Antimicrobial Activities and Phytochemical Profiles of Endemic Medicinal Plants of Mauritius

Abstract

Medicinal plants are assuming widespread use in the primary health care of individuals and communities. In the current study, we investigated the antimicrobial activities of two endemic plants, Antidesma madagascariense. Lam. and Erythroxylum macrocarpum. O. E. Schulz, which form part of the local pharmacopoeia of Mauritius. Aqueous and methanol extracts of the leaves and twigs of both plants were tested in vitro. against three Gram-negative and five Gram-positive strains of bacteria, including a resistant strain of bacteria. Moreover, they were subjected to antifungal assays against Candida albicans. ATCC 10231 and Aspergillus niger. ATCC 16404, a human and a plant pathogenic microorganism, respectively, known to be good indicators of antifungal activity. It was found that aqueous extracts of both plants possess broad-spectrum antibacterial properties against the test organisms compared to methanol extracts, which gave poor inhibition zones. Moreover, the antibacterial substance within these plants seemed to be most prominent in the leaves and least in the twigs, and the best inhibitory activity was observed for Gram-positive bacteria (Staphylococcus aureus. ATCC 25923). Aqueous extracts of both plants do not possess antifungal properties except the methanol extract against A. niger.. Phytochemical screening of the plants showed the presence of at least tannins, phenols, flavonoids, and alkaloids, which are known antimicorbial compounds. In conclusion, the observed antimicrobial properties would tend to further validate the medicinal properties of these commonly used endemic medicinal plants in Mauritius.

Keywords:

• Antimicrobial activity
• endemic plants
• Mauritius
• medicinal plants
• phytochemicals

Introduction

Medicinal plants, since time immemorial, have been used in virtually all cultures as a source of medicine (Ang-Lee et al., 2001; Goldman, 2001). In India and China, herbal medicines are still widely used, and developed countries have rediscovered many of these traditional medicines as cheap sources of complex bioactive compounds (Phillipson, 1994). Modern pharmaceuticals are typically oral dosage forms containing single synthetic chemicals that have potent clinical activity. However, natural products from higher plants continue to be used in pharmaceutical preparations either pure or as extracts (Gogtay et al., 2002). Despite the availability of different approaches for the discovery of therapeutic agents, natural products still remain as one of the best reservoirs of new structural types (Hostettmann, 1999). To this effect, in the constant effort to improve the efficacy and ethics of modern medicinal practice, researchers are increasingly turning their attention to folk medicine as a source of new drugs (Wayne, 1998; Hoareau & Dasilva, 1999).

In Mauritius, the local population has a long-standing tradition in the use of herbal medicine. Many indigenous and endemic plant species of Mauritius have been used in folk medicine to treat various ailments of man (Baumer, 1979; Sussman, 1980; Wong Ting Fook, 1980; Gurib-Fakim et al., 1993, 1996a,b). Available reports tend to show that indigenous folk-medicinal plant preservation and study is vital because such plants are fully adapted to local environments and to conditions compared to introduced species (Qin & Xu, 1998). Pharmacologically active compounds and phytochemicals isolated from such endemic and indigenous plants used in folk medicine have been the center of interest during the past few decades (Farnsworth, 1994; Benzi & Ceci, 1997). In addition, it is also of importance to preserve and maintain knowledge about medicinal uses of plant resources of Mauritius, which is characterized by a rich flora of exotic, endemic, and indigenous plant species. Currently, several kinds of extracts from various exotic, endemic, and indigenous plants are sold as decoctions or “tisanes” in several markets across Mauritius to treat minor ailments (Gurib-Fakim et al., 1993, 1996a,b). This native herbal folk medicinal practice forms an essential part of the heritage of the local pharmacopoeia of Mauritius. Nonetheless, only a few medicinal plants have been validated for their medicinal virtues. Moreover, the biological activity of such medicinal plants against certain human pathogenic bacteria and fungi is poorly investigated, and a literature search indicated limited information about the antimicorbial activity of Mauritian endemic plants (Gurib-Fakim et al., 1993, 1996a,b). Currently, the emergence of resistant pathogens to many of the commonly used antibiotics has provided an impetus for further attempts to search for new antimicrobial agents to combat infections and overcome the problems of resistance to currently available antimicrobial agents (Balandrin et al., 1985; Xu & Lee, 2001). Plant-based antimicrobials and antibacterials represent a vast untapped source for medicines and hence have enormous therapeutic potential (Phillipson, 1994). They are effective in the treatment of infections while mitigating many of the side effects associated with synthetic antimicrobial and antibacterial (Mathews et al., 1999; Bagghi, 2000)

