1) Researchers surveyed 25 locations in South Dakota to study ecological and chemical properties of Achillea (yarrow) species. Plant and soil samples were taken from plots and phenotypic measurements were recorded. 2) Extracts were prepared from two yarrow populations using sequential solvent extractions and tested in an antifungal bioassay against Candida species. Inconclusive results were found due to precipitate formation. 3) Future work includes repeating the bioassay, expanding tests to other microbes, identifying compounds in extracts, and investigating environmental and genetic influences on yarrow phenotypes between populations.

1. Michael R. Hurst, Justin M. Ramsey
Black Hills State University – 1200 University Street, Spearfish, SD 57799
Ecological and Chemical Evaluation of South Dakotan Achillea
I. Introduction
Research and field team:
Kendall Murie, Zak Kay, Darlene Coppe, Dr. Tara Ramsey,
Michael Hurst, Anna Hafele
Research reported in this publication was supported by an Undergraduate Research Fellowship from the
National Center for Research Resources at the National Institutes of Health, as well as a National Science
Foundation CAREER Award, Research Experience for Undergraduates supplement, and student research
assistant funds from South Dakota EPSCoR. Acknowledgement is also made to Dr. Cynthia Anderson, Dr. John
Dixson, Dr. Mark Gabel, Grace Kostel, and Hayden Bender for support and assistance throughout the project.
II. Survey IV. Bioassay
In the scientific sphere, the medicinal chemistry of European species of the
A. millefolium aggregate has been studied over the past two decades,
revealing diverse constituents in chemical analyses as well as strong
antioxidant, antimicrobial, and antifungal effects in biological assays.11 12 13 14
Wild Yarrow is thus regarded as being medicinally significant and a potential
source of remedies derived from natural products.
III. Extraction
V. Future Research
VI. Acknowledgements
Workflow:
- Visit potential sites for Achillea collection and create tentative plant list.
- Re-visit sites in later stage of growing season, marking
presence/absence of species in plots containing Achillea specimens.
Simultaneously measure dimensions of randomly-chosen leaves and
take readings of elevation.
- Considering the prior information, choose two individual sites from
which to collect Achillea material.
- Visit these sites in the latest stage of growing season, when plants are
fully mature but not yet beginning to senesce. Collect leaves from
randomly-spaced specimens to reduce risk of biased sampling.
- Place leaves in drying oven and let sit for 3-4 days.
- Follow laboratory extraction procedure with the two separate
populations of dried, finely-ground leaf material.
- Setup and run a bioassay with two yeast species: Candida albicans and
Candida krusei.
In order to test the medicinal properties of Achillea, an extract was prepared.
This was accomplished by cutting the two populations of plant material into ~2
inch pieces, separately homogenizing them using a standard coffee grinder, and
then employing a sequential gradient extraction procedure. Beginning with
hexanes, this procedure involves increasing the polarity of solvent stepwise to
methylene chloride and then to isopropyl alcohol.
Procedure:
Soak dried, finely-ground leaf material
in hexanes for ~24 hours. Then, pour
hexanes into flask and remove solvent
by rotary evaporation. Repeat these
steps with a second wash of hexanes.
Next, soak material in methylene
chloride for ~24 hours. Pour methylene
chloride into separate flask from
hexanes and remove by rotary
evaporation. Repeat these steps with a
second wash of methylene chloride.
Finally, replicate the procedure using
isopropyl alcohol.
After completing this process for both populations, yielding a total of six
extracts, all were placed on high vacuum for approximately 12 hours to
ensure solvents had been fully removed. The extracts were then weighed:
Hexanes
Methylene
Chloride
Isopropyl
Alcohol
Upper Tinton: 1.114g
% yield by weight: 1.119
Deerfield Lake: 2.331g
% yield by weight: 0.893
Upper Tinton: 2.212g
% yield by weight: 2.221
Deerfield Lake: 9.845g
% yield by weight: 3.772
Upper Tinton: 1.263g
% yield by weight: 1.268
Deerfield Lake: 4.257g
% yield by weight: 1.631
Description:
Hexanes oils seemed to be less viscous
than the others, and lighter in color –
dark golden brown.
Methylene chloride oils were more
viscous than hexanes oils, smelled
strongly of Achillea, and were most
abundant – dark greenish brown.
Isopropanol oils were the most viscous
and darkest – blackish green, tar-like.
Total % yield by weight:
Upper Tinton: 4.607
Deerfield Lake: 6.296
Short-term goals:
- Repeat hexanes bioassay testing and also test remaining Achillea
extracts against Candida albicans and Candida krusei.
- Evaluate the pH and texture of soils collected from surveyed sites.
