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biomedicines
Article
Herbal Tea for the Management of Pharyngitis:
Inhibition of Streptococcus pyogenes Growth and
Biofilm Formation by Herbal Infusions
Niluni M. Wijesundara 1,2 and H. P. Vasantha Rupasinghe 3, 4, *
1Department of Biology, Faculty of Science, Dalhousie University, Halifax, NS 3H 4R2, Canada
2
Department of Animal Science, Faculty of Animal Science and Export Agriculture, Uva Wellassa University,
Badulla 90 000, Sri Lanka
3
Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro,
NS B2N 5E3, Canada
4Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, NS B3H 4R2, Canada
*Correspondence: vrupasinghe@dal.ca; Tel.: +1-902-893-6623; Fax: +1-902-893-1404
Received: 4 August 2019; Accepted: 23 August 2019; Published: 24 August 2019
Abstract:
Herbal teas are becoming popular as functional beverages due to their various health
promotional properties. This study aimed at assessing 13 hot water infusions (HWIs) from different
herbs against streptococcal pharyngitis (strep throat). Licorice root exhibited the lowest minimum
inhibitory concentrations (MIC) of 1.56 mg/mL, followed by barberry root, thyme, and oregano
flowering shoots, with a MIC of 3.13 mg/mL. At their respective minimum bactericidal concentrations
(MBC), licorice showed the bactericidal effect on S. pyogenes within 12 h after exposure while others
need 24 h for a similar outcome. The HWIs exhibited inhibitory activity on biofilm formation, ranging
from 1.56 to 6.25 mg/mL, which confirmed by ruptured cells or clusters of dead cell debris observed
in scanning electron microscope (SEM). Overall, non-toxic concentrations of efficacious HWIs from
licorice root, barberry root, thyme, and oregano flowering shoots may provide potential sources for
developing herbal teas or biomedicine for the management of S. pyogenes infections.
Keywords:
herbal plants; hot water infusions; phytochemicals; natural health product; Streptococcus
pyogenes; mass spectrometry
1. Introduction
For centuries, the therapeutic properties of various spices and herbal plants have been used to
treat several bacterial infections. Streptococcal pharyngitis (strep throat), an acute infection of the
nasopharynx and oropharynx, is one of the common upper respiratory infections, primarily caused by
Streptococcus pyogenes. It accounts for more than 37% of all diagnosed sore throat cases in children and
up to 5–10% in adults reports millions of cases per year worldwide [1,2].
Investigation of natural antimicrobial agents as alternatives to synthetic counterparts have received
significant attention from researchers and natural health product industry. Therefore, plant-derived
new natural antimicrobial agents have been explored against various infectious bacteria, including
S. pyogenes. It has been presumed that herbal remedies and formulations are moderately efficacious
but safer from side effects in contrast to most pharmaceutical agents [
3
]. A World Health Organization
survey reported that around 70–80% of the world population use herbal remedies as their primary
health care [
4
]. Usage of complementary and alternative therapies in Canada has increased at an
exponentially growing pace in recent years, and the estimated use of alternative or herbal remedies in
place of conventional medicine is nearly 30% of the total population in the United States [5].
Biomedicines 2019,7, 63; doi:10.3390/biomedicines7030063 www.mdpi.com/journal/biomedicines
Biomedicines 2019,7, 63 2 of 19
The use of herbal plants for various medicinal purposes by traditional healers in North America
has been reported. Interestingly, over 2500 species of plants have been used by both Native Americans
and Americans of European origin in their traditional medicine systems [
6
]. The First Nations have
used the fresh plant parts or their extracts of herbal plants such as slippery elm, sage, barberry, oregano,
thyme, licorice, and echinacea, and Canadian folk remedial applies for sore throat conditions. Based
on the plant used, type of infection, and the situations of the patient is being treated the preparation
of herbal remedies are different such as infusions (hot teas), decoctions (boiled teas), macerations
(cold-soaking), or tinctures (solvent/water extracts and distillations) [7].
The rapidly expanding natural health product industries keep introducing new herbal supplements,
functional food, and herbs-based energy drinks led by the increased demand for the use of medicinal
remedies from consumers. Drinking herbal tea is gaining popularity as one of the most pleasurable
beverages as well as an efficient herbal remedy due to its health promotional properties. Herbal teas
have been identified and approved as a natural and nonprescription drug in some countries, including
Canada, as a remedy for oral and pharyngeal mucosal irritations with a dry cough.
Due to the potential side effects of long-term administration of traditional herbal medicines
for the management of streptococcal pharyngitis, it is necessary to have proper clinical laboratory
investigations on their activity and safety compared with synthetic antibiotics. However, hot water
infusions (HWIs) of selected plant parts of the current study (Table 1) have not been reported or limited
with emphasizing both growth and biofilm inhibition of S. pyogenes. Therefore, this study aimed
to identify the potential herbals with antibacterial activity and antibiofilm efficacy of 13 HWIs from
different parts of herbal plants against three S. pyogenes strains, to develop specialty herbal tea for
relieving pain and other complications due to streptococcal pharyngitis.
2. Materials and Methods
2.1. Chemicals and Reagents
3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Life
Technologies (Burlington, ON, Canada). Bacteriological agar and brain heart infusion (BHI) media
were purchased from Oxoid Ltd. (Nepean, ON, Canada). Dimethyl sulfoxide (DMSO), penicillin
G sodium salt, phosphate-buffered saline (PBS), sodium chloride (
≥
99.0%, ACS reagent), and other
chemicals were obtained from Sigma-Aldrich (Oakville, ON, Canada).
2.2. Collection of Plant Materials
Eleven different medicinal plants which were commonly used in the Canadian indigenous
medicine were considered in the present study. Their common names, scientific names, and specific
parts used are summerized in Table 1were selected. Geranium (Voucher No: 13010), Sage (Voucher
No: 13011), oregano (Voucher No: 13012), and thyme (Voucher No: 13013) were collected from the
university’s herbal garden, Faculty of Agriculture, Dalhousie University, at GPS location of 45
◦
22’23.3”N
and 63
◦
15’45.2”W during flowering period. Purple coneflower/Echinacea (Voucher No: 13009), was
collected at GPS location of 45
◦
22’20.8”N and 63
◦
15’43.8”W. A taxonomist was authenticated the
herbal plants and specimens were deposited in the A.E. Roland herbarium, Department of Plant, Food,
Environmental Sciences, Faculty of Agriculture, Dalhousie University, Canada. Canadian ginger and
clove flower buds were purchased from the local and supermarket. Fresh plant parts were washed, cut
into small pieces, and dried at 50
◦
C. Ground powders were stored at
−
80
◦
C in airtight containers.
