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Pharm Biol, 2015; 53(6): 800–806
! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.942867

ORIGINAL ARTICLE

Antimicrobial activity of natural products from the flora of Northern

Ontario, Canada
Janique Vandal1, Mamdouh M. Abou-Zaid2, Garry Ferroni3, and Leo G. Leduc1
1
Department of Biology, Laurentian University, Sudbury, Ontario, Canada, 2Natural Resources Canada, Canadian Forest Service, Great Lakes
Forestry Centre, Sault Ste Marie, Ontario, Canada, and 3Division of Medical Sciences, Northern Ontario School of Medicine, Sudbury, Ontario,
Canada

Abstract Keywords
Context: The number of multidrug resistant (MDR) microorganisms is increasing and the Betula, C. albicans, chimaphila, E. coli,
antimicrobial resistance expressed by these pathogens is generating a rising global health Fraxinus, P. aeruginosa, phenolics, S. aureus
crisis. In fact, there are only a few antimicrobial agents left that can be used against MDR
bacteria and fungi. History
Objective: In this study, the antimicrobial activities of selected natural products from the flora of
Northern Ontario against selected microorganisms are reported. Received 22 February 2013
Materials and methods: Plants were collected from Sault Ste. Marie, Ontario, Canada, and Revised 28 April 2014
ethanol extracts were prepared using EtOH:H2O (1:1, v/v). Fungal cultures used in this study Accepted 5 July 2014
were Candida albicans ATCC 10231 and Schizosaccharomyces octosporus. Bacterial cultures Published online 20 February 2015
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 mg/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 used inappropriately. Consequently, there are only a few

antimicrobial agents left that can be used against MDR
The number of multidrug resistant (MDR) microorganisms is
bacteria and fungi (Curcio, 2010).
increasing (Escaich, 2010). Escherichia coli, methicillin-
Because the levels of resistant pathogenic microorganisms
resistant S. aureus (MRSA), Pseudomonas aeruginosa, and
are on the rise, novel and effective antimicrobial agents are
Candida species are but a few among the well-known
needed to contain this epidemic. Recent studies have shown
microorganisms and have shown an evolution in resistance
an interest in the chemical components of plants, especially
to various pharmaceutical drugs (Eppinger et al., 2011;
the secondary phytochemicals, which are non-nutritive plant
Hamouche & Sarkis, 2012; Howden et al., 2011; Riddell &
chemicals known to exhibit antimicrobial activity. Natural
Kauffman, 2008). The antimicrobial resistance expressed by
products obtained from various plant species have been used
these pathogens is generating a rising global health crisis.
for maintaining health as evidenced by their extensive use in
In fact, the misuse of antimicrobials is a leading factor in the
traditional medicine (Lee et al., 2011). In fact, traditional
emergence of antimicrobial resistance (Barah & Gonçalves,
medicine is used by approximately 80% of individuals living
2010), and according to Levy (2002), many antimicrobials are
in the developed countries (Nascimento et al., 2000).
Researchers, such as Jones et al. (2000), have selected
Correspondence: Mamdouh M. Abou-Zaid, Natural Resources Canada, plants based on the ethnobotanical information in correlation
Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste Marie,
Ontario, Canada P6A 2E5. E-mail: mamdouh.abouZaid@nrcan- with their usage by their indigenous population for their
rncan.gc.ca studies. Therefore, the screening of plants for antimicrobial
DOI: 10.3109/13880209.2014.942867 Antimicrobial of Northern Ontario flora 801

