Abstract
Context: The number of multidrug resistant (MDR) microorganisms is increasing and the antimicrobial resistance expressed by these pathogens is generating a rising global health crisis. In fact, there are only a few antimicrobial agents left that can be used against MDR bacteria and fungi.
Objective: In this study, the antimicrobial activities of selected natural products from the flora of Northern Ontario against selected microorganisms are reported.
Materials and methods: Plants were collected from Sault Ste. Marie, Ontario, Canada, and ethanol extracts were prepared using EtOH:H2O (1:1, v/v). Fungal cultures used in this study were Candida albicans ATCC 10231 and Schizosaccharomyces octosporus. Bacterial cultures employed included Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Mycobacterium phlei ATCC 11758, and Streptococcus lactis ATCC 19435. The microplate resazurin assay was used to screen for antimicrobial activity.
Results: Extracts of four plant species Chimaphila umbellata L. (Pyrolaceae), Betula papyrifera Marshall (Betulaceae), Rhus typhina L. (Anacardiaceae), and Fraxinus pennsylvanica Marshall (Oleaceae), and six compounds (gallic acid, ethyl gallate, caffeic acid, sinapic acid, gentisic acid, and chlorogenic acid) demonstrated antibacterial or antifungal activities with MICs ranging from 62.5 to 1000 µg/mL, respectively, for a chemical fraction of an extract from Betula papyrifera against the bacterium S. aureus.
Discussion and conclusion: The present study has shown that certain plant extracts and select fractions and standard chemical compounds exhibit antimicrobial effects. Prince’s Pine, Chimaphila umbellate, White Birch, Betula papyrifera, Staghorn Sumac, Rhus typhina, and Green Ash, Fraxinus pennsylvanica were the principal extracts exhibiting notable antibacterial and/or antifungal activities; while gallic acid, ethyl gallate, and caffeic acid demonstrated antibacterial activities and sinapic acid, gentisic acid, and chlorogenic acid demonstrated antifungal activities.
Introduction
The number of multidrug resistant (MDR) microorganisms is increasing (Escaich, Citation2010). Escherichia coli, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, and Candida species are but a few among the well-known microorganisms and have shown an evolution in resistance to various pharmaceutical drugs (Eppinger et al., Citation2011; Hamouche & Sarkis, Citation2012; Howden et al., Citation2011; Riddell & Kauffman, Citation2008). The antimicrobial resistance expressed by these pathogens is generating a rising global health crisis. In fact, the misuse of antimicrobials is a leading factor in the emergence of antimicrobial resistance (Barah & Gonçalves, Citation2010), and according to Levy (Citation2002), many antimicrobials are used inappropriately. Consequently, there are only a few antimicrobial agents left that can be used against MDR bacteria and fungi (Curcio, Citation2010).
Because the levels of resistant pathogenic microorganisms are on the rise, novel and effective antimicrobial agents are needed to contain this epidemic. Recent studies have shown an interest in the chemical components of plants, especially the secondary phytochemicals, which are non-nutritive plant chemicals known to exhibit antimicrobial activity. Natural products obtained from various plant species have been used for maintaining health as evidenced by their extensive use in traditional medicine (Lee et al., Citation2011). In fact, traditional medicine is used by approximately 80% of individuals living in the developed countries (Nascimento et al., Citation2000). Researchers, such as Jones et al. (Citation2000), have selected plants based on the ethnobotanical information in correlation with their usage by their indigenous population for their studies. Therefore, the screening of plants for antimicrobial properties in combination with traditional medicine could lead to the discovery of new antimicrobials effective against MDR microorganisms.
The province of Ontario contains approximately 2% of the world’s forests (Natural Resources Canada, Citation2011). The Northern Ontario flora, which is mostly constituted by the Great Lakes Forest, the Boreal Forest, and the Hudson Bay Lowlands, provides a great number of vegetative species. Its geographic region lies north of Lake Huron, the French River, Lake Nipissing, and the Mattawa River. This area is mostly dominated by hardwoods, conifers, deciduous species, boreal spruce, balsam fir, jack pine, poplar, birch, and cedar (Natural Resources Canada, Citation2011). First Nations Peoples of Canada use a vast amount of plant species as medicine. Over 400 plants are used in native medicine, of which 105 plants were effective based on the phytochemical constituents and conifers were the most widely used group (Arnason et al., Citation1981). In this article, the antimicrobial activities of selected natural plant products recovered from the flora of Northern Ontario as well as the assessment of various chemical constituents against selected microorganisms are reported.