Barberry root, slippery elm inner bark, licorice root, and olive leaves were obtained as a powder from
Mother Earth Natural Health Inc. (Ottawa, ON, Canada).
2.3. Preparation of Hot Water Infusions (HWIs)
Dried powder of herbal plant parts was infused with distilled water in 1:10 (w/v) ration in a boiling
water bath (ISOTEMP-205, Fisher Scientific, Ottawa, ON, Canada) for 10 min. After infusion period,
Biomedicines 2019,7, 63 3 of 19
mixtures were filtered. Filtrates were frozen overnight and freeze dried at
−
20
◦
C using a freeze dryer
(Kinetics, FTS Systems Inc., Stone Ridge, NY, USA) for 24–48 h. The dried samples were scraped out
and were stored in labeled sterile screw-capped amber bottles, in the freezer at -80
◦
C until used for
further analysis.
2.4. Characterization of Phytochemicals in HWIs
2.4.1. Determination of Total Phenolic and Total Carotenoids Contents
The total phenolic contents and total carotenoids content were assessed for all the HWIs, and
results were previously published by authors [8].
2.4.2. Characterization of Phytochemical Profile
Potential phytochemicals present in HWIs were characterized using ultra-performance liquid
chromatographic-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) as described
in our previous study [
9
]. Briefly, UPLC was directly interfaced with a High Definition MS System
(Waters Xevo TQ-Smicro, Waters Corporation, Milford, USA) with an electrospray ionization (ESI)
source operating in negative ion mode with ionization conditions of capillary voltage of 2.0 V, sampling
cone voltage of 25.0 V and extraction cone voltage of 3.5 V. The optimal temperatures (150
◦
C of source
temperature and 450
◦
C of desolvation gas temperature) and optimal gas flows (100 L/h of cone gas
flow and 1000 L/h of desolvation gas flow) were maintained. Full-scan mass acquisitions in negative
ion mode were made by scanning the m/zrange of 100–1100 Da. Data were collected in centroid
mode. Masses were corrected using an external reference (Lock-Spray
™
) comprising a C
18
column
(UPLC
®
BEH C
18
, Waters Corporation, USA). The filtered samples were auto injected into the column.
A mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (Solvent B)
was used as the mobile phase. The total run time was 12 min (2 min (83.5% A), 2.6 min (83.0% A),
3.1 min (82.5% A), 4 min (81.5% A), 4.7 min (80.0% A), 6.6 min (20.0% A), 8.2 min (20.0% A), and 12 min
(94.0%)), with 94.0% of solvent A flow of 0.3 mL/min. The deprotonated (M–H
+
)
−
ions identified using
the full scan mode was used to generate selected ion monitoring (SIM) channels (51 channels). The
samples were run in SIM mode to confirm the abundance of each recognized compounds matching to
their retention times of SIM mode and full scan mode. These identified phytochemicals were confirmed
with the existing literature (Table 1).
2.5. Bacteria and Growth Conditions
Two American Type Culture Collection (ATCC) of S. pyogenes (ATCC 19615 and ATCC 49399),
as well as a pharyngeal isolate from a streptococcal pharyngitis patient (Queen Elizabeth II Health
Sciences Centre, Halifax, NS, Canada) were used.
Following the manufacturer’s instructions, inoculums were prepared and stored at
−
80
◦
C in 1:1
(v/v) of brain heart infusion (BHI) broth: 40% glycerol. Strains were cultured on BHI agar plates, were
maintained for seven days at 37
◦
C. For experiments, a few colonies were inoculated in BHI broth,
were incubated at 37
◦
C for about 16 h and standardized with saline water (0.85% NaCl, pH =7.0
±
0.1) according to the previously reported method [10].
2.6. Anti-Bacterial Activity
2.6.1. Screening for Inhibitory Antibacterial Effects of HWIs
The spot-on-the-lawn method was used for initial screening to verify the antibacterial effects of
HWIs, with ATCC 19615 and ATCC 49399 bacterial cultures. The samples were spotted, plates were
incubated at 37 ◦C for 24–48 h and examined for the zone of inhibitions.
Biomedicines 2019,7, 63 4 of 19
2.6.2. Determination of Minimum Inhibitory Concentrations (MICs)
Effect of HWIs on bacterial growth was assessed using the standard micro broth-dilution method
recommended by the Clinical and Laboratory Standards Institute [
11
]. Briefly, 100
µ
L volumes of
diluted bacterial suspensions (10
6
CFU/mL) in BHI broth were incubated with the same amount of
serial two-fold diluted HWIs (0.2 to 50 mg/mL), penicillin G (4.0
×
10
−4
to 0.5
µ
g/mL), and BHI media
(diluent control). The assay was carried out in 96-well microplate, and bacteria growth was measured
as absorbance at OD 600 nm after 24 h incubation (37
◦
C). The MIC values corresponded to the lowest
concentration of test compounds that inhibiting visible bacterial growth or showing a significant change
of absorbance compared to the growth of control (in the spectrophotometric method) were recorded.
2.6.3. Determination of Minimum Bactericidal Concentrations (MBCs)
MBCs were determined by subculturing 15
µ
L from non-turbid wells on BHI agar plate, and
colony growth was observed after 24 h incubation at (37
◦
C). The MBC value is the lowest concentration
where no visible colony growth was observed, compared to the control.
2.6.4. Time-Kill Curves
The time taken to show a bactericidal activity by HWIs was measured using time-kill curves as a
previously described method [
10
]. Briefly, the bacterial count was enumerated in 3 h intervals over 24
h incubation periods at 37
◦
C with different concentrations of licorice root, oregano flowering shoot,
thyme flowering shoot HWIs (their own 1/2
×
MIC, MIC, and 2
×
MIC). BHI media alone with bacteria
was used as a diluent control, and penicillin G was as the positive control. The assays were performed
in triplicate, and the results were expressed as log CFU/mL.