properties in combination with traditional medicine could Centre-Sault Ste. Marie herbarium. Taxonomic identification
lead to the discovery of new antimicrobials effective against of the selected plants was authenticated by an acknowledged
MDR microorganisms. authority. Information on each of the plants, including
The province of Ontario contains approximately 2% of location, a description of habitat, notes on their growth,
the world’s forests (Natural Resources Canada, 2011). The and development parameters also recorded.
Northern Ontario flora, which is mostly constituted by the
Great Lakes Forest, the Boreal Forest, and the Hudson Bay
Extractions
Lowlands, provides a great number of vegetative species. Its
geographic region lies north of Lake Huron, the French River, Fresh plant materials (approximately 2 kg fresh weight) were
Lake Nipissing, and the Mattawa River. This area is mostly extracted at room temperature in a two-step process: first,
dominated by hardwoods, conifers, deciduous species, boreal the plant material was steeped in 4 L of 100% ethanol for
spruce, balsam fir, jack pine, poplar, birch, and cedar (Natural 24 h followed by homogenization, filtration, and collection
Resources Canada, 2011). First Nations Peoples of Canada of the filtrate; and second, remaining mulched residue was
use a vast amount of plant species as medicine. Over 400 re-extracted with 4 L of a mixture of EtOH:H2O (1:1, v/v)
plants are used in native medicine, of which 105 plants were for an additional 24 h at which point the mulched extract
effective based on the phytochemical constituents and coni- was filtered and the filtrates from both processes were
fers were the most widely used group (Arnason et al., 1981). combined and evaporated under reduced pressure employing
In this article, the antimicrobial activities of selected natural a rotary evaporator and a water bath set to 40 ! C.
plant products recovered from the flora of Northern Ontario Evaporation of the combined solvents was carried out until
as well as the assessment of various chemical constituents a small amount of extract remained. The residue was freeze-
against selected microorganisms are reported. dried and weighed (Abou-Zaid & Scott, 2012; Abou-Zaid
et al., 2000).
Materials and methods
Plants materials and chemical compounds Table 2. Antimicrobial activity of fractions of extracts against
microorganisms.
Materials consisted of 17 plant species (Table 1), 10 select
fractions (Table 2), and 19 commercially available pure Fractions of extracts
compounds (Table 3). Target plants species and appropriate (1000 mg/mL) E. coli S. aureus P. aeruginosa C. albicans
plant parts were collected from Sault Ste. Marie and Fraction 1 of Extract 3 " + " "
surrounding areas (latitude 46! 320 north and longitude Fraction 2 of Extract 3 + + " +
84! 200 west), during the summer months of July and August Fraction 3 of Extract 3 " + " +
Fraction 4 of Extract 3 " + " "
2009–2010. Pressed voucher specimens of all species were Fraction 2 of Extract 8 " + " "
mounted, labeled, and deposited for permanent accession in Fraction 3 of Extract 8 " + " "
the Natural Products Laboratory, Great Lakes Forestry
+ indicates activity; " indicates no activity.

Table 1. List of plant extracts from Northern Ontario tested for antimicrobial activity.

No. Common name Family Scientific name Plant part

1 Balsam Fir Pinaceae Abies balsamea (L.) Mill. Needles
2 Jackpine Pinaceae Pinus banksiana Lamb. Needles
3a Eastern Hemlock Pinaceae Tsuga canadensis (L) Carriere Needles
3b Eastern Hemlock Pinaceae Tsuga canadensis (L) Carriere Twigs and branches
3c Eastern Hemlock Pinaceae Tsuga canadensis (L) Carriere Twigs and branches
4 Prince’s Pine Ericaceae Chimaphila umbellate (L.) Spreng. Foliage
5 Wintergreen Ericaceae Gaultheria hispidula (L.) Muhl. Foliage
6a Cow Parsnip Apiaceae Heracleum maximum Bart. Stem and leaves
6b Cow Parsnip Apiaceae Heracleum maximum Bart. Flowers and seeds
7 Partridge-berry Rubiaceae Mitchella repens L. Foliage
8a White Birch Betulaceae Betula papyrifera Marsh. Foliage
8b White Birch Betulaceae Betula papyrifera Marsh. Twigs and branches
8c White Birch Betulaceae Betula papyrifera Marsh. Phoem
9a Yellow Birch Betulaceae Betula alleghaniensis Britt. Foliage
9b Yellow Birch Betulaceae Betula alleghaniensis Britt. Twigs and branches
9c Yellow Birch Betulaceae Betula alleghaniensis Britt. Twigs and branches
10a Staghorn Sumac Anacardiaceae Rhus typhina L. Foliage
10b Staghorn Sumac Anacardiaceae Rhus typhina L. Berries
10c Staghorn Sumac Anacardiaceae Rhus typhina L. Berries
11 Green Ash Oleaceae Fraxinus pennsylvanica var. subintegerrima (Vahl) Fern. Foliage
12 White Ash Oleaceae Fraxinus Americana L. Foliage
13 Black Ash Oleaceae Fraxinus nigra Marsh. Foliage
14 Blue Ash Oleaceae Fraxinus quadrangulata Michx. Foliage
15 Pumpkin Ash Oleaceae Fraxinus profunda (Bush) Bush Foliage
16 Manchurian Ash Oleaceae Fraxinus mandschurica Rupr. Foliage
802 J. Vandal et al. Pharm Biol, 2015; 53(6): 800–806