Therefore, the current in vitro. antimicorbial study was aimed at validating the effects of two endemic medicinal plants of Mauritius, Antidesma madagascariense. Lam. (Euphorbiaceae) and Erythroxylum macrocarpum. O. E. Schulz (Erythroxylaceae), for their antimicrobial activity against eight clinical strains of bacteria and two strains of fungi. In addition, we also evaluated their phytochemical profiles to correlate possible biological activities of these plants.

Materials and Methods

Preparation of the medicinal plant extracts

Leaves and twigs of Antidesma madagascariense. and Erythroxylum macrocarpum. used in the study were collected from Maccabe forest. The Curator of the National Herbarium at the Mauritius Sugar Industry Research Institute (MSIRI) confirmed the identity of the plants. Voucher specimens were deposited at the Herbarium collection of the Department of Chemistry, Faculty of Science, University of Mauritius.

Extraction

Crude water extract of the plant material was used in the current study. It was important to assess antimicrobial activities of the crude water extracts in order to validate the medicinal uses of these plants, as this is the way in which the local population in the traditional medicine of Mauritius uses the plants. In addition, methanol extracts of the plants were used for a comparative basis. Moreover, leaves and twigs of both plants were screened for antimicrobial activities, as the local population uses both plant parts as decoctions. Freshly collected plant materials were either air-dried or dried in a drying cabinet at 50°C for 5–7 days. Ten grams of the dried plant materials (leaves and twigs) of the plant species was separately crushed and ground into fine powders using a food blender. Each powered plant material was extracted to exhaustion with water or methanol in a Soxhlet apparatus for 5 h. The solvent was distilled off under reduced pressure to afford crude plant extract. The paste was collected for examination. Percentage yield was calculated, and the paste-like water or methanol suspension was diluted for further experiments.

Test culture

The test organisms used for the screening antimicrobial activity were Bacillus cereus. ATCC 10702, Bacillus subtilis. ATCC 6633, Escherichia coli. ATCC 25922, Staphylococcus aureus., methicillin-resistant Staphylococcus aureus. (MRSA) ATCC 43300, Enterococcus faecalis. ATCC 29212, Pseudomonas aeruginosa. ATCC 27853, and Salmonella typhimurium. ATCC 14028. Stock cultures of the bacteria and fungi used were clinical isolates obtained from the Bacteriology Section of the Central Laboratory Candos, Victoria Hospital. Cultures were maintained as nutrient agar slants in screw-capped bottles and stored at 4°C. All cultures were checked for viability and purity by regular plating. Test cultures were prepared by transferring a loop full of bacteria from stock culture nutrient broth and incubated at 37°C for 24 h. Fungi were transferred into freshly prepared dextrose agar plates and incubated at 25°C for 3 days. The fungi used in the study were Aspergillus niger. and Candida albicans..