2.7. Anti-Biofilm Formation Activity
2.7.1. Determination of Minimum Biofilm Inhibitory Concentrations (MBIC) and Biofilm
Quantification by MTT Assay
The effect of HWIs on biofilm formation of S. pyogenes was examined using a
3-[4–dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay as described
previously [
10
]. As in the MIC assay, two-fold serial dilutions of four HWIs and penicillin G
were prepared in 96-well plates and were inoculated with 100
µ
L of 1
×
10
6
CFU/mL bacterial
suspensions. After three days of incubation at 37
◦
C, the plates were emptied by flipping them over to
remove the planktonic bacteria. Fresh BHI broth (100
µ
L) supplemented with 10
µ
L of 12 mM MTT
was then added into each well, followed by incubation for 3 h at 37
◦
C. DMSO (50
µ
L) was added
after the careful removal of 85
µ
L of BHI broth from each well. Biofilm formation was calorimetrically
quantified by measuring reduction ability of tetrazolium salt (yellow) into a formazan (purple) by
the activity of dehydrogenase enzymes in surviving bacteria in biofilms. Absorbance at 540 nm was
measured using a microplate reader (EpochTM, Biotek, Winooski, VT, USA). Percentage inhibitions
were calculated as follows: [1
−
(A
540
Treatment /A
540
Control)]
×
100. The minimum biofilm inhibition
concentration (MBIC) was defined as the lowest concentration.
2.7.2. SEM Visualization of Biofilms
To examine the effects of selected HWIs on morphology, treated bacterial cells with HWIs at their
respective sub-MIBCs were fixed as described in Wijesundara and Rupasinghe [
10
]. Briefly, treated cells
were centrifuged, washed with PBS, and fixed in 0.1 M sodium cacodylate buffer (pH 7.2) containing
2% glutaraldehyde (2 h) and then in 4% osmium tetroxide (4 h). Biofilms were then dehydrated using
gradient series of ethanol (35%; 50%; 75%; 90%; 100%) and hexamethyldisilazane/ethanol gradient
series (25:75; 50:50; 75:25; 100:0%). The samples were air-dried for 2 h under the fume hood and
were mounted on aluminum sputters. Then, sputters were coated with gold-palladium (15 nm) and
were visualized under SEM (Hitachi FEG-SEM 4700, Hitachi Ltd., Tokyo, Japan) using operational
Biomedicines 2019,7, 63 5 of 19
conditions of 10 kilovolts (kV) of acceleration voltage, 14–16 microamps (
µ
A) of emission current,
10–12 mm working distance and the analysis lens mode. Micrographs were captured at different
magnifications. The experiment was performed in triplicates for three independent times.
2.8. Statistical Analysis
The complete randomized design was used, and all the experiments were performed in triplicates
and three independent times. One-way analysis of variance (ANOVA) was performed using Minitab
statistical software (Version 17.0, Minitab Inc., State College, PA, USA). Tukey’s test was used to
determine the differences among treatments, and significant differences were defined as p<0.05, and
the results were expressed as the mean
±
standard deviation. The time to kill curves were plotted
using GraphPad Prism version 5.0 for Windows (GraphPad Software, La Jolla, CA, USA).
3. Results
3.1. Characterization of HWIs Using UPLC-ESI-MS/MS
We have previously reported the total phenolic content, and total carotenoid content of these
HWIs [
8
]. Potential phytochemicals found in HWIs expressed with their deprotonated molecular
mass and retention time ((M–H
+
)
−
, RT) were summarized in Table 1. The full-scan mode total ion
chromatograms, SIM scan channels, mass spectrum, and molecular structure of the selected major
phytochemicals identified by UPLC-ESI-MS/MS of most effective HWI, licorice root, are presented in
Figure 1. Furthermore, UPLC-ESI-MS/MS identification results of barberry root, oregano flowering
shoots, and thyme flowering shoots are included in supplementary figures section (Figures S1–S3).
Biomedicines 2019,7, 63 6 of 19
Table 1. Potential phytochemical profile of the selected herbal plant parts.
Plant Name Family Parts Used Potential Major Phytochemicals
((M–H+)−, RT in min) References
Common Botanical
Barberry Berberis vulgaris L. Berberidaceae Roots
Gingerol (293.38, 8.06), Caffeic acid (179.16, 3.22),
Naringin (579.54, 5.99), Naringenin (271.26, 7.32), and
Rosmerinic acid (359.32, 2.01).
[12]
Clove Syzygium aromaticum L. Myrtaceae Flower buds
Eugenol (163.20, 4.40), Eugenyl acetate (205.24, 8.43), and
β-Ocimene (135.25, 4.25). [13–15]
Echinacea Echinacea purpurea L. Asteraceae
Flowers
Caftaric acid (311.23, 2.44), Chlorogenic acid (353.31, 2.78),
Caffeic acid (179.16, 3.27), Cynarin (515.46, 6.02),
Echinacoside (785.73, 6.56), Cichoric acid (473.37, 4.78),
and β-Sitosterol (413.70, 4.61). [16–19]
Stems
Quercetin (301.23, 6.12) and Eugenyl acetate (205.24, 8.43).
Leaves Caftaric acid (311.23, 2.44), Cichoric acid (473.37, 4.78),
and Caffeic acid (179.16, 3.27).
Ginger Zingiber officinale L. Zingiberaceae Rhizomes
Gingerol (273.38, 7.18), α-Humulene (203. 24, 2.50),
Gingerol (293.38, 7.18), α-Thujone/β-Thujone/camphor
(151.23, 4.35),
α
or
β
-Caryophyllene (203.35, 2.52), Caffetic
acid (311.23, 10.07), and Liquirtin (419.39, 6.21).
[20,21]
Licorice Glycyrrhiza glabra L. Papilionaceae Roots
Glycyrrhizin (821.94, 6.86), Glabridin (323.97, 7.54),
Thymohydroquinone (165.22, 2.81), Naringenin (271.26,
7.32), Asparegene (131.12, 1.63), Liquirtin (417.39, 6.34), 5
-Methoxyhydnocarpin (493.45, 6.28), Cynarin (515.46,
6.02), Quercetin (301.23, 6.12), p-Cyemene (133.21, 0.82),
Generdiol (153.23, 3.47), α-Humulene (203.35, 2.53), and
Kaempferol (285.23, 6.42).