Table 3. List of compounds tested for antimicrobial activity. analytical column from Waters (Milford, MA) housing a
Van Guard BEH C18, and 1.7 mm reverse-phase pre-column.
Compound Source
A linear gradient chromatographic technique was employed at
Benzoic acid B-3250, Sigma (Ronkonkoma, NY) room temperature with the following solvent system: Solvent
4-Hydroxy-3-methoxybenzoic H36001, Aldrich (St. Louis, MO) A ¼ 0.1% formic acid (aqueous); Solvent B ¼ acetonitrile,
acid
2,6-Dihydroxybenzoic acid D10, 960-6, Aldrich (St. Louis, MO) starting at 95% A:5% B and ending 12 min later with 20%
4-Methoxycinnamic acid M1, 380.7, Aldrich (St. Louis, MO) A:80% B, and a flow rate set at 0.5 mL/min. Two fixed
3,4-Dihydroxybenzoic acid P-5630, Sigma (Ronkonkoma, NY) detection wavelengths (280 nm and 350 nm) were used to
4-Hydroxy-3-methoxy- 382051-1G, Aldrich (St. Louis, MO)
monitor the eluting peaks. Resolved peaks were scanned by
cinnalamaldehyde
Ellagic acid E-2250, Sigma (Ronkonkoma, NY) the photodiode array detector from 240 to 460 nm.
Gallic acid G-7384, Sigma (Ronkonkoma, NY)
Methyl gallate G-1140, Sigma (Ronkonkoma, NY) Solutions
Ethyl gallate 48640, Fluka (Newport News, VA)
Caffeic acid C-0625, Sigma (Ronkonkoma, NY) All freeze-dried extracts were dissolved in 10% (v/v) DMSO–
Ferulic acid F-3500, Sigma (Ronkonkoma, NY) Milli Q water and were prepared in glass test tubes to obtain
o-Coumaric acid C-4400, Sigma (Ronkonkoma, NY)
m-Coumaric acid C-5017, Sigma (Ronkonkoma, NY)
final concentrations of 1000 mg/mL and 10 000 mg/mL (w/v).
p-Coumaric acid C-9008, Sigma (Ronkonkoma, NY) All fractions and compounds were dissolved in 10% (v/v)
Sinapic acid 78867, Fluka (Newport News, VA) DMSO and were prepared in glass test tubes to obtain a final
Gentisic acid G-5254, Sigma (Ronkonkoma, NY) concentration of 1000 mg/mL.
Vanillin V1104-2G, Aldrich (St. Louis, MO)
Chlorogenic acid C-3686, Sigma (Ronkonkoma, NY)
Reagent solutions and media
The resazurin solution used as a redox indicator was prepared
by dissolving 270 mg of resazurin (Sigma-Aldrich, Ontario,
Fractionation Canada) in 40 mL of sterile distilled water. The iso-sensitest
The ethanol freeze-dried extracts were the following four broth (ISB) (Oxoid, Ottawa, Canada) solution was prepared
species: Prince’s Pine, White Birch, Staghorn Sumac, and according to the manufacturer’s recommendations. The
Green Ash, F. pennsylvanicawere adsorbed onto polyvinyl- sterility of the final solutions was assured by utilizing the
polypyrrolidone (PVPP) powder (Sigma, Ronkonkoma, NY) appropriate microbiology aseptic techniques.
and packed in a Buchner funnel. Solvent elution (fraction-
ation) was carried out at a slow rate, initially with water Fungal cultures
followed by aliquots of increasing concentrations (20, 50, 70, The primary fungus used in this study was the yeast
and 100%) of ethanol. Fractions were monitored and selected C. albicans ATCC 10231. Strains of this species are known
for further analysis on the basis of their bioassay activity. to be MDR. The secondary fungus used was S. octosporus.
Final clean-up of the compounds of interest was achieved by All microbial cultures were provided by the American Type
employing a Sephadex LH-20 column (2 # 50 cm), using Culture Collection, with the exception of S. octosporus, which
methanol as the eluting solvent (Abou-Zaid & Scott, 2012; was obtained from the Laurentian University Culture
Abou-Zaid et al., 2000). Collection.

