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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