[22,23]
Oregano Origanum vulgare L. Lamiaceae Flowering shoots
Rosmerinic acid (359.32, 6.20), Oleanolic acid (455.71,
7.23), ρ-Cymene (133.21, 1.04), and
5-Methoxyhydnocarpin (493.45, 6.25).
[24,25]
Olive Olea europeus L. Oleaceae Leaves
Hydroxytyrosol, Rutin, Luteolin-7-glucoside, Oleuropein
glucoside, Luteolin-40-glucoside, Oleuropein, and
Oleuropein aglycon.
[26–28]
Rose geranium
Pelargonium graveolens L.
Geraniaceae Leaves Geraniol (151.24, 6.62) [29]
Biomedicines 2019,7, 63 7 of 19
Table 1. Cont.
Plant Name Family Parts Used Potential Major Phytochemicals
((M–H+)−, RT in min) References
Common Botanical
Sage Salvia officinalis L. Lamiaceae Leaves
1,8-Cineole (153.24, 2.18 Borneol and/or Linalool and/or
α-Terpineol and/or β-Pinene (153.24, 4.01), β-carotene
(535.87, 3.46), γ-Terpinene and/or Mycrene and/or
β-Pinene and/or α-Pinene (135.24, 4.24), Asparegene
(131.12, 3.65), and α-Terpine (135.24, 4,42).
[25,30,31]
Slippery elm Ulmus rubra Muhl. Ulmaceae Inner barks Ursolic acid /Betulinic acid (455.71, 9.5) and β-carotene
(535.87, 5.29) [32]
Thyme Thymus vulgaris L. Lamiaceae Flowering shoots
Thymol and Carvacrol (149.21, 6.65),
Thymohydroquinone (165.22, 7.17), γ-Terpinene,
Myrcene, and α-Pinene (135.24, 4.24), Gingerol (293.38,
7.68), and Kaempferol (285.23, 6.42)
[25,33]
(M–H+)−: deprotonated molecular mass; RT: retention time.
Biomedicines 2019,7, 63 8 of 19
Biomedicines 2019, 7, x FOR PEER REVIEW 8 of 19
Figure 1. The full scan mode total ion chromatogram of licorice root hot water infusion from a
negative mode of UPLC-ESI-MS/MS analysis (A). The SIM channels (B–E) and mass spectra of the
full-scan of selected four major phytochemicals; thymohydroquinone (b), liquiritin (c), kaemferol (d),
and glabridin (e) are shown for m/z of deprotonated ions of 165.22, 417.39, 285.23, and 323.97,
respectively. The identified phytochemical name and chemical structure are presented. SIM: Selective
ion monitoring scan; TIC: Total ion chromatograms, UPLC-ESI-MS/MS: Ultra-performance liquid
chromatographic-electrospray ionization-tandem mass spectrometry.
3.2. HWIs Inhibits S.Pyogenes Planktonic Growth
The antibacterial activities of 13 HWIs have assessed against three strains of S. pyogenes (ATCC
19615, ATCC 49399 and a clinical isolate) and MIC and MBC values shown in Table 2. Zone of
inhibitions around the spotted HWIs were detected in the initial screening and shown in Figure 2A.
The HWI of licorice root exhibited greater activity against S. pyogenes planktonic growth with the
lowest MIC of 1.56 mg/mL followed by barberry root, thyme, and oregano flowering shoots, as
indicated by the relatively lower MIC of 3.13 mg/mL. Colony growth of sub-cultured four HWIs on
BHI agar, which demonstrated significant distinct bactericidal effects shown in Figure 2B.
Figure 1.
The full scan mode total ion chromatogram of licorice root hot water infusion from a
negative mode of UPLC-ESI-MS/MS analysis (
A
). The SIM channels (
B
–
E
) and mass spectra of the
full-scan of selected four major phytochemicals; thymohydroquinone (b), liquiritin (c), kaemferol
(d), and glabridin (e) are shown for m/zof deprotonated ions of 165.22, 417.39, 285.23, and 323.97,
respectively. The identified phytochemical name and chemical structure are presented. SIM: Selective
ion monitoring scan; TIC: Total ion chromatograms, UPLC-ESI-MS/MS: Ultra-performance liquid
chromatographic-electrospray ionization-tandem mass spectrometry.
3.2. HWIs Inhibits S.Pyogenes Planktonic Growth
The antibacterial activities of 13 HWIs have assessed against three strains of S. pyogenes (ATCC
19615, ATCC 49399 and a clinical isolate) and MIC and MBC values shown in Table 2. Zone of
inhibitions around the spotted HWIs were detected in the initial screening and shown in Figure 2A. The
HWI of licorice root exhibited greater activity against S. pyogenes planktonic growth with the lowest
MIC of 1.56 mg/mL followed by barberry root, thyme, and oregano flowering shoots, as indicated by
the relatively lower MIC of 3.13 mg/mL. Colony growth of sub-cultured four HWIs on BHI agar, which
demonstrated significant distinct bactericidal effects shown in Figure 2B.
Biomedicines 2019,7, 63 9 of 19
Table 2.
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of hot water infusions against three strains of Streptococcus
pyogenes. ATCC: American Type Culture Collection.
Plant-Source, Plant Part ATCC 19615 ATCC 49399 Clinical Isolate
MIC (mg/mL) MBC (mg/mL) MBC/MIC MIC (mg/mL) MBC (mg/mL) MBC/MIC MIC (mg/mL) MBC (mg/mL) MBC/MIC
Clove FB 12.50 25.00 2 12.50 25.00 2 12.50 25.0 2
Sage L 12.50 25.00 12.50 25.00 12.50 25.0
Ginger-Canada Rh 50.00 >50.00 - 50.00 >50.00 - NA NA -
Ginger-Chinese Rh 50.00 >50.00 - 50.00 >50.00 - NA NA -
Oregano FB 3.13 6.25 2 3.13 6.25 2 3.13 6.25 2
Thyme FB 3.13 6.25 2 3.13 6.25 2 3.13 6.25 2
Licorice R 1.56 3.13 2 1.56 3.13 4 3.13 6.25 2
Barberry R 3.13 6.25 2 3.13 6.25 2 3.13 6.25 2
Echinacea L 50.00 >50.00 - 50.00 >50.00 - NA NA -
Echinacea S 6.25 12.50 2 6.25 12.50 2 6.25 12.50 2
Echinacea F 50.00 >50.00 - 50.00 >50.00 - 50.00 >50.00 -
Geranium L 25.00 50.00 2 25.00 50.00 2 NA NA -
Slippery elm IB >50.00 >50.00 - >50.00 >50.00 - NA NA -
Olive L >50.00 >50.00 - >50.00 >50.00 - NA NA -
Penicillin G 0.0078 0.0156 2 0.0078 0.0156 2 0.0078 0.0156 2
FB: Flowering buds; F: Flowers; FS: Flowering shoots; Rh: Rhizome; R: Roots; IB: Inner bark; L: Leaves; NA: Not analyzed.
