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A Chemical Extraction from Sanguinaria canadensis (Bloodroot) and its Potential as an Antibacterial Agent
Anna R. Wille
Department of Biology, Warren Wilson College, Asheville, NCNatural Science Undergraduate Research Sequence, Fall 2015
Committee: Dr. Dana Emmert, Dr. Langdon Martin, Dr. Jeffrey Holmes
INTRODUCTION
The advent of antibiotics in the 1940s revolutionized and defined modern
American medicine. By dropping the mortality rate from common infections,
advances could be made in surgery and medical fields that necessitate
immunocompromisation, which includes chemotherapy and common arthritis
medication (Childress, 2013). However, due to high costs and difficulty of
development, pharmaceutical companies have all but dropped antibiotic research.
As a consequence, the FDA has only approved of 11 new antibiotics in the last 17
years; in that same amount of time, antibiotic resistance in hospitals has increased
from 15 to 60 percent (Kranz, 2015). The escalation of resistance is in part due to
the fact that the few new drugs produced were not technically of a new class—the
last of which, the Lipopeptides, was introduced in 1987. This means that no new
antibiotic has used a new mode of action or changed the spectrum of bacteria
targeted since the 1990s (Gallagher, 2015).
Sensing an impending crisis, congress passed the GAIN (Generating Antibiotic
Incentives Now) Act, signed into law by President Barack Obama in July of 2012.
The GAIN Act is intended to create a financial incentive for pharmaceutical
companies to develop new antibiotics, with a large focus on smaller companies who
risk less in early developmental stages (Kranz, 2014). As a result, a few
pharmaceutical companies are getting more creative in their approach, including
developing new methods to culture bacteria found in soil samples, and a few new
potential antibiotics are now in the later stages of development (Gallagher, 2015).
Even so, new antibiotics will have to be developed and introduced at a much higher
rate if the medical field is going to keep ahead of the steadily rising antibiotic
resistance (Childress, 2013).
Sanguinaria candaensis, also known as Bloodroot, is a small herbaceous
perennial found in varying quantities along the east coast of North America and as
far west as the Rockies, with a high concentration in the Appalachian Mountains of
Virginia and North Carolina. S. canadensis has been listed as “Exploitably
Vulnerable” in the State of New York and of “Special Concern” in the State of Rhode
Island, but is available from commercial growers across the country. The plant has
been known for centuries as a medicinal herb, used as early as my mid-18th century
by the Cherokee people as an external salve to treat breast cancer (U.S. National
Parks Service, 2015). The bioactive alkaloid, sanguinarine, has been found to be an
anti-inflammatory (Li et al, 2014), anti-tumoral (Ahmad et al, 2000), and
antimicrobial agent. Sanguinarine is a defensive chemical with cytotoxic effect that
can be found in the root of the low-growing herb, giving it the aspect that it is
“bleeding” when cut (Campbell, 2007).
Image 1. North American range of Bloodroot Plant, according to the NRCS Plants Database
Image 2. Chemical structure of Sanguinarine
The aim of this study is to confirm and elaborate on previous findings of
sanguinarine’s antibacterial properties by testing the extract from the rhizome of
Sanguinaria canadensis against a broad spectrum of bacterial strains.
METHODS
CHEMICAL EXTRACTION
Rhizomes of S. canadensis were obtained from Dr. David Ellum. Most of the
rhizomes were purchased from Moonbranch Botanicals of Robbinsville, NC, and
some were gathered in late summer from woodland areas near Warren Wilson
College. The fresh rhizomes were dried in a Labcare America precision oven to a
constant mass, and then, combined with the purchased rhizomes, were ground in a
Mr. Coffee® spice and coffee grinder until a homogenous powder was obtained. The
resulting powder, a 5:1 ratio of purchased to gathered rhizomes, was stored in an
amber bottle at less than 20 ºC to prevent degradation.
Of the powder, 20 mg was immersed in 200 mL of methanol and placed in an
orbital shaker at 100 rpm for at least 24 hours. The resulting mixture was vacuum
filtered and the solids discarded. The liquid phase was then rotary-evaporated to
reduce the mixture to a concentrated extract. The resulting sample was viscous,
deep red, and massed at 2.852 grams. The extract was then diluted with methanol to
50 mL.
