toxicity to the brine shrimp Artemia salina (Chariandy, 1997). In this

0378-8741/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-8741(98)00130-5

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paper, the extracts from 29 plants are evaluated for antibacterial activity; and the extracts from eight of these plants also have been tested for toxicity towards Aedes aegypti, one of the mosquito species responsible for the transmission of dengue haemorrhagic fever in the Caribbean region. The goal of these tests is to choose the most promising plants for further phytochemical evaluation.

Trinidad. They were grown on a medium consisting of nutrient broth (CMI 1) solidied with agar (CMI 39) (Oxoid, Basingstoke, UK) and incubated at 37C for 24 h.

2.4. Antibacterial assays

The antibacterial activity of each extract was determined by the agar dilution streak method (Mitscher et al., 1972). Each dried plant extract was dissolved in 0.2 ml methanol and then incorporated in the agar medium to obtain a concentration of 1000 v g/ml in each plate. For inoculation of the micro-organisms, small strokes of the cultures were applied to the surface of the agar medium. Each plant extract was tested in triplicate. The negative control was methanol (2% in the agar medium). The plates were incubated at 37C for 24 h after which the results were rated by the unaided eye as: 1. Strong inhibition (no bacterial growth) 2. Partial inhibition (less growth than normal) 3. No inhibition (full growth) The results are shown in Table 1.

2. Materials and methods

2.1. Plant material

The plants were collected by C. Chariandy in the eastern rural areas of Trinidad between March 1992 and March 1994 and identied by Staff at the National Herbarium housed at the University of the West Indies, St. Augustine in Trinidad. Voucher specimens are deposited in the abovenamed herbarium. Plant parts were air-dried at ambient temperature and afterwards powdered in preparation for analysis.

2.2. Extraction
Extraction of each plant part material was done using an ethanol/dichloromethane (1:1) solvent mixture. After ltration the solution was evaporated in vacuo and the non-volatile material was progressively extracted with petroleum ether, then ethyl acetate. The separated fractions were evaporated in vacuo, yielding two dried extracts one of petroleum ether solubles (P) and the other of ethyl acetate solubles (E) from each selected plant part. These dried plant extracts were used for the biological assays.

2.5. Mosquitoes and the lar6icidal test

The larvae of Aedes aegypti mosquitoes were bred in chlorine-free tap water and fed a 5% yeast suspension. The test solutions used were a negative control of 2% methanol in water, or the plant extracts at levels of 0.05, 0.20 and 0.50 mg/ml in water containing 2% methanol. A slightly modied version of the standard WHO test (Busvine, 1971) was employed in which a single fourth-stage larva was put into each of ten vials containing 5 ml of the test solution. Half of the vials were covered and left at 27C for as many days as needed for 100% mortality to be observed in the larvae. The ve remaining vials were covered and left at 27C for 1 h only, after which the larvae were returned to vials containing clean, stale tap water where they were observed until mortality occurred. Larvae were considered dead when they did not react to touching with a needle.

2.3. Micro -organisms and culture methods

The bacteria used: Escherichia coli, Pseu domonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis were provided by the Caribbean Epidemiological Centre (a regional centre for the Pan American Health Organisation of the WHO of the UN) in Port-of-Spain,

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Table 1 Inhibitory activity towards six bacterial species shown by the extracts of various parts of 29 plant species in Trinidad and Tobago Botanical name (voucher number) Plant part (extract) Antibacterial activity toward Ec Acantahaceae J. pectoralis Jacq. (TRIN 30005) Amaryllidaceae Hymenocallis tubiora Salisb. (TRIN 30964) Annonaceae Annona muricata L. (TRIN 31241) Ps Sa Se Ef St

A (E) A (P) A (E)

1 3 3

3 3 3

3 0 0

1 3 3

2 3 0

3 3 3

Annona squamosa L. (TRIN 32031) Apocynaceae Catharanthus roseus (L.) G.Don f. albus Pich (TRIN 31362) Bignoniaceae Tabebuia serratifolia (Vahl.) Nicholson (TRIN 28111) Bixaceae Bixa orellana L. (TRIN 31833) Caricaceae Carica papaya L. (TRIN 7885) Chenopodiaceae Chenopodium ambrosioides L. (TRIN 29821) Compositae Bidens pilosa L. (TRIN 17665) Eclipta alba (L.) Hassk (TRIN 32595) Neurolaena lobata (L.) Cass (TRIN 31864)

L (E) L (P) Sd (E) Sd (P) L (E) L (P) L (E) L (P)