Identification of purified compounds Bacterial cultures

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
b-glucosidase using an acetate buffer (pH 5), hydrogen
peroxide oxidation; and by (ii) physical analyses: UV
spectroscopy; 1H-NMR; 13C-NMR, and (positive and nega-
tive) FAB-mass spectroscopy (Andersen & Markham, 2006;
Dey & Harborne, 1989; Harborne, 1994), 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, !). The UPLC was
equipped with an ACQUITY UPLC BEH C18 (Waters Corp,
Milford, MA), 1.7 mm (2.1 # 50 mm i.d.), reverse-phase
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 octos-
porus 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 men-
tioned. Serial dilutions of the microbial suspensions were
DOI: 10.3109/13880209.2014.942867
made using sterile saline until the optical density in the range
of 0.500–0.599 was obtained as determined using a spectro-
photometer at 500 nm.
Microplate resazurin assay
To screen for antimicrobial activity, 96-well plates were
prepared according to the methods of Sarker et al. (2007). 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 mL of ciprofloxacin (antibacterial agent; 3000 mg/mL
(w/v) for E. coli, 6000 mg/mL (w/v) for S. aureus, 384 mg/mL
(w/v) for P. aeruginosa, 5000 mg/mL (w/v) for M. phlei, and
1000 mg/mL (w/v) for S. lactis) or amphotericin B (antifungal
agent; 786 mg/mL (w/v) for C. albicans and 384 mg/mL (w/v)
for S. octosporus) dissolved in 10% DMSO was pipetted. 10%
DMSO (100 mL) 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 mL of resazurin
was pipetted into each well, 10 mL of 0.9% sterile saline
was pipetted into the wells of the sterile control columns, and
10 mL of microbial suspension (OD of 0.500–0.599) was
pipetted into each well, with the exception of the sterile
control columns. 30 mL of 3.3 # strength ISB was added to
each well to ensure a final volume of 100 mL. Below is an
example of resazurin microtiter assay result.
C" C+ 1 2 3 C1
Antimicrobial 10% Test Test Test Test
agent DMSO substance substance substance substance
3.3 # ISB 3.3 # ISB 3.3 # ISB 3.3 # ISB 3.3 # ISB 3.3 # ISB
Resazurin Resazurin Resazurin Resazurin Resazurin Resazurin
Microbial Microbial Microbial Microbial Microbial Sterile saline
suspension suspension suspension suspension suspension
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 mg/mL were further fractio-
nated. The fractions were tested for antimicrobial activity
employing the same protocol as described above against the
same primary microorganisms. The compounds were identi-
fied based on the methods of Dey and Harborne (1989) and
Antimicrobial of Northern Ontario flora 803
Harborne (1994). Final separation, purification, and quanti-
fication 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 mg/mL, only extracts
#4 (Prince’s Pine) and #8a (White Birch) demonstrated signs
of antimicrobial effects against S. aureus with MICs of
250 mg/mL and 1000 mg/mL, respectively. These extracts
were further separated into fractions and tested against only
the primary microorganisms at initial concentrations of
1000 mg/mL. Fractions 1–4 of plant extract #4 demonstrated
positive antimicrobial activities against S. aureus with
varying MICs (Tables 2 and 4). 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 mg/mL and 62.5 mg/mL, respectively (Tables 2 and 4).
Starting with an initial concentration of 10 000mg/mL,
extract #4 (Table 5). Extracts #10b and #10c were positive
against the three primary bacteria and extracts #8b, 8c, and 11
were positive against the fungi, C. albicans (Table 5).
In addition, all extracts had an antimicrobial effect against
S. aureus at this concentration with varying MICs (Table 6).
With an initial concentration of 1000 mg/mL, gallic acid,
ethyl gallate, and caffeic acid were positive against S. aureus
with MICs of 500, 1000, and 1000 mg/mL, respectively
(Tables 7 and 8). Gallic acid was also positive against
M. phlei with an MIC of 1000 mg/mL. Moreover, sinapic acid,
gentisic acid, and chlorogenic acid were positive against
C. albicans and S. octosporus, all with MICs of 1000 mg/mL.
The known pharmaceutical antimicrobial agents, cipro-
floxacin (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 mg/mL, all extracts were positive against
S. aureus, suggesting that this species of bacterium had
little defense against the natural substances present in the
804 J. Vandal et al. Pharm Biol, 2015; 53(6): 800–806