Antimicrobial bioassay procedure

The antibacterial and antifungal activities were determined using the agar disk diffusion method (Xu & Lee, 2001; Mahasneh, 2002). This method is highly effective for rapidly growing microorganisms, and the activities of the test extracts are expressed by measuring the diameter of the zone of inhibition. Sterilized filter paper disks (6 mm in diameter) were impregnated in appropriate concentration of each plant extract. The disks (made from Whatman no. 1) were allowed to absorb the plant extracts as described by Mahasneh (2002). Plates of Mueller-Hinton sensitivity agar (Oxoid, UK) were aseptically inoculated with broth cultures for the test organisms using sterile Pasteur pipette. The plates were allowed to dry. The disks containing the plant extract were transferred using flamed but cooled forceps onto the surface of the seeded agar plates. They were sufficiently spaced to prevent the resulting zones of clearing from overlapping. The extractive solvent (water) was used as a negative control. The plates with the organisms were incubated for 24 h. After incubation, the growth inhibition rings were quantified by measuring the diameter for the zone of inhibition to the nearest millimeter from the lower surface of the Petri dishes. Inhibition zone values were corrected, that is, disk diameter was subtracted from the value of the inhibition zone. As the diameter of the disk was 6 mm, inhibition zones of less than 7 mm were not evaluated (Hong et al., 2004) Negative control disks contained the solvent. Standard antibiotic (ampicillin) was used as positive control for comparison. The experiments were performed three-times to minimize errors.

Determination of minimum inhibitory concentration (MIC) values

The MIC of each plant extract was determined by a slight modification of the tube dilution method (Omoregbe et al., 1996). The MIC is defined as the lowest concentration of that extract in a medium without visible growth of the test organisms. In a set of 10 sterile capped microtubes using the extraction solvent as diluent, serial dilutions were made from the different extracts to yield graded concentrations in mg/ml and a tube containing only diluent as the sensitivity control. Sterile filter paper disks (6 mm in diameter) were impregnated with the different dilutions of the plant extract and aseptically transferred to the surface of the inoculated plates using flamed but cooled forceps. The disks were sufficiently spaced to avoid overlapping of zones of inhibition. The MIC of the different plant extracts that inhibited the growth of the test organism other than inhibition due to the diluent was taken as the MIC.

Phytochemical screening of A. madagascariense. and E. macrocarpum.

Both leaves and twigs were subjected to a thorough phytochemical screening using standard (Narod, 2002) protocols to detect the presence of the following secondary metabolites: alkaloids, coumarins, terpenes, anthraquinones, tannins, phenols, leucoanthocyanins, flavones, and saponins.

Results

Comparative antimicrobial screening

Results obtained for the antimicrobial tests performed on aqueous and methanol extracts of A. madagascariense. and E. macrocarpum. are presented in Table 1. Our results showed that the aqueous extract of both plants showed a broad spectrum of activity, being active to both the Gram-positive and Gram-negative organisms in the antimicrobial assays. The zones of inhibition ranged from 3 to 12 mm, and 3 to 9 mm for aqueous extract of leaves and twigs of A. madagascariense., respectively. For E. macrocarpum., the inhibitory zone ranged from 4 to 13 mm and 3 to 10 mm for leaves and twigs, respectively. Aqueous leaf extract of both endemic plants were found to exhibit more antibacterial activity (100%). The methanol extract of leaves and twigs of both plants gave poor inhibition zones (3–10 mm). The leaf extract was also found to be most active against Gram-positive bacteria (inhibition zone 6–12 mm) compared to Gram-negative strains (inhibition zones 5–8 mm). On the other hand, 87% for twigs of A. madagascariense. and 50% for twigs of E. macrocarpum., showed antimicrobial activities against the test organisms. Comparative inhibitory zones to standard antibiotic ampicillin was observed for aqueous leaf extract of E. macrocarpum. against S. aureus. and twigs of A. madagascariense. against E. faecalis.. It was also found that aqueous leaf extract of both endemic plants showed moderate antibacterial activities against a resistant strain of S. aureus. (MRSA). Neither aqueous leaf nor twigs extract of the two plants possess antifungal activities against A. niger. and C. albicans.. However, a methanol extract of A. madagascariense. showed antifungal activities against A. niger..

Table 1 Antimicrobial activities of A. madagascariense. and E. macrocarpum. against the test microorganisms.

