The antibacterial and antifungal analysis of crude extracts from the leaves and bark of Pimenta species found in Jamaica

Due to the rapid increase in the number of antibiotic-resistant bacteria, medicinal plants are being studied as new and promising alternatives to conventional antibiotic treatment. The crude hexane, ethyl acetate and methanol extracts from Pimenta dioica, Pimenta jamaicensis and Pimenta racemosa were quantitatively assessed to determine their antimicrobial susceptibility and potency using zones of inhibition methods, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations minimum (MBC) or fungicidal concentrations concentration (MFC) against Streptococcus A, Streptococcus B, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Salmonella species, Proteus mirabilis and Candida albicans. The crude ethyl acetate extract of the P. dioica was most active against Candida albicans (MFC of 1.3 mg/mL and MIC of 0.63 mg/mL) while the crude hexane extract of P. jamaicensis was most active against Streptococcus A (MBC of 0.63 and MIC of 1.3 mg/mL). The crude ethyl acetate extract of P. racemosa was most active against Streptococcus A and Salmonella (MBC of 2.5 mg/mL and MIC of 1.3 mg/mL; MBC of 0.63 and MIC of 0.63 mg/mL, respectively). Extracts from selected species of Pimenta may potentially provide a source of new antimicrobial agents for the treatment of infectious diseases.


INTRODUCTION
The spice of Jamaican cuisine is often times completed with a dash of Pimenta, also known as allspice, Jamaican pepper, new spice or myrtle pepper.Its distinct flavor results from its unique blend of terpenes and other *Corresponding author.E-mail: lowebiotech@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License secondary metabolites.These compounds have also contributed to the medicinal properties of the plant and therefore used to treat various ailments in Jamaican folk medicine.The genus Pimenta contains about 14 species from the Myrtaceae family, all of which are indigenous to the Caribbean and Central America; Jamaica has 6 endemic species found within the genus (Adams, 1976).The species that may be found in Jamaica include Pimenta dioica (Allspice), Pimenta racemosa (bay rum), Pimenta jamaicensis (wild pimento), Pimenta obscura, Pimenta richardii and a species not yet identified (Institute of Jamaica Identification number: P22536B).These flowering plants may be monoecious or dioecious and contain many active ingredients.Pimenta is entrenched in many folklore remedies and used to treat diabetes, malaria and other chronic and infectious diseases.
The chemical profile of the Pimenta genus is distinct and contributes to the diversity in the use of the plant.The active components found in P. dioica, P. obscura and P. richardii are reported to be methyl eugenol, caryophyllene, and limonene identified via chemical analysis (Zhang et al., 2012).The medicinal properties for P. dioica and P. racemosa have been studied extensively.The essential oils and extracts from these plant species have been found to contain antimicrobial and antifungal properties (Martinelli et al., 2017;Hammer et al., 1999;Zabka et al., 2009), anti-nociceptive properties as well as anti-cancer activity against prostate cancer cell lines (Garcıa et al., 2004).P. dioica also has significant cytotoxic, anti-oxidant, analgesic and antihypertensive effects (Suárez et al., 1997;Marzouk et al., 2007).The aqueous extract of the berries showed significant antitumor activity, while ethanolic extracts from the leaves and berries can also be used as a food preservative (Sessou et al., 2012).The macerated leaves of P. dioica have previously shown some antimicrobial activity with crude hexane and alcoholic extracts against Pseudomonas fluorescens, Bacillus megaterium, Aspergillus niger and Penicillium species (Boyd et al., 2014).Other studies have shown that essential oils obtained from P. dioica have the potential of being used as a natural pesticide and fungicide (Seo et al., 2009).
Essential oils of P. racemosa possess several types of bioactivity that have been validated experimentally.Alitonou et al. (2012) reported that the oil has low antiinflammatory activity based on the lipoxygenase test results, as well as, antiradical, acaricidal and antimicrobial activity against both bacteria and fungi.It was postulated that the oils can be exploited industrially for its antioxidant activity to delay the degradation of fatty substances.Neither of the crude extracts (hexane, ethyl acetate and chloroform) of P. racemosa was found to have any significant effect as a cytotoxic agent.In a research conducted to assess the antimicrobial activity of 52 plants, P. racemosa was one of the three plants that showed anti-microbial activities on all the following organisms: Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp.enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method (Hammer et al., 1999), which also formed a part of the basis for this study.Nesrin et al. (2017) have demonstrated that the most abundant component of essential oil obtained from P. racemose is methyleugenol and suggested that the essential oil holds great promise as natural preservative and antimicrobial agents.P. jamaicensis contains eugenol and limonene which contributes to its characteristic smell and properties; other compounds include p-cymene, 1, 8-cineole, βcaryophyllene, and 4-terpineol as the main constituents (Tucker et al., 1992).
The potential for new antimicrobial agents is dependent on their experimental validation when extracts or compounds are exposed to different microorganisms.Plant-based secondary metabolites have immense potential for combating a broad spectrum antibioticresistant bacterium with little or no side effects (Chandra et al., 2017).The literature suggests that Pimenta species could hold significant promise as an antibiotic agent.In this study, the antifungal and antimicrobial activity of various crude extracts (hexane, methanol and ethyl acetate) was evaluated for P. dioca, P. racemose and P jamaicensis.