Materials and methods
Plants materials and chemical compounds
Materials consisted of 17 plant species (), 10 select fractions (), and 19 commercially available pure compounds (). Target plants species and appropriate plant parts were collected from Sault Ste. Marie and surrounding areas (latitude 46°32′ north and longitude 84°20′ west), during the summer months of July and August 2009–2010. Pressed voucher specimens of all species were mounted, labeled, and deposited for permanent accession in the Natural Products Laboratory, Great Lakes Forestry Centre-Sault Ste. Marie herbarium. Taxonomic identification of the selected plants was authenticated by an acknowledged authority. Information on each of the plants, including location, a description of habitat, notes on their growth, and development parameters also recorded.
Table 1. List of plant extracts from Northern Ontario tested for antimicrobial activity.
Table 2. Antimicrobial activity of fractions of extracts against microorganisms.
Table 3. List of compounds tested for antimicrobial activity.
Extractions
Fresh plant materials (approximately 2 kg fresh weight) were extracted at room temperature in a two-step process: first, the plant material was steeped in 4 L of 100% ethanol for 24 h followed by homogenization, filtration, and collection of the filtrate; and second, remaining mulched residue was re-extracted with 4 L of a mixture of EtOH:H2O (1:1, v/v) for an additional 24 h at which point the mulched extract was filtered and the filtrates from both processes were combined and evaporated under reduced pressure employing a rotary evaporator and a water bath set to 40 °C. Evaporation of the combined solvents was carried out until a small amount of extract remained. The residue was freeze-dried and weighed (Abou-Zaid & Scott, Citation2012; Abou-Zaid et al., Citation2000).
Fractionation
The ethanol freeze-dried extracts were the following four species: Prince’s Pine, White Birch, Staghorn Sumac, and Green Ash, F. pennsylvanicawere adsorbed onto polyvinylpolypyrrolidone (PVPP) powder (Sigma, Ronkonkoma, NY) and packed in a Buchner funnel. Solvent elution (fractionation) was carried out at a slow rate, initially with water followed by aliquots of increasing concentrations (20, 50, 70, and 100%) of ethanol. Fractions were monitored and selected for further analysis on the basis of their bioassay activity. Final clean-up of the compounds of interest was achieved by employing a Sephadex LH-20 column (2 × 50 cm), using methanol as the eluting solvent (Abou-Zaid & Scott, Citation2012; Abou-Zaid et al., Citation2000).
Identification of purified compounds
Structural elucidation was achieved both by (i) chemical analyses: acid hydrolysis in 2 M and 0.1 M HCl (mild hydrolysis) at 100 °C for 60 min, enzymatic hydrolysis with β-glucosidase using an acetate buffer (pH 5), hydrogen peroxide oxidation; and by (ii) physical analyses: UV spectroscopy; 1H-NMR; 13C-NMR, and (positive and negative) FAB-mass spectroscopy (Andersen & Markham, Citation2006; Dey & Harborne, Citation1989; Harborne, Citation1994), among others. Confirmation of the structure by comparison with authentic samples was also carried out whenever possible.
Ultra performance liquid chromatography conditions
Ultra performance liquid chromatography (UPLC) analysis was performed using a Waters ACQUITY UPLC equipped with a computer and Masslynx software (Waters Corp, Milford, MA), a binary solvent manager, a sample manager, and an autoscan photodiode array spectrophotometer detector (Waters Corp, Milford, MA) (PDA, λ). The UPLC was equipped with an ACQUITY UPLC BEH C18 (Waters Corp, Milford, MA), 1.7 μm (2.1 × 50 mm i.d.), reverse-phase analytical column from Waters (Milford, MA) housing a Van Guard BEH C18, and 1.7 μm reverse-phase pre-column. A linear gradient chromatographic technique was employed at room temperature with the following solvent system: Solvent A = 0.1% formic acid (aqueous); Solvent B = acetonitrile, starting at 95% A:5% B and ending 12 min later with 20% A:80% B, and a flow rate set at 0.5 mL/min. Two fixed detection wavelengths (280 nm and 350 nm) were used to monitor the eluting peaks. Resolved peaks were scanned by the photodiode array detector from 240 to 460 nm.