CHEMICAL ANALYSIS
The extract was analyzed by a Shimadzu LC-10AT HPLC coupled with UV-VIS
and fluorometer against known quantities of pure sanguinarine chloride (purchased
from Tocris Bioscience, Bristol, UK). The UV-VIS detector was set to detect
absorbance at 335 nm. The fluorometer was set to excite at 335 nm and detect
fluorescence at 587 nm. The column used to perform the separation was a ProntoSIL
120-c18-ace-EPS (5.0 µm, 4.6x150) made by MAC-MOD Analytical. The separation
methods were modified from Reinhart et al (Campbell, 2007) using a multistep
gradient mobile phase beginning with a 10:90 ratio of acetonitrile to 50% Methanol,
50% DI water acidified with 0.1% trifluoric acid. The concentration of acetonitrile
was increased to a 50:50 ratio over ten minutes and the results were recorded using
LoggerPro software. The fluorescence data was then used to calculate a standard
curve and approximate the sanguinarine content in the extraction.
BACTERIAL ANALYSIS
The bacterial strains were chosen for diversity of type and availability,
resulting in 7 strains to be tested: Bacillus subtilis, Bacillus thuringiensis,
Corynebacterium xerosis, Escherichia coli DH5α, Providencia alcalifaciens,
Pseudomonas fluorescens, and Moraxella species. These strains were subjected to
Kirby-Bauer disk diffusion susceptibility tests (Hudzicki, 2009) with varying extract
dilutions containing from 7 to 7,000 ppm sanguinarine. A list of the concentrations
used and number of replicates made can be found below, in Table 1. The assays
were prepared by dropping 5 µl of each sanguinarine concentration onto disks
(prepared from Whattman qualitative filter paper cut to 6 millimeter diameters using
a standard paper hole-punch), which were then placed evenly on a bacterial lawn
(prepared by growing the bacteria strains overnight in a vial of 5 ml tryptic soy
broth, gently shaken) spread on a tryptic soy agar plate. The plates were placed in
an incubator at 28 ºC for exactly 24 hours, at which point any visible diameters were
measured using a digital caliper.
Table 1. Bacterial Assay DescriptionsASSAY
CONCENTRATIONS (PPM) REPLICATES
1 7, 114, 1828, 3648 1 of each bacterial strain
2 37, 114, 456, 1828, 7296 2 of each bacterial strain
3 114, 228, 456, 912, 1828 2 of each bacterial strain
DATA & RESULTS
The standard curve graphed from the HPLC fluorescence data for sanguinarine
revealed a linear regression trend-line with R2 value of 0.998, seen below in Figure 1.
The amount of sanguinarine in the extract was calculated to be 364.8 mg. This is
12.8% of the extract mass, and 1.8% of the 20-gram rhizome sample from which it
was extracted.
Figure 1. HPLC fluorescence data for sanguinarine standards
The bacterial analysis showed six of the seven bacteria tested were responsive
to the extract treatment, to varying degrees (see Image 3, below). The results were
analyzed by t-tests comparing halo diameters to the control to determine the
minimum comparable concentration at which the bacteria respond. Non-
responsiveness was recorded as 6 mm, the diameter of the filter paper disks and
therefore the limit of detection. A summary of this analysis may be found below in
Table 2, and the averages of the comparable concentrations in Figure 2. A line of
best fit was calculated for the diameters of the halos compared to concentration for
the four most responsive bacterial strains: M. species, C. xerosis, B. thuringiensis,
and P. alcalifaciens to examine the behavior. These calculations may be seen below,
in Figures 3.