Plant collection and extraction
The plant materials were obtained and verified by the taxonomist at The University of the West Indies, Mona Herbarium and samples were given reference numbers 35922, 35923 and 35924 for P. dioica, P. racemosa, and P. jamaicensis, respectively.The samples (leaves and bark) were dried at room temperature and milled into a powder prior to solvent extraction with hexane, ethyl acetate and methanol to obtain crude extracts of each species.The first solvent, hexane, was percolated through the plant material for 24 h, filtered, concentrated in vacuum under reduced pressure using rotary evaporator and dried in a desiccator to obtain the crude hexane extract.The same plant material was then treated with ethyl acetate and methanol sequentially for the same time period to obtain the crude ethyl acetate and methanol extracts.A sample of 0.1 g of the plant extracts was dissolved in 10 ml of dimethyl sulphoxide (DMSO) and a serial dilution was carried out to obtain 6 extract concentrations (10,000, 5000, 2500, 1250, 625, and 312.5 µg/mL) which was then used in the microbial test.

Anti-microbial assays
All the extracts were screened to assess their antibacterial and antifungal properties using agar-well diffusion method (Perez et al., 1990).The samples were further tested at 1 mg/mL (the maximum concentration at which an extract may be considered active controls) (DeMarsh et al., 2001).The minimal inhibitory concentration (MIC) of the active samples was determined using extracts obtained from leaves and bark of the respective Pimenta spp.

Well diffusion assay
Suspensions made from 24 h bacterial or fungal cultures were standardized using 0.5 McFarland Standard (ca. 10 8 CFU/mL) and inoculated on the surface of Mueller-Hinton agar (bacteria) and Sabouraud dextrose agar (fungi) plates using sterile cotton swab (Baker et al., 1983).The diluted crude extract and the control (100 µl of each) were then introduced into the wells of 6 mm diameter that were created in the agar.The plates were kept at 4°C for 1 h to permit diffusion of the extracts, then incubated at 37°C for 24 (bacteria) and 48 h (fungi) and the diameter of the zone of growth inhibition was then measured using a caliper.The experiment was repeated for reliability.