3 3 3 3 3 3 3 3

3 0 2 3 3 3 0 0

3 2 3 0 0 2 2 0

3 3 3 3 3 3 3 3

3 2 3 2 1 2 1 2

3 3 3 3 3 3 3 3

Fl (P)

Sd (E)

L (E) L (P) A (P)

3 3 1

3 0 3

0 2 3

3 2 3

2 3 3

3 2 3

L (P) Fl (E) Sd (P) Fl (E) Fl (P) L (E) L (P) L (E)

3 3 3 0 3 0 3 3

0 3 3 3 3 2 3 3

2 2 1 1 3 1 2 3

3 3 3 3 0 2 3 3

3 3 0 0 3 0 3 3

3 2 3 3 3 2 3 3

Pluchea odorata of S. Moore (TRIN 32561) Ehretiaceae Cordia curassa6ica (Jacq) Roemer and Schultes (TRIN 32217)

Fl (E)

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Table 1 Inhibitory activity towards six bacterial species shown by the extracts of various parts of 29 plant species in Trinidad and Tobago Botanical name (voucher number) Plant part (extract) Antibacterial activity toward Ec Euphorbiaceae Croton conduplicatus H.B.K. (TRIN 30273) Hura crepitans L. (TRIN 30295) Jafropha curcas L. (TRIN 31220) Jatropha gossypifolia L. (TRIN 30966) M. utilissima Pohl (TRIN 22776) Ps Sa Se Ef St

L (E) L (P) L (P) L (E) L (P) L (E) L (P) A (E) A (P) L (E) L (P) A (E)

3 3 3 3 3 3 3 2 3 3 3 3

3 3 2 3 3 2 3 2 2 3 3 3

3 3 3 3 3 3 2 0 0 0 0 1

3 0 2 3 3 2 3 2 3 2 3 3

3 3 3 3 3 3 3 0 3 2 3 0

3 0 3 3 3 2 3 2 2 3 3 3

Labiatae Ocimum micranthum Willd. (TRIN 31597) Leguminosae Cassia fruticosa Mill (TRIN 32683) Cassia occidentalis L. (TRIN 32787) Entada polystachya (L.) DC (TRIN 32452) Moghania strobilifera (L.) J.St.-Hil. (TRIN 31867) Mellaceae Swietenia mahagoni (L.) Jacq. (TRIN 19026) Myrtaceae Psidium guaja6a L. (TRIN 32118) Verbenaceae Citharexylum fruticosum L. (TRIN 29084) Lantana camara L. (TRIN 9190) Stachytarpheta jamaicensis (L.) Vahl. (TRIN 30091)

Fl (E) Fl (P) Sd (E) Ba (E) Ba (P) Fl (E) Fl (P) Sd (E) Sd (P) L (P)

2 3 3 3 3 3 3 3 2 3

2 3 3 3 3 3 3 3 3 3

2 3 3 1 3 3 3 1 0 0

2 3 3 3 3 3 3 3 2 3

2 3 3 1 3 2 3 0 2 2

2 2 3 2 3 3 3 3 2 3

Sd (E) L (E) L (P) A (P)

0 3 3 3

3 0 0 3

2 0 2 3

0 2 3 3

0 3 3 3

3 2 3 3

Plant parts: A, aerial; Ba, bark; Fl, ower; L, leaf; and Sd, seed. Solvents: E, ethyl acetate; and P, petroleum ether. The bacteria are: EC, E. coli; Ps, P. aeruginosa; Sa, S. aureus; Se, S. epidermidis; Ef, E. faecalis ; and St, S. typhimurium. Antibacterial levels: 1, 100% inhibition; 2, B100% inhibition; 3, no inhibition; and 0, not tested.

C.M. Chariandy et al. / Journal of Ethnopharmacology 64 (1999) 265270 Table 2 Plant species, tissues and fractions which produced 100% mortality in IV-stage Aedes aegypti mosquito larvae after brief (1 h) exposure at two concentrations of the test fractions Plant species, tissue and fraction C. roseus leaf, Ethyl acetate E. alba aerial, Petroleum ether H. crepitans leaf, Ethyl acetate H. crepitans leaf, Petroleum ether J. pectoralis leaf, Ethyl acetate J. pectoralis leaf, Petroleum ether M. utillissima leaf, Ethyl acetate S. jamaicensis leaf, Ethyl acetate Concentration mg/ml 0.50 0.05 0.50 0.05 0.50 0.05 0.50 0.05 0.50 0.05 0.50 0.05 0.50 0.05 0.50 0.05 Time to mortality (days) 12.0 23.3 10.4 28.0 10.0 24.3 15.0 18.6 2.0 4.7 9.0 12.0 7.2 13.4 7.3 14.7