Biomedicines 2019,7, 63 10 of 19
Biomedicines 2019, 7, x FOR PEER REVIEW 10 of 19
Figure 2. (A) Antibacterial effect of HWIs (100 mg/mL) spotted on brain heart infusion (BHI) agar
plates inoculated with the Streptococcus pyogenes ATCC 19615. HWIs: Hot water infusions; a: barberry
roots; b: licorice roots; c: thyme flowering shoots; d: oregano flowering shoots; e: echinacea leaves; f:
echinacea stems; g: echinacea flowers; h: clove flower buds; i: ginger rhizomes, and j: sage leaves. (B)
Sub-culturing for minimum bactericidal concentration on BHI agar plates of four most efficient hot
water infusions against S. pyogenes ATCC 19615 followed by the micro-broth dilution assay. a: licorice
roots; b: barberry roots; c: oregano flowering shoots; d: thyme flowering shoots.
3.3. Time to Kill Analysis of HWIs Against S. Pyogenes
HWIs from licorice roots, barberry roots, thyme flowering shoots, and oregano flowering shoots
were selected for time-kill analysis based on their significantly low MIC and MBC values. Time taken
to achieve 99.99% planktonic S. pyogenes kill by the HWIs were assessed, and results are shown in
Figure 3. Results showed that licorice HWI exhibits the complete bactericidal effect on S. pyogenes
within 12 h after exposure (Figure 3), whereas barberry, thyme, and oregano required a longer time
of 24 h, at their respective MBC. No regrowth was observed after an additional 24 h incubation.
Figure 2.
(
A
) Antibacterial effect of HWIs (100 mg/mL) spotted on brain heart infusion (BHI) agar
plates inoculated with the Streptococcus pyogenes ATCC 19615. HWIs: Hot water infusions; a: barberry
roots; b: licorice roots; c: thyme flowering shoots; d: oregano flowering shoots; e: echinacea leaves; f:
echinacea stems; g: echinacea flowers; h: clove flower buds; i: ginger rhizomes, and j: sage leaves. (
B
)
Sub-culturing for minimum bactericidal concentration on BHI agar plates of four most efficient hot
water infusions against S. pyogenes ATCC 19615 followed by the micro-broth dilution assay. a: licorice
roots; b: barberry roots; c: oregano flowering shoots; d: thyme flowering shoots.
3.3. Time to Kill Analysis of HWIs Against S. Pyogenes
HWIs from licorice roots, barberry roots, thyme flowering shoots, and oregano flowering shoots
were selected for time-kill analysis based on their significantly low MIC and MBC values. Time taken
to achieve 99.99% planktonic S. pyogenes kill by the HWIs were assessed, and results are shown in
Figure 3. Results showed that licorice HWI exhibits the complete bactericidal effect on S. pyogenes
within 12 h after exposure (Figure 3), whereas barberry, thyme, and oregano required a longer time of
24 h, at their respective MBC. No regrowth was observed after an additional 24 h incubation.
Biomedicines 2019,7, 63 11 of 19
Biomedicines 2019, 7, x FOR PEER REVIEW 11 of 19
Figure 3. Time-kill curves of the hot water infusions of (a) licorice root, (b) barberry root, (c) oregano
flowering shoots, and (d) thyme flowering shoots on the growth of Streptococcus pyogenes ATCC 19615.
A viable count was performed for different folds of their respective minimum inhibitory
concentrations (MICs) at 0.08, 3, 6, 9, 12, and 24 h incubation time points. = 2 × MIC; = MIC;
= ½ × MIC; = BHI media (diluent) control. Each data point represents mean from three independent
experiments performed in triplicate.
3.4. HWIs Possesses Anti-Biofilm Formation Activity
The HWIs were shown biofilm inhibition activities at the range of 1.56 to 6.25 mg/mL
concentrations where licorice root infusion had the most active antibiofilm activity among the tested
HWIs. The inhibition effects of sub-inhibitory concentrations of HWIs of licorice root, barberry root,
oregano flowering shoots, and thyme flowering shoots on biofilm formation over 72 h incubation
(Minimum biofilm inhibitory concentration; MBIC) of the three S. pyogenes strains were quantified
by MTT staining (Table 3).
Table 3. Minimum biofilm inhibitory concentration (MBIC) against Streptococcus pyogenes strains.
Hot Water Infusions MBIC (mg/mL)
ATCC 19615 ATCC 49399 Clinical
Licorice Roots 1.56 (1 × MIC) 6.25 (4 × MIC) 3.13 (2 × MIC)
Barberry Root 6.25 (2 × MIC) 6.25 (2 × MIC) 6.25 (2 × MIC)
Oregano Flowering shoots 6.25 (2 × MIC) 6.25 (2 × MIC) 6.25 (2 × MIC)
Thyme Flowering shoots 6.25 (2 MIC) 6.25 (2 × MIC) 6.25 (2 ×MIC)
Penicillin G 0.0156 (2 × MIC) 0.0625 (8 × MIC) 0.0625 (8 × MIC)
MIC: Minimum inhibitory concentration.
Figure 3.
Time-kill curves of the hot water infusions of (
a
) licorice root, (
b
) barberry root, (
c
) oregano
flowering shoots, and (
d
) thyme flowering shoots on the growth of Streptococcus pyogenes ATCC 19615.
A viable count was performed for different folds of their respective minimum inhibitory concentrations
(MICs) at 0.08, 3, 6, 9, 12, and 24 h incubation time points.