			Diameter of zone of inhibiting (mm)^b.
			A. madagascariense.		E. macrocarpum.
Test microorganisms	Gram stain+/−	Standard antibiotic (Ampicillin)^a.	Leaves	Twigs	Leaves	Twigs
Bacillus cereus.	G+	14	10	9	7	3
			(5)	(3)	(−)	(−)
Bacillus subtilis.	G+	12	7	8	8	4
			(3)	(4)	(−)	(−)
Escherichia coli.	G−	10	8	7	6	−
			(8)	(5)	(−)	(−)
Staphylococcus aureus.	G+	13	12	4	13	10
			(10)	(4)	(10)	(6)
MRSA	G+	0	3	−	5	−
			(−)	(−)	(−)	(−)
Enterococcus faecalis.	G+	7	6	7	4	−
			(3)	(−)	(−)	(−)
Pseudomonas aeruginosa.	G−	9	5	6	6	6
			(2)	(4)	(−)	(−)
Salmonella typhimurium.	G−	10	7	9	8	−
			(5)	(5)	(3)	(5)
Aspergillus niger.	F	0	−	−	−	−
			(5)	(7)	(−)	(−)
Candida albicans.	F	0	−	−	−	−
			(−)	(−)	(−)	(−)

−, complete lack of activity. G+, Gram-positive; G−, Gram-negative; F, fungi; MRSA, methicillin-resistant Staphylococcus aureus.. () inhibition zone of methanolic extract.

^a.Tested at a concentration of 10 µg/disk (Oxoid).

^b.Zone of inhibition; average of 3 independent experiments.

Minimum inhibitory concentration

The extracts showing antibacterial activities by the disk diffusion method were further tested by the broth dilution assay to determine the MICs. The results of the MICs of the different plant extracts are shown in Table 2. The MIC value for aqueous extract of leaves and twigs of A. madagascariense. ranged from 2 to 32 mg/ml, respectively; 0.5 to 32 mg/ml for leaves and twigs of E. macrocarpum., respectively. The lowest MIC value (0.5 mg/ml) was recorded for E. macrocarpum. against the Gram-positive strain of S. aureus.. However, for the MRSA strains, high concentrations of the leaves extracts of both endemic plants were needed to inhibit growth of the bacteria. On the other hand, the methanol extract of both plants showed high MIC values except for leaves of E. macrocarpum. against S. aureus. (MIC of 1 mg/ml).

Table 2. Minimum inhibitory concentrations (mg/ml) of A. madagascariense. and E. macrocarpum. leaves and twigs for the 10 strains of microorganisms.

		Minimum inhibitory concentration (mg/ml)
		A. madagascariense.		E. macrocarpum.
Test microorganisms	Gram stain+/−	Leaves	Twigs	Leaves	Twigs
Bacillus cereus.	G+	2	8	16	16
		(4)	(8)	(−)	(−)
Bacillus subtilis.	G+	4	16	8	8
		(8)	(16)	(−)	(−)
Escherichia coli.	G−	8	16	10	−
		(8)	(4)	(−)	(−)
Staphylococcus aureus.	G+	2	4	0.5	0.5
		(4)	(8)	(1)	(4)
MRSA	G+	32	−	16	−
		(−)	(−)	(−)	(−)
Enterococcus faecalis.	G+	4	10	8	−
		(8)	(−)	(−)	(−)
Pseudomonas aeruginosa.	G−	8	8	4	8
		(8)	(8)	(−)	(−)
Salmonella typhimurium.	G−	8	8	4	−
		(8)	(8)	(8)	(8)
Aspergillus niger.	F	−	−	−	−
		(16)	(16)	(−)	(−)
Candida albicans.	F	−	−	−	−
		(−)	(−)	(−)	(−)

−, complete lack of activity. G+, Gram-positive; G−, Gram-negative; F, fungi; MRSA, methicillin-resistant Staphylococcus aureus.. () MIC of methanolic extract.