Solutions
All freeze-dried extracts were dissolved in 10% (v/v) DMSO–Milli Q water and were prepared in glass test tubes to obtain final concentrations of 1000 µg/mL and 10 000 µg/mL (w/v). All fractions and compounds were dissolved in 10% (v/v) DMSO and were prepared in glass test tubes to obtain a final concentration of 1000 µg/mL.
Reagent solutions and media
The resazurin solution used as a redox indicator was prepared by dissolving 270 mg of resazurin (Sigma-Aldrich, Ontario, Canada) in 40 mL of sterile distilled water. The iso-sensitest broth (ISB) (Oxoid, Ottawa, Canada) solution was prepared according to the manufacturer’s recommendations. The sterility of the final solutions was assured by utilizing the appropriate microbiology aseptic techniques.
Fungal cultures
The primary fungus used in this study was the yeast C. albicans ATCC 10231. Strains of this species are known to be MDR. The secondary fungus used was S. octosporus. All microbial cultures were provided by the American Type Culture Collection, with the exception of S. octosporus, which was obtained from the Laurentian University Culture Collection.
Bacterial cultures
The primary bacteria used in this study were S. aureus ATCC 29213, E. coli ATCC 25922, and P. aeruginosa ATCC 27853. Strains of these species are known to be MDR. The secondary bacteria used were M. phlei ATCC 11758 and S. lactis ATCC 19435.
Preparation of microbial cultures
Staphylococcus aureus, E. coli, P. aeruginosa, and C. albicans were grown on iso-sensitest agar (ISA) (Oxoid, Ottawa, Canada) and incubated for 24 h at 37 °C for 24 h for the bacteria and for 48 h at 30 °C for 48 h for the fungus, respectively. Streptococcus lactis was grown on brain heart infusion agar (ATCC medium 44) for 48 h at 37 °C. Mycobacterium phlei was grown on glycerol agar (ATCC medium 27) for 48 h at 37 °C. Schizosaccharomyces octosporus was grown on Sabouraud Dextrose Agar (SDA) for 48 h at 30 °C. Single colonies were subcultured in 150 mL of iso-sensitest broth (ISB) 1 × concentration. The flasks were incubated once again under the conditions previously mentioned. Serial dilutions of the microbial suspensions were made using sterile saline until the optical density in the range of 0.500–0.599 was obtained as determined using a spectrophotometer at 500 nm.
Microplate resazurin assay
To screen for antimicrobial activity, 96-well plates were prepared according to the methods of Sarker et al. (Citation2007). All natural substances were tested in triplicate (columns 1, 2, and 3) and all plate assays included a sterile control (C1), a negative control (C−), and a positive control (C+) (see figure below). In the first row of the positive control wells, 100 µL of ciprofloxacin (antibacterial agent; 3000 μg/mL (w/v) for E. coli, 6000 μg/mL (w/v) for S. aureus, 384 μg/mL (w/v) for P. aeruginosa, 5000 μg/mL (w/v) for M. phlei, and 1000 μg/mL (w/v) for S. lactis) or amphotericin B (antifungal agent; 786 μg/mL (w/v) for C. albicans and 384 μg/mL (w/v) for S. octosporus) dissolved in 10% DMSO was pipetted. 10% DMSO (100 µL) was pipetted into the first row of the negative control wells. One hundred microliters of the relevant test substance was pipetted into the first row wells labeled 1, 2, and 3 and into the first row wells of C1, the sterile control. Fifty microliters of 3.3 × strength ISB was added to all other wells. Subsequently, a two-fold serial dilution was performed using a multi-channel pipette. Next, 10 µL of resazurin was pipetted into each well, 10 µL of 0.9% sterile saline was pipetted into the wells of the sterile control columns, and 10 µL of microbial suspension (OD of 0.500–0.599) was pipetted into each well, with the exception of the sterile control columns. 30 µL of 3.3 × strength ISB was added to each well to ensure a final volume of 100 µL. Below is an example of resazurin microtiter assay result.
