Antimicrobial Activity of 23 Endemic Plants in Madagascar

Purpose: To screen the crude methanol extracts obtained from 23 endemic plants in Madagascar for antimicrobial activity. Methods: In order to assess the antimicrobial properties of the extracts, their minimum inhibitory concentrations (MICs) were obtained using the broth microdilution method. The six test pathogenic species used were Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa and Candida albicans. Bioautography agar overlay test and phytochemical screening were also performed on the most active extracts. Results: From the 23 plants tested, 16 of which are used in traditional medicine, Poivrea phaneropetala (Combretaceae), Koehneria madagascariensis (Lythraceae) and Rhopalopilia perrieri (Opiliaceae) exhibited the broad spectrum of activity, being active against all the test organisms, while Monoporus clusiifolius (Myrsinaceae) showed the strongest antifungal activity against Candida albicans with a minimal inhibitory concentration of 0.250 mg/ml. Bioautography and phytochemical analysis of the five active extracts against bacterial strains and of one active extract against C. albicans indicate that the active compounds responsible for antimicrobial activity may be mainly flavonoids and/or terpenes. Conclusion: These preliminary results are the first antimicrobial studies on these plants and lend support for the use of some of them in traditional medicine.


INTRODUCTION
Madagascar is host to approximately 12,000 vegetable species, more than 80 % of which are endemic to the island [1]. Owing to environmental degradation, increasing deforestation, slash and burn agriculture in primary forest, this unique patrimony is threatened by extinction. Only 9 % of the original areas are currently available and the country is among 25 most critical regions for plant life protection in the world [2].
In Madagascar, herbal medicines are often used as the first line of treatment of various diseases. The practice of traditional medicine is well-embedded in the lifestyle of the eighteen indigenous tribes of Madagascar. Previous investigations on Madagascan flora mainly dealt with ethnobotanical practices of plants in folk medicine [3][4][5]. Screening surveys have already shown some biological activities such as antiplasmodial [6], antiviral [7], and cytotoxic activities [8]. However, very few scientific studies have been carried out on the putative antimicrobial properties of endemic plants in the island although many of them have been claimed by local traditional healers to be effective in the treatment of infectious diseases.
The aim of this study was to investigate the antimicrobial properties of 23 endemic plants obtained from different parts of the country and to identify the phytochemical class of active components. Plant names and their folkoric use are given in Table 1. Dried plant materials were ground into fine powders and preserved at the IMRA herbarium.

Preparation of plant extracts
Dried and powdered plants (10 g) were macerated with agitation in 50 ml methanol (MeOH) overnight at room temperature. After filtration, the solvent was eliminated by evaporation at reduced pressure and crude extracts were dried at 45 °C using a speedvac concentrator (Savant), and stored at the IMRA bank at 4 °C.

Microorganisms
The bacterial strains used for the investigation were Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa, and were all obtained as clinical isolates from patients at the Microbiology Department, IMRA. The yeast strain, Candida albicans (MUCL 31360), was obtained from the Mycothèque de l'Université Catholique de Louvain (MUCL), Belgium. Stock cultures were maintained at 4 °C on slopes of nutrient agar (Difco) for bacteria and on Sabouraud dextrose agar (SDA, Difco) for the yeast, prior to their use.

Determination of minimum inhibitory concentration (MIC)
The broth microdilution method was used to assess the MIC of the different plant extracts in a 96-well microplate using a modified method [9]. Microbial suspensions were first prepared from an overnight culture of bacterial and fungal cells grown in flasks, each containing 10 ml of Mueller-Hinton Broth (Oxoid) for bacteria and Sabouraud dextrose broth (Difco) for yeast at 37 °C and 30 °C, respectively. The turbidity of the microbial suspensions was adjusted to 0.5 McFarland using Densicheck (BioMerieux). Stock solutions of the different extracts were prepared by re-suspending the crude methanol extracts in 10 % dimethyl sulfoxide (DMSO) to produce concentrations in the range of 1.25 -160 mg/ml. These solutions were further diluted 10-fold with water and then sterilized by filtration through 0.22 µm membrane filters. A known volume (100 µl) of each solution was deposited in the wells. This was followed by the addition of 100 µl of the inoculum (approximately 10 6 CFU/ml for bacteria and 10 5 CFU/ml for C. albicans) was added to each well. The microplates were incubated overnight at 37 °C for bacteria and 30 °C for 48 h for C. albicans. After incubation, 40 µl of 0.2 mg/ml aqueous solution of methylthiazoyltetrazolium chloride (MTT) was added to each well and further incubated for 30 min at room temperature. MIC was defined as the lowest concentration in which no transformation of MTT was observed. Streptomycin sulfate and nystatin were used as positive controls and their MICs were determined using the same process. All samples were tested in triplicate and the tests were repeted twice.