Tube dilution assay
The extracts that showed antimicrobial activity were further tested to determine the MIC for each bacterial or fungicidal sample. 1 mL of each extract was added to 1 mL of sterile brain heart infusion broth and used to prepare two-fold broth micro-dilutions of the various extracts (Ferraro, 2000).The tubes were inoculated with a drop of microbial suspension and incubated for at 37°C for 24 h, after which the MICs were measured macroscopically to assess the efficacy and effectiveness of the extracts in comparison with the sterility and growth controls which were included for each assay (DeMarsh et al., 2001).All the tubes were evaluated for growth indicated by turbidity.The samples at each concentration, sterile and growth controls were then seeded onto blood agar plates and incubated at 37°C for 24 h after which the minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) was recorded.The MBC or MFC was defined as the lowest concentration of the extract associated with no bacterial or fungal growth on a freshly inoculated agar plate.

RESULTS
The antibacterial and antifungal activity of P. jamaicensis, P. dioica and P. racemosa are outlined (Tables 1 and 2).All the leaf extracts of the investigated Pimenta spp.showed significant antimicrobial activity against one or more microbes, except for the crude hexane extract of P. dioica and crude methanol extract of P. racemosa (Table 1).P. racemosa was the most active of the three Pimenta spp., with the ethyl acetate extract being most active against Salmonella and Streptococcus B and the hexane extract being most active against C. albicans.The ethyl acetate extract of P. dioca and hexane extract of P.

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jamaicensis were most active against C. albicans and Streptococcus A, respectively.There was no significant antimicrobial activity observed for E. faecalis, E. coli, P. mirabilis and K. pneumonia using any of the extracts.The MBCs and MICs exhibited by the extract against test bacteria ranged between 0.63 and 2.5 mg/mL and 0.63 and 1.3 mg/mL, respectively (Table 2).The growth of C. albicans was inhibited by P. dioica leaf ethyl acetate extracts giving a MFC of 1.3 mg/mL and MIC of 0.63 mg/mL.Of the three plant species, the P. racemosa leaf ethyl acetate extracts were the most potent antibacterial agents against the Gram-positive bacteria, Streptococcus B (MBC of 0.63 mg/mL and MIC of 0.63 mg/mL).This extract also showed an antibacterial effect against the Gram-negative bacteria, Salmonella spp., but was less potent (MBC of 2.5 mg/mL and MIC of 1.3 mg/mL).

