The plant extracts listed in Table 1 were studied for their antimicrobial activities against Staphylococcus saprophyticus (ATCC 15305), Staphylococcus aureus (ATCC 6341), Pseudomonas aeruginosa (ATCC 7700), Proteus vulgaris (ATCC 6896), Escherichia coli (ATCC 4157), Haemophilus influenza (ATCC 8142), and the fungus Candida albicans (ATCC 752). Of the 13 plant extracts tested, data corresponding to the extracts that showed microbial growth inhibition in the disc diffusion assays will be discussed. These extracts include G. barbadense fruit juice and the decoctions of P. calomelanos, T. ananassae, and S. jambos (Figure 1). The bacterial species inhibited by these extracts were S. saprophyticus (ATCC 15305), S. aureus (ATCC 6341), P. aeruginosa (ATCC 7700) and P. vulgaris (ATCC 6896).

Figure 1 Herbal remedies in the Caribbean that showed antimicrobial activities. Inhibition was calculated as percentages relative to the positive control, Streptomycin- treated discs. A) G. barbadense juice; B) P. calomelanos decoction; C) T. ananassae decoction and D) S. jambos decoction. Full size image

Juice prepared from unripe G. barbadense fruit exhibited more than 25% growth inhibition of the two Gram-positive bacteria S. aureus (ATCC 6341) and S. saprophyticus (ATCC 15305) relative to Streptomycin treatment which showed 100% inhibition at 5 mg/mL. S. aureus (ATCC 6341) has been isolated from the discharge produced during an otitis media infection, which provides a rationale for the use of G. barbadense juice (juice from the unripe fruit) as an herbal remedy for this infection [17]. The antimicrobial activity was observed at a concentration of 7 mg/mL, the lowest concentration tested (Figure 1A). The MIC values that were determined in this study for G. barbadense against S. aureus (ATCC 6341) and S. saprophyticus (ATCC 15305) did not meet the p-value of 0.001 or less used in our analyses. This result contrasts with a study of medicinal plants from Yemen in which aqueous extracts from G. barbadense were not active (MIC values > 1,000 μg/mL) against S. aureus and E. coli and showed low activity (MIC = 1000 μg/mL) against P. aeruginosa and other bacterial strains [18]. In our study, no measureable MBC was observed for either bacterial species. Treatment for otitis media using herbal remedies has been very difficult. A study using an ethanolic extract of the commonly used herb for infectious diseases, Echinacea purpurea roots and seeds, did not show a decrease in the risk of otitis media infection [19]. Nevertheless, naphthofuran analogues and biologically active terpenoids from G. barbadense are known to exhibit growth-inhibitory and antibacterial activities [20, 21]. In addition, the essential oil has been reported to show moderate activities against S. aureus, E. coli and the fungus C. albicans[22]. Although the antimicrobial activity in the oil might be due to the major components or to synergy between major and minor components of the oil, α-pinene, which is a terpene that comprises 12.8% of the oil, has shown efficient antimicrobial properties.

The decoction of P. calomelanos (at the lowest concentration of 37.5 mg/mL) inhibited the growth of P. aeruginosa (ATCC 7700), S. aureus (ATCC 6341) and S. saprophyticus (ATCC 15305) by 2%-20% relative to Streptomycin which showed 100% inhibition at 5 mg/mL (Figure 1B). The MIC for P. aeruginosa (ATCC 7700) was 2 mg/mL (p = 0.0001); however, for S. aureus (ATCC 6341) and S. saprophyticus (ATCC 15305), the MIC values were not statistically significant (Table 2). None of the concentrations that were tested showed bactericidal activity. P. calomelanos is traditionally used to treat kidney stones. The bacterium P. aeruginosa has been associated with biofilm formation on kidney stones, which leads to urinary infections [23]. The use of P. calomelanos decoctions to treat kidney stones could be related to the growth inhibition of P. aeruginosa, which may justify the use of this treatment as an herbal medicine. Studies that focused on several fern families have demonstrated that ethanol and methanol extracts from these plants exhibit antibacterial properties [24, 25]. Recently, a study reported the antimicrobial and modulatory antibiotic activity of the ethanol extract and the methanolic fraction from the leaves of P. calomelanos[26]. Neither preparation demonstrated antimicrobial activity that was clinically relevant to fungi or bacteria (MIC > 1024 μg/mL). However, in the modulation assay, both extracts modulated most of the antibiotics tested against S. aureus. The present study only tested aqueous extracts as a way to validate the traditional remedies employed by the surveyed population and is the first to report the antimicrobial activities of P. calomelanos decoctions. Phytochemical analysis of aqueous extracts of P. calomelanos has revealed the presence of carbohydrates and coumarin [27]. Although data regarding the specific antibiotic properties of coumarins are scarce, antimicrobial activity against bacteria, fungi and viruses has previously been reported for this class of phytochemicals [3]. Conversely, polysaccharides are commonly more effective as inhibitors of pathogen adsorption and would not be identified in the screening techniques commonly used.