Resazurin is a blue-colored redox indicator. Reactions indicating positive results were represented by blue-colored solutions, indicating inhibition of microbial growth. When the oxygen within the medium is limited, indicating microbial growth, the resazurin is reduced and the color changes from blue to pink. The wells showing no microbial growth were tested with a micro-probe to verify the pH (buffer 7) of their contents. Furthermore, the change in color from blue to pink will permit the determination of the minimum inhibitory concentration (MIC).
Microplates were incubated for 22 h at 37 °C for bacteria and for 46 h at 30 °C for yeasts, respectively.
Fractionation
Plant extracts that demonstrated antimicrobial activity using initial concentrations of 1000 µg/mL were further fractionated. The fractions were tested for antimicrobial activity employing the same protocol as described above against the same primary microorganisms. The compounds were identified based on the methods of Dey and Harborne (Citation1989) and Harborne (Citation1994). Final separation, purification, and quantification of the individual components found in the crude extracts were carried out using high-performance liquid chromatography (HPLC) equipped with a photodiode array spectrophotometric detector measuring peak areas at 280 and 350 nm. The fractions were used for antimicrobial activity testing. Fractions were tested using the same protocol as described above against the same primary microorganisms.
Results
With an initial concentration of 1000 µg/mL, only extracts #4 (Prince’s Pine) and #8a (White Birch) demonstrated signs of antimicrobial effects against S. aureus with MICs of 250 µg/mL and 1000 µg/mL, respectively. These extracts were further separated into fractions and tested against only the primary microorganisms at initial concentrations of 1000 µg/mL. Fractions 1–4 of plant extract #4 demonstrated positive antimicrobial activities against S. aureus with varying MICs ( and ). Interestingly, fractions 2 and 3 of extract #4 also showed antimicrobial activities against E. coli and C. albicans. Fractions 2 and 3 of plant extract #8a were positive against only S. aureus with MICs of 500 µg/mL and 62.5 µg/mL, respectively ( and ).
Table 4. MIC (µg/mL) of fractions of extracts against microorganisms.
Starting with an initial concentration of 10 000µg/mL, extract #4 (). Extracts #10b and #10c were positive against the three primary bacteria and extracts #8b, 8c, and 11 were positive against the fungi, C. albicans (). In addition, all extracts had an antimicrobial effect against S. aureus at this concentration with varying MICs ().
Table 5. Antimicrobial activity of extracts against microorganisms.
Table 6. MIC (µg/mL) of extracts against microorganisms.
With an initial concentration of 1000 µg/mL, gallic acid, ethyl gallate, and caffeic acid were positive against S. aureus with MICs of 500, 1000, and 1000 µg/mL, respectively ( and ). Gallic acid was also positive against M. phlei with an MIC of 1000 µg/mL. Moreover, sinapic acid, gentisic acid, and chlorogenic acid were positive against C. albicans and S. octosporus, all with MICs of 1000 µg/mL.
Table 7. Antimicrobial activity of compounds against microorganisms.
Table 8. MIC (µg/mL) of compounds against microorganisms.
The known pharmaceutical antimicrobial agents, ciprofloxacin (antibacterial) or amphotericin B (antifungal), as expected inhibited the growth of the target microorganisms. The pH values of all antimicrobial extracts, fractions, and pure compounds were measured and indicated that they were within the neutral zone, demonstrating that the inhibition of microbial growth was not influenced by either acidic or alkaline conditions.
Discussion
Given the growing concern and problems associated with the issues of MDR microorganisms in medicine, it is important and imperative to find new and effective antimicrobial agents to manage such problems. Many plants are known to produce substances that show antimicrobial activity. The diverse flora of Northern Ontario forests offers the possibility of finding phytochemicals that have antimicrobial properties. It was the purpose of this study to examine the antimicrobial activities of selected natural products from the flora of the Boreal and Great Lakes Forests.
