1. A ChemicalExtraction from Sanguinariacanadensis
(Bloodroot) and its Potential as an AntibacterialAgent
Anna R. Wille
DepartmentofBiology, Warren Wilson College, Asheville, NC
NaturalScience UndergraduateResearch Sequence,Fall 2015
Committee: Dr. Dana Emmert, Dr. Langdon Martin, Dr.Jeffrey Holmes

2. 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, pharmaceuticalcompanies 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,

3. 2014), anti-tumoral (Ahmad et al, 2000), and antimicrobial agent. Sanguinarine is a defensive chemicalwith
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

4. 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.

5. 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 susceptibilitytests
(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 Descriptions
ASSAY 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.

6. Figure 1. HPLC fluorescence data for sanguinarine standards
The bacterial analysisshowed 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.

7. 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
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. thuringiensis
P. alcalifaciens
M. species
B. subtilis
C. xerosis
E. coli DH5α P. fluorescens
A B C D
E F G H

8. 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

9. A B
C D
Figure 3. Effect of bloodroot extract with known sanguinarineconcentrationson 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

10. 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 successfullytreated (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 sensitiveto 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

11. 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 Pseudomonasfluorescens 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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