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ity. These three are among the better known traditional medicinal species in the Caribbean region. As noted by earlier researchers (Grosvenor et al., 1995), S. aureus was the most easily inhibited of all the bacteria exposed to plant extracts. For the anti-mosquito tests, eight plants were selected on the basis of their performances in the anti-bacterial assays, or from their anti-insect activities as reported in the literature (Roark, 1947; Patterson et al., 1975; Secoy and Smith, 1983; Yang and Tang, 1988). In these tests, the effects of selected plant extracts were observed on the growth and development of IV-stage larvae of Aedes aegypti mosquitoes. The times required to produce 100% larvicidal activity by brief (1 h) exposure to concentrations of 0.05 and 0.50 mg/ ml of these plant extracts are shown in Table 2. The extracts of J. pectoralis were found to be the most toxic of all. Larval survival without development was observed for 2 weeks up to 4 weeks following brief (1 h) exposure of the insects to the six other plant extracts listed in Table 2. It is suggested that these six plant extracts showed growth retardant and/or anti-feedant activities. Overall, the results show that a proportion of Caribbean traditional medicinal plants have antibacterial and insecticidal activity. These plants are the subject of further evaluation to elucidate the constituents responsible for observed activities.

3. Results and discussion The results of the antibacterial screening are shown in Table 1. Starting with 29 medicinal plants, the extracts from ve plant species inhibited E. coli, eight inhibited P. aeruginosa, 13 inhibited S. typhimurium, 23 inhibited S. aureus, 11 inhibited S. epidermidis and 15 inhibited E. fae calis. None of the negative controls used had antibacterial activity. Signicant activity was shown by the extracts of seven plants which scored 1 or 2 against at least two micro-organisms, neither being S. aureus. Extracts of Neuro laena lobata, Manihot utilissima and Justicia pectoralis showed the highest antibacterial activ-

Acknowledgements We wish to thank Y. Comeau the Curator and Staff at the National Herbarium at St. Augustine, Trinidad for plant identications. The authors are grateful also for nancial support provided by LARC-Rothamsted Experimental Station at Harpenden in Hertfordshire and the British Technology Group of London, England.

References
Busvine, J.R., 1971. A critical review of the techniques for testing insecticides. Commonwealth Agricultural Bureau, Dorset Press, London.

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C.M. Chariandy et al. / Journal of Ethnopharmacology 64 (1999) 265270 Patterson, B.D., Wahba-Khalil, S.K., Schermeister, L.J., Qurashi, M.S., 1975. Plant-insect interactions I. Biological and phytochemical evaluation of selected plants. Lloydia 38 (5), 391 403. Recio, M.C., Rios, J.L., Villar, A., 1989. A review of some antimicrobial compounds isolated from medicinal plants reported in the literature 1978 1988. Phytotherapy Research 3, 117 125. Roark, R.C., 1947. Some promising insecticidal plants. Economic Botany 1 (4), 437 445. 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. Seaforth, C.E., Adams, C.D., Sylvester, Y., 1985. A Guide to the Medicinal Plants of Trinidad and Tobago. Commonwealth Secretariat, London. Secoy, D.M., Smith, A.E., 1983. Use of plants in control of agricultural and domestic pests. Economic Botany 37 (1), 28 57. Yang, R.Z., Tang, C.S., 1988. Plants used for pest control in China: A literature review. Economic Botany 42 (3), 376 406.

Chariandy, C.M., 1997. Investigation into the potential of local plants as sources of bioactive agents. Master of Philosophy Thesis, The University of the West Indies, Trinidad. Grainge, M., Ahmed, S., 1988. Handbook of Plants with Pest Control Properties. Wiley, New York. Grosvenor, P.W., Supriono, A., Gray, D.O., 1995. Medicinal plants from Riau Province, Sumatra, Indonesia. Part 2: antibacterial and antifungal activity. Journal of Ethnopharmacology 45, 97111. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L., 1982. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Medica 45, 3134. Mitscher, L.A., Leu, R.P., Bathala, M.S., Wu, W.W., Beal, J., White, R., 1972. Antimicrobial agents from higher plants. I. Introduction, rationale and methodology. Lloydia 35, 157 166. Mitscher, L.A., Drake, S., Gollapudi, S.R., Okwute, S.K., 1987. A modern look at folklore use of antiinfective agents. Journal of Natural Products 50, 10251040. Morton, J.F., 1981. Atlas of Medicinal Plants of Middle America. Charles C. Thomas Publishers, Springeld, IL, USA.