Table 2 MIC (p-value 0.001 or less) and MBC of extracts that showed inhibition in the disc diffusion assays a Full size table

The decoction of T. ananassae at 15.3 mg/mL, the lowest concentration tested, inhibited the growth of two Gram-positive and two Gram-negative bacterial strains by 22%-30% compared to the 100% inhibition of Streptomycin at 5 mg/mL (Figure 1C). Specifically, the growth inhibition values of P. aeruginosa (ATCC 7700), S. saprophyticus (ATCC 15305), S. aureus (ATCC 6341) and P. vulgaris (ATCC 6896) were 29%, 22%, 32% and 24%, respectively. The MIC values for T. ananassae were 0.5 mg/mL for P. aeruginosa (ATCC 7700), and P. vulgaris (ATCC 6896) (p = 0.0002 and 0.0001, respectively), 0.015 mg/mL for S. aureus (ATCC 6341) (p = 0.001) and 2.0 mg/mL for S. saprophyticus (ATCC 15305) (p = 0.0004) (Table 2). T. ananassae displayed bactericidal activity against P. vulgaris (ATCC 6896) and S. saprophyticus (ATCC 15305) at concentrations of 2.0 mg/mL for both species (Table 2). Our study showed that T. ananassae inhibits the growth of pathogens associated with opportunistic infections of burns and skin infections such as P. aeruginosa and S. aureus, respectively [28]. P. aeruginosa has become an important cause of infection, especially in immunocompromised patients, such as diabetic and cancer patients [29]. The antibiotic activities of T. ananassae are important because people with diabetes are vulnerable to infections as a result of low blood flow to the extremities [30] and because of certain types of neuropathy that cause dry and cracked skin. Additional antimicrobial activity has been reported for T. ananassae against the Gram-negative bacterium Helicobacter pylori[31].

Decoctions of Syzygium jambos leaves inhibited S. aureus (ATCC 6341) and S. saprophyticus (ATCC 15305) by 34% and 11%, respectively, at the lowest concentration studied (10.5 μg/μL) relative to Streptomycin which showed 100% inhibition at 5 mg/mL. S. jambos at 20.9 mg/mL also inhibited the Gram-negative P. vulgaris (ATCC 6896) by 24% relative to Streptomycin which showed 100% inhibition at 5 mg/mL (Figure 1D). The MIC value for S. aureus (ATCC 6341) and S. saprophyticus (ATCC 15305) were 0.5 mg/mL (p = 0.00001 and 0.001, respectively), and the MIC value for P. vulgaris (ATCC 6896) was 0.031 mg/mL (p = 0.001) (Table 2). S. jambos was the only extract that showed bactericidal activity against all of the isolates that were tested. The MBC value for both S. aureus (ATCC 6341) and P. vulgaris (ATCC 6896) was 1.0 mg/mL, and the MBC value for S. saprophyticus (ATCC 15305) was 2.0 mg/mL (Table 2). Previously, Lin et al. reported that aqueous and acetone extracts from the bark of Syzygium jambos had activity against several species of S. aureus with MIC values that ranged from 0.50 to 0.75 mg/mL and suggested that more active inhibitory compounds are found in the leaves (0.21 to 0.83 mg/mL) than in the bark [32]. Additionally, methanolic extracts of Syzygium aromaticum showed inhibition against S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Salmonella enterica serovar Typhi and P. aeruginosa with MICs ranging from 31.25-250 μg/mL [33]. We did not find antimicrobial activity against P. aeruginosa, which suggests that variation in the polarities of the extraction solvents and the preparation methods used to make the extracts that were studied may account for the difference in the activities of these two species.

For nearly all of the plant species that have significant uses but that did not show antimicrobial activity, there is evidence that their activity varies significantly with the polarity and the concentration of the extracts, which in turn determines the phytochemicals responsible for the antibiotic effects [3, 34]. For instance, the antimicrobial effects of hydroalcoholic extracts from the leaves of Plectranthus amboinicus have been reported against S. aureus with a MIC of 9.3 mg/mL [35]. In another study [36], seven out of ten S. aureus strains obtained from exudates of otitis externa were sensitive to P. amboinicus extracts at concentrations ranging from 1.25 to 5.0 mg/mL. In particular, the essential oil of P. amboinicus was active against two of the strains tested at 4% and 8% (w/V). This result is noteworthy because the decoction, which corresponds to the traditional preparation that is used, significantly resembles the fraction obtained from a steam distillation, which carries the essential oils that would have been inactive in solution at the concentrations obtained in this study (5.3 – 21.3 μg/μL). The authors do not report the polarity of the extracts, although the use of hydroalcoholic extracts was suggested.