N
=2
×
MIC;
#
=MIC;
Biomedicines 2019, 7, x FOR PEER REVIEW 11 of 19
Figure 3. Time-kill curves of the hot water infusions of (a) licorice root, (b) barberry root, (c) oregano
flowering shoots, and (d) thyme flowering shoots on the growth of Streptococcus pyogenes ATCC 19615.
A viable count was performed for different folds of their respective minimum inhibitory
concentrations (MICs) at 0.08, 3, 6, 9, 12, and 24 h incubation time points. = 2 × MIC; = MIC;
= ½ × MIC; = BHI media (diluent) control. Each data point represents mean from three independent
experiments performed in triplicate.
3.4. HWIs Possesses Anti-Biofilm Formation Activity
The HWIs were shown biofilm inhibition activities at the range of 1.56 to 6.25 mg/mL
concentrations where licorice root infusion had the most active antibiofilm activity among the tested
HWIs. The inhibition effects of sub-inhibitory concentrations of HWIs of licorice root, barberry root,
oregano flowering shoots, and thyme flowering shoots on biofilm formation over 72 h incubation
(Minimum biofilm inhibitory concentration; MBIC) of the three S. pyogenes strains were quantified
by MTT staining (Table 3).
Table 3. Minimum biofilm inhibitory concentration (MBIC) against Streptococcus pyogenes strains.
Hot Water Infusions MBIC (mg/mL)
ATCC 19615 ATCC 49399 Clinical
Licorice Roots 1.56 (1 × MIC) 6.25 (4 × MIC) 3.13 (2 × MIC)
Barberry Root 6.25 (2 × MIC) 6.25 (2 × MIC) 6.25 (2 × MIC)
Oregano Flowering shoots 6.25 (2 × MIC) 6.25 (2 × MIC) 6.25 (2 × MIC)
Thyme Flowering shoots 6.25 (2 × MIC) 6.25 (2 × MIC) 6.25 (2 ×MIC)
Penicillin G 0.0156 (2 × MIC) 0.0625 (8 × MIC) 0.0625 (8 × MIC)
MIC: Minimum inhibitory concentration.
=
1
2×
MIC;
=
BHI media (diluent) control. Each data point represents mean from three independent experiments
performed in triplicate.
3.4. HWIs Possesses Anti-Biofilm Formation Activity
The HWIs were shown biofilm inhibition activities at the range of 1.56 to 6.25 mg/mL concentrations
where licorice root infusion had the most active antibiofilm activity among the tested HWIs. The
inhibition effects of sub-inhibitory concentrations of HWIs of licorice root, barberry root, oregano
flowering shoots, and thyme flowering shoots on biofilm formation over 72 h incubation (Minimum
biofilm inhibitory concentration; MBIC) of the three S. pyogenes strains were quantified by MTT staining
(Table 3).
Table 3. Minimum biofilm inhibitory concentration (MBIC) against Streptococcus pyogenes strains.
Hot Water Infusions MBIC (mg/mL)
ATCC 19615 ATCC 49399 Clinical
Licorice Roots 1.56 (1 ×MIC) 6.25 (4 ×MIC) 3.13 (2 ×MIC)
Barberry Root 6.25 (2 ×MIC) 6.25 (2 ×MIC) 6.25 (2 ×MIC)
Oregano Flowering shoots 6.25 (2 ×MIC) 6.25 (2 ×MIC) 6.25 (2 ×MIC)
Thyme Flowering shoots 6.25 (2 ×MIC) 6.25 (2 ×MIC) 6.25 (2 ×MIC)
Penicillin G 0.0156 (2 ×MIC) 0.0625 (8 ×MIC) 0.0625 (8 ×MIC)
MIC: Minimum inhibitory concentration.
Biomedicines 2019,7, 63 12 of 19
3.5. HWIs Cause Morphological Changes of S. pyogenes Biofilms
The inhibition of biofilm was significantly effective, along with four HWIs when compared to
controls, as shown in Figure 4. Mainly, biofilm reduction by 1/2
×
MIC and MIC values of HWIs
was about 87.4% and 99.1% for licorice root, 36.6% and 97.0% for barberry root, 37.9% and 95.8%
for oregano, and 35.3% and 94.0% for thyme flowering shoot, respectively. Surface structure and
morphology changes of biofilms formed with or without HWIs treatment at their sub-inhibitory
MIBC, S. pyogenes ATCC 19615 are shown in Figure 5. The SEM analysis revealed that the HWIs
cause noticeable cellular lysis and morphological alterations compared to untreated cells. Biofilms of
untreated control showed a typical multi-layer bacterial colony growth while a significant reduction in
microcolonies was observed in HWI-treated samples. Even at sub-inhibitory concentrations, penicillin
G eradicated biofilm leaving a few of dead cell debris. Interestingly, HWIs of thyme and oregano were
also showed significant destruction of biofilm by leaving either a few bacteria or cell debris. Dead
cell debris resulted from substantial biofilm inhibition by barberry root HWI treatment resulting in a
cluster, as shown in Figure 5. However, active disruption morphologies of cells such as ruptured shape
were observed. Therefore, our findings provide evidence that HWIs has an intense antimicrobial action
against S. pyogenes biofilm formation.
Biomedicines 2019, 7, x FOR PEER REVIEW 12 of 19
3.5. HWIs Cause Morphological Changes of S. pyogenes Biofilms
The inhibition of biofilm was significantly effective, along with four HWIs when compared to
controls, as shown in Figure 4. Mainly, biofilm reduction by 1/2 × MIC and MIC values of HWIs was
about 87.4% and 99.1% for licorice root, 36.6% and 97.0% for barberry root, 37.9% and 95.8% for
oregano, and 35.3% and 94.0% for thyme flowering shoot, respectively. Surface structure and
morphology changes of biofilms formed with or without HWIs treatment at their sub-inhibitory
MIBC, S. pyogenes ATCC 19615 are shown in Figure 5. The SEM analysis revealed that the HWIs cause
noticeable cellular lysis and morphological alterations compared to untreated cells. Biofilms of
untreated control showed a typical multi-layer bacterial colony growth while a significant reduction
in microcolonies was observed in HWI-treated samples. Even at sub-inhibitory concentrations,
penicillin G eradicated biofilm leaving a few of dead cell debris. Interestingly, HWIs of thyme and
oregano were also showed significant destruction of biofilm by leaving either a few bacteria or cell
debris. Dead cell debris resulted from substantial biofilm inhibition by barberry root HWI treatment
resulting in a cluster, as shown in Figure 5. However, active disruption morphologies of cells such as
ruptured shape were observed. Therefore, our findings provide evidence that HWIs has an intense
antimicrobial action against S. pyogenes biofilm formation.