At 10 000 µg/mL, all extracts were positive against S. aureus, suggesting that this species of bacterium had little defense against the natural substances present in the extracts at such high concentrations. However, it is also consistent with the fact that Gram-positive bacteria, such as S. aureus, are usually more sensitive to antibiotics than Gram-negative bacteria, like E. coli and P. aeruginosa (Cos et al., Citation2006).
As previously mentioned, the extract of Prince’s Pine was effective against all primary microorganisms starting with an initial concentration of 10 000 µg/mL. Prince’s Pine is a common plant used as a traditional medicine by North American First Nations people mostly for its antimicrobial, anti-inflammatory, alterative, diuretic, astringent, and urinary antiseptic properties (Caldecott, Citation2010; Moerman, Citation2004).
The extracts made from the berries of Staghorn Sumac also proved to be effective against the three primary bacteria when using an initial concentrations of 10 000 µg/mL. These results are similar to the findings by Borchardt et al. (Citation2008), where the berries of Rhus typhina demonstrated clear inhibition zones against S. aureus, E. coli, and P. aeruginosa. However, a partial inhibition zone was also observed for C. albicans. Borchardt et al. (Citation2008) tested the drupes of the plant both with and without the pericarp. Only the extract containing the pericarp showed antimicrobial activity, suggesting that the phytochemicals responsible for this activity were to be found in the fruits and hairs surrounding the seeds. Interestingly, the Staghorn Sumac has been used in healing and in ceremonies by some native communities in Canada (Arnason et al., Citation1981).
The extracts of the twigs and branches, and phloem of White Birch, and the extract of the foliage of Green Ash exhibited antifungal activity against C. albicans using initial concentrations of 10 000 µg/mL. In a study conducted by Omar et al. (Citation2000), the bark and wood extract of B. papyrifera showed a positive antibacterial activity against methicillin sensitive S. aureus (MSSA). The genus Fraxinus has been used in traditional medicine. In fact, many natural compounds have been identified in the Fraxinus species that possess anti-inflammatory, immunomodulatory, antimicrobial, antioxidative, skin regenerating, photodynamic damage prevention, liver protecting, diuretic, and anti-allergic activities (Kostova & Iossifova, Citation2007).
The fact that P. aeruginosa and E. coli were resistant to the fractions of extracts 3 and 8 is probably a reflection of the complex nature of the Gram-negative cell wall. In other words, it is quite possible that some substances, because of their size, shape, or other extenuating factors, simply cannot cross the outer membranes of such bacteria and, therefore, cause no effect.
As previously mentioned, gallic acid, ethyl gallate, and caffeic acid exhibited antimicrobial activity against S. aureus. Since S. aureus was the only Gram-positive bacterium of the primary bacteria, those three compounds were believed to affect only Gram-positive bacteria. Therefore, the aforementioned compounds were tested against two other Gram-positive bacteria, namely M. phlei and S. lactis. Of the three compounds tested, only gallic acid demonstrated antimicrobial activity against M. phlei.
Gallic acid is a well-known natural compound, and, when tested alone or in combination with other natural products, has been shown to exhibit both antimicrobial and antiviral activities. Sato et al. (Citation1997) have shown that gallic acid, isolated from Terminalia chebula RETS, is effective against E. coli and P. aeruginosa with MICs of >1000 µg/mL. This study also demonstrated its antibacterial activity against 20 strains of MRSA and seven strains of MSSA. An increase in our initial concentration of gallic acid may have reflected the results observed by Sato et al. (Citation1997).
It is also important to note that gallic acid is a compound found in R. typhina (Borchardt et al., Citation2008). The extracts of R. typhina were not active against the microorganisms when using an initial concentration of 1000 µg/mL. However, when increasing the initial concentration to 10 000 µg/mL, all R. typhina extracts showed positive activity ( and ).