Table 4. MIC (mg/mL) of fractions of extracts against microorganisms. Table 6. MIC (mg/mL) of extracts against microorganisms.

E. S. P. C. E. S. P. C.
Fractions of extracts coli aureus aeruginosa albicans No. Extract coli aureus aeruginosa albicans
Fraction 1 of Extract 3 – 1000 – – 1 Balsam Fir – 2500 – –
Fraction 2 of Extract 3 1000 250 – 1000 2 Jackpine – 2500 – –
Fraction 3 of Extract 3 – 250 – 1000 3a Eastern Hemlock – 10 000 – –
Fraction 4 of Extract 3 – 1000 – – 3b Eastern Hemlock – 5000 – –
Fraction 2 of Extract 8 – 500 – – 3c Eastern Hemlock – 5000 – –
Fraction 3 of Extract 8 – 62.5 – – 4 Prince’s Pine 2500 310.25 10 000 2500
5 Wintergreen – 10 000 – –
6a Cow Parsnip – 10 000 – –
6b Cow Parsnip – 5000 – –
Table 5. Antimicrobial activity of extracts against microorganisms. 7 Partridgeberry – 2500 – –
8a White Birch – 620.5 – –
Extract 8b White Birch – 10 000 – 10 000
No. (10 000 mg/mL) E. coli S. aureus P. aeruginosa C. albicans 8c White Birch – 10 000 – 10 000
9a Yellow Birch – 10 000 – –
4 Prince’s Pine + + + + 9b Yellow Birch – 10 000 – –
8b White Birch " + " + 9c Yellow Birch – 2500 – –
8c White Birch " + " + 10a Staghorn Sumac – 1250 – –
10b Staghorn Sumac + + + " 10b Staghorn Sumac 5000 1250 5000 –
10c Staghorn Sumac + + + " 10c Staghorn Sumac 5000 1250 10 000 –
11 Green Ash " + " + 11 Green Ash – 10 000 – 10 000
12 White Ash – 10 000 – –
+ indicates activity; "indicates no activity. 13 Black Ash – 2500 – –
14 Blue Ash – 5000 – –
15 Pumpkin Ash – 5000 – –
extracts at such high concentrations. However, it is also 16 Manchurian Ash – 2500 – –
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 liver protecting, diuretic, and anti-allergic activities (Kostova
(Cos et al., 2006). & Iossifova, 2007).
As previously mentioned, the extract of Prince’s Pine was The fact that P. aeruginosa and E. coli were resistant to
effective against all primary microorganisms starting with the fractions of extracts 3 and 8 is probably a reflection of
an initial concentration of 10 000 mg/mL. Prince’s Pine is a the complex nature of the Gram-negative cell wall. In other
common plant used as a traditional medicine by North words, it is quite possible that some substances, because of
American First Nations people mostly for its antimicrobial, their size, shape, or other extenuating factors, simply cannot
anti-inflammatory, alterative, diuretic, astringent, and urinary cross the outer membranes of such bacteria and, therefore,
antiseptic properties (Caldecott, 2010; Moerman, 2004). cause no effect.
The extracts made from the berries of Staghorn Sumac also As previously mentioned, gallic acid, ethyl gallate, and
proved to be effective against the three primary bacteria when caffeic acid exhibited antimicrobial activity against S. aureus.
using an initial concentrations of 10 000 mg/mL. These results Since S. aureus was the only Gram-positive bacterium of the
are similar to the findings by Borchardt et al. (2008), where primary bacteria, those three compounds were believed to
the berries of Rhus typhina demonstrated clear inhibition affect only Gram-positive bacteria. Therefore, the aforemen-
zones against S. aureus, E. coli, and P. aeruginosa. However, tioned compounds were tested against two other Gram-
a partial inhibition zone was also observed for C. albicans. positive bacteria, namely M. phlei and S. lactis. Of the three
Borchardt et al. (2008) tested the drupes of the plant both with compounds tested, only gallic acid demonstrated antimicro-
and without the pericarp. Only the extract containing the bial activity against M. phlei.
pericarp showed antimicrobial activity, suggesting that the Gallic acid is a well-known natural compound, and, when
phytochemicals responsible for this activity were to be found tested alone or in combination with other natural products,
in the fruits and hairs surrounding the seeds. Interestingly, the has been shown to exhibit both antimicrobial and antiviral
Staghorn Sumac has been used in healing and in ceremonies activities. Sato et al. (1997) have shown that gallic acid,
by some native communities in Canada (Arnason et al., 1981). isolated from Terminalia chebula RETS, is effective against
The extracts of the twigs and branches, and phloem of E. coli and P. aeruginosa with MICs of 41000 mg/mL. This
White Birch, and the extract of the foliage of Green Ash study also demonstrated its antibacterial activity against 20
exhibited antifungal activity against C. albicans using initial strains of MRSA and seven strains of MSSA. An increase in
concentrations of 10 000 mg/mL. In a study conducted by our initial concentration of gallic acid may have reflected the
Omar et al. (2000), the bark and wood extract of B. papyrifera results observed by Sato et al. (1997).
showed a positive antibacterial activity against methicillin It is also important to note that gallic acid is a compound
sensitive S. aureus (MSSA). The genus Fraxinus has been found in R. typhina (Borchardt et al., 2008). The extracts of
used in traditional medicine. In fact, many natural compounds R. typhina were not active against the microorganisms when
have been identified in the Fraxinus species that possess anti- using an initial concentration of 1000 mg/mL. However, when
inflammatory, immunomodulatory, antimicrobial, antioxida- increasing the initial concentration to 10 000 mg/mL, all
tive, skin regenerating, photodynamic damage prevention, R. typhina extracts showed positive activity (Tables 5 and 7).
DOI: 10.3109/13880209.2014.942867 Antimicrobial of Northern Ontario flora 805
Table 7. Antimicrobial activity of compounds against microorganisms.