Figure 4. Inhibition of biofilm formation of Streptococcus pyogenes 19615 by hot water infusions at
various concentrations. (a) licorice roots, (b) barberry roots, (c) oregano flowering shoots, and (d)
thyme flowering shoots. Each data point represents mean ± SD from three independent experiments
performed in triplicate.
Figure 4.
Inhibition of biofilm formation of Streptococcus pyogenes 19615 by hot water infusions at
various concentrations. (
a
) licorice roots, (
b
) barberry roots, (
c
) oregano flowering shoots, and (
d
)
thyme flowering shoots. Each data point represents mean
±
SD from three independent experiments
performed in triplicate.
Biomedicines 2019,7, 63 13 of 19
Biomedicines 2019, 7, x FOR PEER REVIEW 13 of 19
Figure 5. Scanning electron micrographs of biofilms of Streptococcus pyogenes formed on the
microplate surface with the presence of hot water infusions of licorice roots, oregano flowering shoots,
thyme flowering shoots, barberry root and penicillin G at their sub-inhibitory concentrations. HWI,
hot water infusion.
4. Discussion
Herbaceous plants are a source of complementary and alternative remedies to conventional
medications in treating several bacterial infectious diseases, including streptococcal pharyngitis
(strep throat). Previous studies have demonstrated that numerous herbal extracts could act as
antibacterial agents against S. pyogenes [10,34–38]. In the present study, we focused on 13 HWIs,
known to Canadian traditional and indigenous healers who used them against sore throat. The goal
Figure 5.
Scanning electron micrographs of biofilms of Streptococcus pyogenes formed on the microplate
surface with the presence of hot water infusions of licorice roots, oregano flowering shoots, thyme
flowering shoots, barberry root and penicillin G at their sub-inhibitory concentrations. HWI, hot
water infusion.
4. Discussion
Herbaceous plants are a source of complementary and alternative remedies to conventional
medications in treating several bacterial infectious diseases, including streptococcal pharyngitis (strep
throat). Previous studies have demonstrated that numerous herbal extracts could act as antibacterial
agents against S. pyogenes [
10
,
34
–
38
]. In the present study, we focused on 13 HWIs, known to Canadian
traditional and indigenous healers who used them against sore throat. The goal is to identify herbal
Biomedicines 2019,7, 63 14 of 19
plants as sources for specialty herbal tea to use in the management of streptococcal pharyngitis.
Furthermore, we identified the major bioactive components in those HWIs that may contribute to their
overall antimicrobial activities against S. pyogenes.
Spices and herbs (flavor foods) comprise a large number of phytochemicals [
39
], which have
been revealed to possess antimicrobial properties. For example, anti-S. pyogenes ability of phenolic
compounds [
40
,
41
], flavonoids [
35
], alkaloids [
42
], terpenoids [
43
], and tannins [
35
] have been reported.
Qualitative UPLC analysis in the present study for HWI revealed the presence of polyphenols,
flavonoids, alkaloids, terpenoids, steroids, and tannins. The phytochemical constituents of the different
plants, as well as different parts of the same plant, showed phytochemical diversity, which partially
explains the different antimicrobial potential established as a range of MIC and MBC values.
When comparing MIC and MBC values of the present study, licorice root, barberry root, oregano
flowering shoot, and thyme flowering shoot infusions were identified as the most efficient HWIs.
Similar antimicrobial potential of various varieties of thyme and oregano from different countries was
also reported against Streptococcus species [
43
,
44
]. The Anti-bacterial efficacy of licorice root infusion
was the most active extracts which showed the lowest MIC. Although HWIs of barberry, thyme, and
oregano showed a concentration- and time-dependent bacteriostatic effect, a significant bactericidal
effect was observed in licorice root infusion within 12 h of exposure at the concentration of 2
×
MIC.
The existence of antibacterial activities could be due to one or a few of phytochemicals specific to
particular plant species or a particular part of a plant used. The chemical characterization of licorice
infusion suggested the presence of specific phytochemicals such as glycyrrhizin, glabridin, naringenin,
asparagine, and 5-methoxyhydnocarpin. Previous studies have also demonstrated that isoflavonoid
compounds, such as glabridin, glycyrrhizin, glabriol, and hispaglabridin in the extracts purified from
licorice roots, can act as bactericidal agents against various microorganisms [45].
Biofilm formation in S. pyogenes infections is one of the significant defensive mechanisms during
pharyngitis infections where microbial cells tightly arranged and covered with extra polymeric
substances [
46
,
47
]. Usually, biofilms make bacteria more resistant to antibiotics than their planktonic
cells. Several other studies have shown that extractions or infusions from medicinal plants inhibit the
biofilm formation in different bacteria, including S. pyogenes [
41
,
48
,
49
]. Our findings suggest that these
HWIs of licorice roots, barberry roots, oregano, and thyme flowering shoots could be used against
drug resistant-S. pyogenes. The present study investigated morphological changes such as structural
alterations of S. pyogenes after exposure to the effective infusion treatments at their sub-MIBCs. The
destruction of bacterial biofilms and cells shown in the SEM images are compatible with biofilm
quantification findings of the MTT assay. Similar SEM observations against S. pyogenes have been
reported [40,50].
Due to the recognition of significant healing power in traditional medicine systems, herbal
medicines from indigenous pharmacopeia are being used as home remedies. On the other hand,
public perception of using functional foods as biomedicines has begun to expand, especially in the
Western world. Therefore, opportunities exist for innovation of efficacious, safe, and convenient herbal
products as functional foods/biomedicines such as herbal teas and herbal tonics. However, some of
the pharmacologically active compounds in herbs may cause side effects; thus, proper ethnomedical
verification of efficacy and safety is required. On the other hand, no universal regulatory system
ensures the efficacy and safety of plant remedies. Moreover, different countries have their policies
and classification for natural health products. For example, herbal teas are categorized under “herb
and plant-based remedies” in the Licensed Natural Health Products Database (LNHPD) in Canada
and trade as an over-the-counter (OTC) product according to the report by consumer health product
Canada [
51
], while herbal teas are traded as a dietary supplement in the United States. Therefore,
extensive evidence for the
in vitro
,
in vivo
, or clinical efficacies, as well as pharmacological mechanisms
of their phytochemical constituents, is necessary for the new product development.