In the study conducted by Sato et al. (Citation1997), ethyl gallate, isolated from the fruits of Terminalia chebula RETS, proved to be effective against 20 different strains of MRSA and seven different strains of MSSA with MICs varying from 31.3 to 62.5 μg/mL. Additionally, ethyl gallate has been shown to have a synergistic effect when combined with tetracycline, mupirocin, and fusidic acid against specific MRSA and MSSA strains (Sow et al., Citation2010).
Varying results have been obtained when studying the antimicrobial properties of caffeic acid. On one hand, according to Eppinger et al. (Citation2011), this compound was effective against S. aureus with an MIC of <200 μg/mL, and also against E. coli with an MIC of 100 µg/mL when isolated from the hairy roots of Salvia miltiorrhiza. On the other hand, a study conducted by Rauha et al. (Citation2000) has shown that caffeic acid (C-0625) was not effective against S. aureus, E. coli, and C. albicans when using a 500 µL volume of the compound (1000 µg/mL) in the hole-plate diffusion method. It also demonstrated a slight antibacterial activity against P. aeruginosa, although the results regarding antimicrobial activity were somewhat variable. Future studies should include increased concentrations of this compound to verify its activity.
As previously mentioned, sinapic acid, gentisic acid, and chlorogenic acid were the only three compounds effective against C. albicans and consequently tested against another fungus, S. octosporus, to assess their antifungal activity. All three compounds demonstrated an antimicrobial activity against the latter fungus.
A study conducted by Ayaz et al. (Citation2008) demonstrated that a phenolic fraction (ester-bound) extracted from the seeds of kale (Brassica oleraceae L. var. acephala DC) was effective against C. albicans. This specific extract contained 2505 ng/g of sinapic acid, which was the most abundant compound found in the extract. In regard to gentisic acid, the results of this present study concur with the results of the study conducted by Pinheiro et al. (Citation2003), which found this compound to be inactive against E. coli, S. aureus, and P. aeruginosa. Moreover, gentisic acid is currently being used in combination with fludioxonil for treatments against fungi because it increases the activity of this fungicide (Kim et al., Citation2007). Chlorogenic acid has been known to possess antifungal activities. In fact, novel chlorogenic acid-based antifungal agents have been developed and exhibit novel mechanisms of action exhibiting low toxicity (Daneshtalab, Citation2008).
Along with the background history and applied uses of traditional medicine, the pharmacological screening of phytochemicals found in plant extracts is leading to the discovery of novel and effective antimicrobial agents. The present study has shown that certain plant extracts and compounds have antimicrobial effects/activities. Prince’s Pine, White Birch, Staghorn Sumac, and Green Ash were the principal extracts exhibiting notable antibacterial and/or antifungal activities. Gallic acid, ethyl gallate, and caffeic acid were the compounds exhibiting antibacterial activities. Moreover, sinapic acid, gentisic acid, and chlorogenic acid were the compounds demonstrating antifungal activity. Although many of the extracts tested demonstrated antimicrobial activity at relatively high concentrations, it is possible that some could be useful for certain topical applications in ointments and creams to control infections of the skin or mucus membranes. Also, although MDR microorganisms were not used in this study, MDR strains of the species selected typically exist. It is imperative, and important, that subsequent work in this area should include MDR strains of microorganisms to assess the efficacy of the selected extracts, fractions, and chemical compounds tested within this study. It would also be important to include experiments using plating techniques to test whether the active extracts are bactericidal or bacteriostatic.
Conclusion
The results of the present study, in accordance with the results of previously mentioned studies, demonstrate that extracts of Prince’s Pine, Staghorn Sumac, White Birch, and Green Ash, in addition to certain pure compounds, have the potential of being developed into new antimicrobial drugs or to be used in combination with other drugs. The discovery and development of novel and effective phytomedicines are urgently needed as the incidence of antimicrobial resistance in pathogenic microorganisms is increasing.
Declaration of interest
The authors report that they have no conflicts of interest. We acknowledge the financial support of the Natural Resources Canada, Canadian Forest Service – Canadian Wood Fibre Centre and the Government of Ontario for the project “Forest FAB: Applied Genomics for Functionalized Fibre and Biochemicals” (ORF-RE-05-005).
Related Research Data
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