DISCUSSION
Pimenta spp.have shown significant promise based on the preliminary tubal broth dilution assays and subsequent inhibitory and bactericidal tests conducted.The MIC indicates the lowest concentration that can inhibit growth, while the MBC determines the concentration at which a compound or extract will kill the bacteria being tested.The MIC and MBC are complementary in that the extract or compound would be most effective as the MIC determines if it is antiproliferative and then the MBC determines if it is also bactericidal.
The results reveal that both Gram-positive and Gramnegative bacteria and the fungi C. albicans were susceptible to the effects of the extracts obtained from Pimenta plant species.As outlined in Table 1, all three crude leaves extracts of the endemic P. jamaicensis showed antibacterial activities against microbes such as Streptococci which are known to cause numerous ailments.Streptococcus A (Group A Streptococcus, GAS) was susceptible to the effect of the P. jamaicensis crude extracts, however, the hexane extract was the most active, while the crude ethyl acetate and methanol extracts were partially active against Streptococcus B. Streptococcus A is usually spread via mucus from the nose, throat or via skin, and is often treated with antibiotics.Over 500,000 deaths have been reported per year for GAS related diseases (Cohen-Poradosu and Kasper, 2007).The P. jamaicensis leaf hexane extract could be an effective natural astringent or disinfectant which could aid with growth control of Streptococcus A. Streptococcus B or Streptococcus agalactiae (Group B Streptococcus, GBS) are bacteria also found commonly in the human body that may cause infections, especially with pregnant women and newborns.The highest cases of GBS infections were observed in non-pregnant women above 65 years old, with the incidence decreasing exponentially with age.The P. racemosa ethyl acetate extract was active against Streptococcus B with MBC of 0.63 mg/mL and MIC of 0.63 mg/mL which is comparable to that of fluconazole (Barchiesi et al., 1994), while the hexane extract showed partial activity.The P. jamaicensis ethyl acetate leaf and methanol extracts and P. dioica methanol leaf extract also showed partial activity for GBS.All three Pimenta species crude leaves extracts were partially active against P. aeruginosa, which has multiplemechanisms of resistance to antibiotics and can only be treated by a selected number of anti-pseudomonal agents.This gram-negative bacterium has about 13 different strains, which is often implicated in ailments such as pneumonia, urinary tract infections, gastrointestinal infections, hemorrhage, and nosocomial infections.It thrives on moisture and as such is often found in persons hospitalized for over a week via cross infection due to catheters and other medical equipment.Its presence inside the body can be fatal when associated with some of the major organs such as the kidney and lungs.P. aeruginosa growth may be reduced or retarded by the P. racemosa hexane leaf extract, P. dioica ethyl acetate leaf extract, and the P. jamaicensis ethyl acetate leaf extract.
Interestingly, the gram-negative Salmonella spp.which is often associated with gastrointestinal diseases, are susceptible to the effect of P. diocia and P. racemosa which are often added to food to enhance the flavor.The crude methanol extract of P. dioica was partially active while the crude ethyl acetate P. racemosa was active against Salmonella spp.This bacteria has been implicated in over one million cases of food poisoning annually (Centre for Disease Control and Prevention, 2016).According to the Centre for Disease Control and Prevention, Salmonella infection can be extreme and cause death, as reported in 2013 where over 19,000 patients were hospitalized for salmonellosis, from which 378 died.These infections are preventable with simple hands hygiene techniques, the separating of uncooked and cooked foods, cooking food properly and keeping foods refrigerated.Nonetheless, these crude extracts may have applications as an antimicrobial agent, to reduce Salmonella growth and as such reduce the prevalence of food borne illnesses.
C. albicans is a yeast-like fungus that forms a part of the normal intestinal microbiota of 70% of the human population.Its overgrowth may cause infections when there is favorable change in the internal conditions of the body, which results in candidiasis making this fungus the fourth leading cause of nosocomial infection (Pfaller and Diekema, 2007).Infections involving skin or mucosal surfaces are usually combated with oral or topical agents.C. albicans may, however, become invasive, traveling throughout the bloodstream causing damage to major organs requiring parenteral antifungal therapy.The P. racemosa and P. dioica were the most effective against C. albicans with the crude hexane leaf extract and the crude ethyl acetate leaf extract, respectively showing significant activity in the initial screening of the microbes.In the second stage of antimicrobial confirmation, the MICs of the P. jamaicensis ethyl acetate crude leaf extract was 99% (0.63 mg/mL) against C. albicans which was comparable fluconazole, antifungal, which usually has MIC of 92% against this organism (Barchiesi et al., 1994).

Conclusion
The use of Pimenta spp.has scientific credential as an antimicrobial agent.This is particularly important as globally there has been an increase in antimicrobial resistance, while fewer antimicrobial agents are being made, creating a possible scenario where there are no options for treatment.Most significant activity for P. jamaicensis was against the Streptococcus A bacteria, P. dioica was active against C. albicans and P. racemosa was active against Salmonella, Streptococcus B and C. albicans.These Pimenta species showed significant inhibitory and antimicrobial properties against the respective microbes and may be further studied in order to see whether they can be effectively incorporated into disinfecting, antimicrobial or antiseptic formulations to aid in combating diseases caused by these microbes.
Proteus mirabilis (7002) and fungal isolate (C.albicans; ATCC 14053)) used for this study were obtained from The University of the West Indies, Mona (Microbiology Department).

Table 1 .
Antibacterial and antifungal activity of Pimenta dioica, Pimenta jamaicensis and Pimenta racemosa leaf extracts at 1 mg/mL through well diffusion method.

Table 2 .
The MBCs/MFCs and MICs of the most effective crude extract of P. jamaicensis, P. dioica and P. racemosa using tube dilution assay.