The polar methanolic extracts from the leaves and bark of Aristolochia trilobata showed activity against E. coli with a MIC value of 2.5 mg/mL [37]. This MIC value is comparable to the concentration of the decoctions tested in this study, which were 3.22 and 3.53 mg/mL for the stem and leaves, respectively. It is known that methanol extractions are more exhaustive than those of the aqueous or less polar organic and non-polar organic solvents. Therefore, the compounds that are responsible for the observed activity could be absent from the aqueous decoctions. However, the authors previously reported that the traditional use of this plant species has to be discouraged due to the mutagenic and possibly human carcinogenic properties of the aristolochic acids present in the methanol extracts and possibly in the water preparations of aerial parts of the plant.

Serrulatane quinonoid biflorin, which is isolated from the root tissue of Capraria biflora, has been reported to exhibit antimicrobial activities against Gram-positive bacteria [38]. This activity is consistent with the traditional use to treat conjunctivitis, which may be associated with infections by S. aureus, S. epidermidis and Propionibacterium spp., among others. Thus far, the activity of aqueous extracts from the leaves has not been reported.

Aqueous extracts from the rhizomes of Costus speciosus have been reported to exhibit antimicrobial activity against S. aureus at a concentration of 200 mg/mL [39]. The report describes the traditional system of administration studied herein, but given that the concentrations tested in our study varied from 11.8 – 47.3 μg/μL, a continuous administration of the decoction would be required to explain the traditional use for kidney infections.

Although there are several reports of the broad spectrum antimicrobial activity of Cucurbita moschata oil and seed extracts, no reports were found on the activity of decoctions from the leaves [40]. Likewise, only ethanol and acetone:ethyl acetate (1:1) extracts of the aerial parts of H. rosa-sinensis have been studied previously [41, 42]. The reported MIC and MBC values for the ethanolic extract of the flowers against S. aureus were 20 mg/mL. Thus, although the activities reported indicate that antimicrobial compounds are present in more polar solvents, higher concentrations than that found in the traditional remedy are needed to assess the antibacterial activity.

The results for Plantago major are similar to those described above. That is, at a concentration of 2 mg/mL in the hydroalcoholic extract, which is higher than those found in the decoctions (7.2 – 28.7 μg/μL), the authors reported MIC values of 1 mg/mL for S. aureus and > 1 mg/mL against E. coli, P. aeruginosa, C. albicans and others [43]. Furthermore, the antibacterial properties of lyophilized and fresh water extracts of Abelmoschus esculentus were effective against all bacterial strains tested, including S. aureus, E. coli and P. aeruginosa. The fresh extract displayed better antibacterial properties than the lyophilized extract [44]. The MIC values for peeled and unpeeled A. esculentus were 6.4 and 12.8 mg/mL for S. aureus and P. aeruginosa, respectively. In this case, the lipid fraction of A. esculentus was found to be responsible for the antibacterial properties, whereas the protein and polysaccharide fractions displayed no antimicrobial activity. The concentration of the A. esculentus decoction that was prepared in this study according to the traditional use reported during the ethnopharmacological survey was 12.6 mg/mL.

This study did not find antimicrobial activity that validates the traditional use of several herbal decoctions that showed significant use during TRAMIL ethnopharmacological surveys. Two separate analyses, the disc diffusion and the MIC assays, demonstrated that fewer than 33% of the herbal remedies that were examined exhibited antibacterial activities. This result is not discouraging because several factors, in addition to those studied, could determine the effectiveness of the extracts. First, the extracts could contribute to the treatment of disease, not necessarily by exerting an antibacterial response but by promoting an anti-inflammatory response. This effect was shown by Yang and collaborators, who demonstrated the antimicrobial and anti-inflammatory properties of the essential oils from Citrus sunki[45]. In addition, aqueous extracts are typically less potent formulations than those prepared with organic solvents such as methanol [46]. The solvent and the extraction system may modify the final results as described in the study of Argemone mexicana[47]. In this study, although the various solvent extracts examined were effective, the methanolic extracts showed maximum inhibition against the tested microorganisms, followed by hot aqueous extracts and cold aqueous extracts.