Destruction of the cell wall, loss of cell membrane integrity due to disruption of phospholipid
bilayer and inhibition of cell wall, membrane, protein, and DNA/RNA synthesis are considered as
Biomedicines 2019,7, 63 15 of 19
potential mechanisms of phytochemicals on their antibacterial and antibiofilm activities. However,
since these HWIs contain a large number of different phytochemicals they may responsible for these
activities through different mechanisms insole or combine. However, the present study does not
have sufficient evidence to conclude the exact mechanism/s of these herbal extracts. Among the four
most efficacious extracts identified, only licorice HWI shows moderately quick (12 h) bactericidal
effect at its MBC value. The SEM images show clear cell destructions by the four HWIs when the
bacterial cultures were exposed to longer period at their sub-inhibitory concentrations. Leakage of
intracellular components followed by the loss of integrity of phospholipid bilayer and ultimately
leading to cell death may be the reason for observed lesser bacterial density and deformed cells/debris.
Licorice root, sage leaves, oregano, and thyme HWIs show bacteriostatic effects at lower concentrations,
which suggest they may not have immediate cell wall or membrane destruction effect. The chemical
structure of individual antimicrobial phytochemicals and their abundance in the efficacious HWIs
are important for their mechanisms of action. Licorice root extract contains flavonoids, isoflavones,
saponins, and coumarins, which may regulate the expression of genes responsible for bacterial virulence
of streptococcus species have been reported [
52
,
53
]. Different species of thyme and oregano were
reported to possess some antimicrobial activities [
44
]. Both of plant leaves or young shoots are used
in various food preparations as flavor enhancers as well as in herbal remedies. Although the modes
of action of the extracts are not clearly recognized, it may be due to the major bioactive compounds,
including thymol, terpenes, eugenol, flavones, glycosides of phenolic monoterpenoids, and aliphatic
alcohols, among others [44,54].
Herbal teas provide relief for pharyngeal inflammation in patients with streptococcal pharyngitis.
Anti-inflammatory activities of herbal extracts and infusions have been shown by several authors
previously [
55
,
56
]. Our recently published data on anti-inflammatory properties of these extracts have
proven the ability of all four efficacious HWIs for suppression of pro-inflammatory cytokines released
from inflamed human tonsil epithelial cells (HTonEpiCs) induced by antigens (lipoteichoic acid and
peptidoglycan) of S. pyogenes [
8
]. Following the herbal treatment, production of IL-8, hBD-2, ENA-78,
and CGP-2 was suppressed in HTonEpiCs. However, to use them as biomedicine such as herbal
tea with antibacterial and anti-inflammatory compounds, the suppressive effect of pro-inflammatory
cytokines production by HWIs is expected only to happen in inflamed cells without affecting to the
healthy pharynx epithelium cells. Therefore, we have previously performed cell viability assay to
assess the cytotoxicity of these extracts on HTonEpiCs and found that all HWIs showed no cytotoxicity
to the HTonEpiCs
in vitro
[
8
]. Therefore, use of these HWIs seems to be safer but further validations
using
in vivo
models are required. The dry weight-based extraction yield of licorice, barberry, oregano,
and thyme were 19.5% 6.0%, 8.2%, and 18.6%, respectively [
8
] which could be further enhanced by the
improved technology such as subcritical water extraction.
Moreover, the water-based extraction process used in this study is acceptable for functional
beverage applications as no chemical was used in the preparation and allows incorporation of
the extracts in natural health products without concern of potential toxic solvent residuals [
57
].
Additionally, the preparation of infusions is simple, fast, and economical, and their applicability in
commercial product development is straightforward. Therefore, it is possible that the use of non-toxic
concentrations of licorice, barberry, thyme, or oregano infusions to develop into functional beverages,
biomedicine, and natural health products for the management of streptococcal pharyngitis.
5. Conclusions
We report the inhibitory effect on the growth and formation of biofilm of S. pyogenes by
phytochemical-rich HWIs of selected Canadian traditional herbal plants. The water-based extraction
process is economical, environment- and consumer-friendly as well as allows the incorporation of
the extracts in functional foods without concern of solvent residuals. Therefore, we conclude that
HWIs of licorice, barberry, thyme, or oregano at their non-toxic concentrations as safe and efficient
symptomatic treatments for the management of discomfort conditions associated with strep throat of
Biomedicines 2019,7, 63 16 of 19
streptococcal pharyngitis patients. Potential mechanisms of antibacterial and antibiofilm properties of
these efficacious infusions may be due to the direct destruction of cell wall/membrane, inhibition of
biosynthesis of cell wall/membrane, and inhibition of protein synthesis involved growth, adherence,
and biofilm formation. However, further investigations are required to determine the impact of HWIs
on cell wall/membrane leakage and specific gene expression to reveal the mechanisms of cell disruption,
adhesion, and biofilm formation by presumed phytochemicals.
Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9059/7/3/63/s1.
Author Contributions:
H.P.V.R. (principal investigator) and N.M.W. designed the experiments; N.M.W. performed
the sample collection, sample preparation, conducted the experiments, and data analysis. Both authors wrote
the manuscript.
Funding:
This research was funded by the Collaborative Research and Development Grant program (CRDPJ
448052) of the Natural Sciences and Engineering Research Council (NSERC) and Canada and Island Abbey Foods,
Charlottetown, PEI, Canada.
Acknowledgments:
The authors acknowledge the assistance of JeffMorton (Department of Plant, Food, and
Environmental Sciences, Faculty of Agriculture, Dalhousie University) for sample collection and authentication,
Ryan Gibbs and Cory Murphy (Canadian Food Inspection Agency, Dartmouth, NS, Canada) for UPLC-ESI-MS/MS
analysis, and Ross J. Davidson (Director of Bacteriology, Division of Microbiology, Queen Elizabeth II Health
Sciences Centre, Halifax, NS, Canada) for providing a bacterial clinical isolate as a gift.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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