Phytochemical screening

The phytochemical components of both plants are summarized in Table 3. Tannins, phenols, flavonoids, and alkaloids were present in leaves and twigs of both E. macrocarpum. and A. madagascariense..

Table 3. Phytochemical components of A. madagascariense. and E. macrocarpum. leaves and twigs (+, presence; −, absence).

Plants	Part used	Alkaloids	Flavonoid	Tannins	Leucoanthocyanins	Anthraquinones	Terpenes	Phenols	Coumarins	Saponins
A. madagascariense.	Leaf	+	+	+	+	−	−	+	−	+
	Twigs	+	+	+	+	−	−	+	−	+
E. macrocarpum.	Leaf	+	−	+	+	−	−	+	−	−
	Twigs	+	+	+	+	−	+	+	−	−

Discussion

Interest in higher plant extracts exhibiting antimicrobial activity has increased in recent years, and several reports on this subject have been published (Farnsworth, 1994; Benzi & Ceci, 1997; Bagghi, 2000). Both endemic plants studied in the current work showed antibacterial activities against the test microorganisms. However, the plant parts differ significantly in their activity against the test microorganisms. The differences observed in the antimicrobial activities assays suggest the susceptibility of the test microorganisms to various secondary metabolites present in these endemic plants. The composition of these secondary metabolites in turn varies from species to species, climatic conditions, and the physiological state of developments of the endemic plants (Hussain & Deeni, 1991). Moreover, the antibacterial substance within these plants in the current study seemed to be most prominent in the leaves and least in the twigs, and the best inhibitory activity was observed for Gram-positive bacteria (S. aureus.). This may be attributed to the fact that cell wall in Gram-positive bacteria consists of a single layer, whereas Gram-negative cell wall is a multilayered structure bounded by an outer cell membrane (Yao & Moellering, 1995). On the other hand, both plants were not effective against the test fungi except for methanol extract of A. madagascariense.. Such results indicate that antibacterial agents are more common in the plants studied than antifungal agents. This may be attributed to structural differences between prokaryotic bacteria and eukaryotic fungal agents; antimicrobial agents should bind to sterols in eukaryotic membrane so as to exhibit their action, whereas this step is not needed for bacterial cells (Medoff & Kayashi, 1993; Ali-Stayeh et al., 1998). Results from the current study also show that methanol extracts of both plants gave poor inhibition zones. Indeed, Cragg et al. (1994) reported that there are great differences between the activity of aqueous and organic extracts in their anti-HIV screening: about 34% of the plant was active in aqueous extract and only 4% in organic extract.

Differences in antimicrobial activity of medicinal plants are obviously related to differences in their contents of active compounds (Boakye-Yiadom & Konning, 1975). Available reports tend to show that alkaloids and flavonoids are the responsible compounds for the antimicrobial activities in higher plants (Cordell et al., 2001). Moreover, it is also claimed that secondary metabolites such as tannins and other compounds of phenolic nature are classified as active antimicrobial compounds (Mitscher et al., 1972; Rojas et al., 1991). Interestingly, phytochemicals screening of the current investigation has revealed that extracts from both endemic plants possess at least four of the following classes of secondary metabolites: tannins, phenols, flavonoids, and alkaloids. Therefore, the presence of these phytochemicals could to some extent justify the observed antimicrobial activities in the current study. Moreover, because the current antimicrobial study was done using the same aqueous preparation as prescribed by the traditional healers, these results would support the way people use these herbal remedies. To this effect, it is possible that these two endemic medicinal plant extracts can be used as antibacterial agents in food or other ingredients. However, the exact mechanism of antibacterial effects from A. madagascariense. and E. macrocarpum. needs to be further examined for potential uses. In conclusion, the current study advocates the need for continuing screening for antimicrobial agents from local endemic plants of Mauritius.

Acknowledgment

The authors are grateful to the Tertiary Education Commission and the University of Mauritius for Financial support.