Compound (1000 mg/mL) E. coli S. aureus P. aeruginosa C. albicans M. phlei S. lactis S. octosporus
Gallic acid " + " " + "
Ethyl gallate " + " " " "
Caffeic acid " + " " " "
Sinapic acid " " " + +
Gentisic acid " " " + +
Chlorogenic acid " " " + +

+ indicates activity; " indicates no activity.

Table 8. MIC (mg/mL) of compounds against microorganisms.

Compound (1000 mg/mL) E. coli S. aureus P. aeruginosa C. albicans M. phlei S. lactis S. octosporus
Gallic acid – 500 – – 1000 –
Ethyl gallate – 1000 – – – –
Caffeic acid – 1000 – – – –
Sinapic acid – – – 1000 1000
Gentisic acid – – – 1000 1000
Chlorogenic acid – – – 1000 1000

In the study conducted by Sato et al. (1997), 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 mg/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., 2010).
Varying results have been obtained when studying the
antimicrobial properties of caffeic acid. On one hand,
according to Eppinger et al. (2011), this compound was
effective against S. aureus with an MIC of 5200 mg/mL, and
also against E. coli with an MIC of 100 mg/mL when isolated
from the hairy roots of Salvia miltiorrhiza. On the other hand,
a study conducted by Rauha et al. (2000) has shown that
caffeic acid (C-0625) was not effective against S. aureus,
E. coli, and C. albicans when using a 500 mL volume of the
compound (1000 mg/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. (2008) 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. (2003), 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.,
2007). 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, 2008).
Along with the background history and applied uses of
traditional medicine, the pharmacological screening of phyto-
chemicals 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 com-
pounds 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 rela-
tively 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
806 J. Vandal et al.
combination with other drugs. The discovery and develop-
ment 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).
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