RESULTS
A total of 23 methanol plant extracts of Madagascan endemic species belonging to 20 different botanical families, among which 16 are used in traditional medicine (Table 1), were tested for their antimicrobial activities against 5 bacterial species and one yeast. The results of these tests are summarized in Table 2

DISCUSSION
This study showed that all but two plant extracts possessed some degree of activity against at least one tested microorganism, at the highest tested dose of 8 mg/ml. Overall, Gram positive bacteria (B.subtilis and S. aureus) were more sensitive to plant extracts than Gram negative bacteria (E. coli, S. typhi and P.aeruginosa). Among them, P. phaneropetala, K. madagascariensis and R. perrieri demonstrated a broad spectrum of activity against all the tested organisms while M. clusiifolius was the most active against C. albicans. R. perrieri showed the most promising antibacterial activity, confirming its use in traditional medicine as antiseptic and wound healing. The present investigation also confirmed the anti-infective property of H. coriacea in the treatment of vaginitis and urethritis and that of D. oblongifolia which is used to treat diarrhoea and dysentery in folk medicine. In contrast, the use of Leptolaena pauciflora Baker (Sarcolaenaceae) as antiinfective agent for the treatment of venereal diseases was not supported by our investigation. Furthermore, the oral use of this plant containing several phenolic compounds could be toxic [12].
Based on this investigation, we are unable to say categorically that extracts having the same Rf values contained the same bioactive compounds. It is possible that the observed inhibition was likely due to one or more compounds sharing the same Rf in the solvent system used, particularly for low Rf spots. Preliminary phytochemical screening revealed the presence of flavonoids, alkaloids and terpenes as the probable active compounds present in the crude extracts. It is well known that numerous members of these phytochemical groups have already demonstrated antimicrobial activity [13].   To the best of our knowledge, no previous antimicrobial survey has been reported on these plants. However, some of them were previously reported for other biological activities. Literature searches indicate that Evonymopsis longipes (H. Perrier) H. Perrier (Celastraceae) was particularly potent and could completely inactivate the Herpes Simplex Virus at a concentration lower than 25 µg/ml [7]. Peddiea involucrata Baker (Thymelaeaceae) and Piptadenia pervillei Vatke (Fabaceae) were said to possess antimalarial activity [15][16]. From this latter was isolated (+)-catechin 5-gallate and (+)catechin 3-gallate which demonstrated, in vitro, high activity against the chloroquineresistant strain FcB1 of Plasmodium falciparum as well as (+)-catechinant ethylgallate which were less effective [16]. With regard to antibacterial activity, howerver, these plants were less effective, being active only against B. subtilis with MIC ranging from 2 to 4 mg/ml. While no ethnomedicinal practices were reported to date for 7 of the 23 plants, the study revealed that A. densiflora and K. madagascariensis were found to possess effective antibacterial activity; in addition, the extract from M. clusiifolius may be a good candidate in the search for potential antifungal compounds. Further stuydies on these plants are being undertaken in our laboratory to isolate and elucidate the compounds responsible for the observed anti-candidiasis and antibacterial activities.

CONCLUSION
This survey contributes new data to the existing knowledge of antimicrobial activity of the endemic flora of Madagascar and may serve as a basis for further pharmaceutical investigation. The findings also lend support for the use of some of these plants in traditional medicine in Madagascar.
antifungal compounds from higher plants.