Image 3. Bacterial response to bloodroot extract: a) control; b) Bacillus subtilis;
c) Bacillus thuringiensis; d) Corynebacterium xerosis; e) E. coli DH5a; f) Providencia alcalifaciens; g) Pseudomonas fluorescens; h) Moraxella species
Table 2. Summary of bacterial response to extract with known sanguinarine concentrations
A B C D
E F G H
P≤0.05 for concentrations 114 ppm and above:
P≤0.05 for concentrations 456 ppm and above:
P≤0.05 for concentrations 1828 ppm and above:
Not responsive to extract treatment at any concentration:
B. thuringiensisP. alcalifaciens M. species
B. subtilisC. xerosis
E. coli DH5α P. fluorescens
A B
C D
E F
G
Figure 2. Comparisons of average ring diameter to known sanguinarine concentrations for: a) Moraxella
species; b) Bacillus thuringiensis; c) Providencia alcalifaciens; d) Corynebacterium xerosis; e) Bacillus subtilis; f) Escherichia coli DH5a; g) Pseudomonas fluorescens
A B
C D
Figure 3. Effect of bloodroot extract with known sanguinarine concentrations on bacterial strains
DISCUSSION
In HPLC analysis by UV-VIS, the peaks were not consistently distinguishable
between sanguinarine and other compounds absorbing at 335 nm. One such
compound could be the known bloodroot product and similarly-structured
benzophenathridine alkaloid chelerythrine (Graf et al, 2007). Chelerythrine is known
to significantly change absorbance behavior at different pH levels (Absolínová et al,
2010), and thus may have affected the consistency of the absorbance peaks by HPLC
UV-VIS. Although the compound sanguinarine has also shown to change structurally
according to pH (Bashmakova et al, 2009), its behavior by fluorescence detection is
relatively stable (Urbanová et al, 2009) and therefore testing by HPLC fluorometry
yielded much more consistent results. By these extraction and detection methods,
the amount of sanguinarine in extract consisted of 1.8% of the total rhizome mass,
slightly lower than published values. The yields in literature have been anywhere
from 2 to upwards of 4 mg per 100 mg dried rhizome from wildcrafted and cultivated
bloodroot plants (Graf et al, 2007).
Dried bloodroot rhizomes are commercially available by the pound on the
market for anywhere between $60 and $100. By the extraction results in this study,
up to 165.6 grams of sanguinarine can be obtained from a pound of bloodroot
rhizomes, and even more using more precise or costly extraction methods. The
plants, seeds, and roots can also be bought for at-home cultivation at costs as low as
$5. A lab-synthesized sample of sanguinarine, in contrast, can cost between $150
and $500 for less than 0.01 grams. It is therefore worth noting that for many
different kinds of uses, from laboratory studies to natural home remedies, it would be
much more cost effective to use the bloodroot rhizome. Further, since many of the
commercial sources are cultivated rather than wildcrafted, the use of bloodroot plant
for testing should not continue to adversely affect local bloodroot populations.
Of the bacteria tested, Moraxella species showed the largest halos at any
individual concentration, but also had some of the highest variances for halo size. M.
species is a gram-negative bacteria that is generally sensitive to antibiotics, and
rarely a cause of infection in humans (Berrocal, 2003), though in one case an
uncharacteristically penicillin-resistant Moraxella species infection was successfully
treated (Cox, 1994). The bacterial strain that is arguably the next-most responsive to
treatment by bloodroot extract, though highly variable at lower concentrations, was
Corynebacterium xerosis, an opportunistic gram-positive bacterium commonly found
on human skin. C. xerosis has been found to cause a large number of severe post-
operative infections, and though it is usually sensitive to most antibiotics, has shown
the potential to quickly become multiply resistant to antibiotics (Lortholary, 1993).
Bacillus thuringiensis was also highly susceptible at low concentrations, though
gram-positive B. thuringiensis is not a known pathogen and is, in fact, used as a
biological pesticide (Ibrahim, 2010).
The responsiveness found in Providencia alcalifaciens is of more significance
medically as it is a member of the family Enterobacteriaceae, a group of consistently
dangerous pathogens. P. alcalifaciens, though generally more susceptible to
antibiotics than its relatives, is a known cause for diarrhea in travelers and children
(Albert, 1992). The other gram-negative bacteria tested, Escherichia coli DH5α and
Pseudomonas fluorescens are neither pathogenic to humans nor very responsive to
treatment by bloodroot extract. A strain that responded somewhat better, gram-
positive Bacillus subtilis, can be purchased as a probiotic for humans and often
purposefully developed to have antibiotic resistance.
The concentration of sanguinarine at which the bacteria responded in these
assays was lower than 114 ppm in most cases, and the bacteria that responded were
of a large range in respect to type, ecology, and function. This leads to an optimistic
view on the possibility for use of bloodroot extract as a future antibiotic. Studies on
the mode of action used (Beuria et al, 2005) and comparing cytotoxicity versus action
have already been attempted for sanguinarine (Ahmad et al, 2000), but further
examination in these areas would be necessary before drawing any conclusions.
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