- Complete final measurements of Achillea stems and inflorescences in
the field.
Study sites were established at 25 locations across South Dakota, including
habitats in six counties and spanning ~5,000 feet in elevation (Fig. 1)
Tinton Deerfield
Elevation 6100 feet 5960 feet
Avg. leaf area ~130 cm2 ~40cm2
Collected 99.611g 261.000g
To evaluate the medicinal properties of yarrow, an antifungal bioassay was
ran using Candida albicans and Candida krusei against hexanes extracts.
Cultures:
Load petri dishes with stock cultures
and leave to incubate for ~24 hours at
45o C. Then, use grown cultures to
create solutions matching ½ McFarland
Standard.
Dilute solutions with RPMI by 1/100
and then by 1/20 to create final
culture dilutions equal to 1/2000 of
½ McFarland Standard.
Treatment:
Find maximum solubility of extracts in
dimethyl sulfoxide (DMSO) – found to
be 80mg/mL. Using this result, mix a
stock solution of each extract in DMSO.
Properly dilute this stock solution
down to produce working solutions of
32mg/mL, 16mg/mL, 8mg/mL, and
4mg/mL.
As a positive control, also create
16mg/mL and 1.6mg/mL solutions of
Amphotericin B, an agent known to kill
Candida species.
Plating:
Using clear costar 96-well plates -
leaving outside wells empty due to
potential effect on plate reader - fill
plates to match the developed
template.
End with a total of four plates, with
one corresponding to each yarrow
population tested against each
Candida species.
Take readings on plate reader set to
530nm absorbance at 0, 19, 24, and 48
hours. Replicate every scan to ensure
consistent results.
Finally, export data to Microsoft Excel
for analysis.
Results:
At 19 hours, precipitate was seen
forming in treatment wells, darkening
color and skewing read results. Thus,
results are inconclusive and testing will
be repeated.
Some possible causes of this result are
user error, treatment’s response to
heat, or interactive effect.
Wild Yarrow (Achillea millefolium aggregate, Asteraceae) is a taxonomic
complex of ~15 plant species noteworthy for its circumpolar distribution,
frequent incidence of polyploidy, robust secondary chemistry, and use in
traditional folk medicine.1 One species (A. borealis) is endemic to North
America , and varieties of it exist in many ecotypes.2 3 4 These plants show
morphological and phenological adaptations to their environments and
represent a classical example of ecological diversification.5 6 7 The system
has recently emerged as a model for testing the significance of genome
duplication to phenotypic evolution and incipient speciation.8 9 10
1 Ehrendorfer F., Oesterreichische botanische Zeitschrift 1973, 122(3): 133-143.
2 Lawrence W.E., American Journal of Botany 1947, 34: 538-545.
3 Nobs M., Achillea in Illustrated Flora of the Pacific States 1960, Stanford University Press.
4 Tyrl R.J., Brittonia 1975, 27: 287-296.
5 Clausen et al., Carnegie Institution of Washington Publication 1940, 520.
6 Clausen et al., Carnegie Institution of Washington Publication 1948, 581.
7 Hiesey W.M. and M. Nobs, Botanical Gazette 1970, 131(3): 245-259.
8 Ramsey J., Heredity 2007, 3(98): 143-150.
9 Ramsey J., Proceedings of the National Academy of Sciences USA 2011, 17(108): 7096-7101
10 Ramsey J. and T. Ramsey, Philosophical Transactions of the Royal Society of London 2014, 369(1648): 20130352
11 Falconieri et al., Natural Product Communications 2011, 6(10): 1527-1530.
12 Stojanovic et al., Journal of Ethnopharmacology 2005, 1-3(101): 185-190.
13 Candan et al.,Journal of Ethnopharmacology 2003, 2-3(87): 215-220
14 Vitalini et al., Acta Biochimica Polonica 2011, 58(2): 203-209
Despite this promise, however, the North American subspecies – A. borealis
– has not been widely studied. As the biologically active properties of
European Achillea become more well-supported, it becomes increasingly
pertinent to apply similar study methods to find the medicinal effects of A.
borealis. Moreover, the ecology and systematics of the species are poorly
known outside of the U.S. and Canada. In summer 2015, the Ramsey Lab
initiated comprehensive studies of Achillea in western South Dakota to
address the following questions:
(1) What is the environmental distribution of wild yarrow in the Black Hills
and adjoining Great Plains, as inferred by vegetation and soil analyses?
(2) What is the occurrence of the European invader A. millefolium sensu
strictu, as inferred by flow cytometry and cpDNA sequencing?
(3) How do populations phenotypically diverge amongst environments,
based on field measurements of plant size and architecture?
(4) What are the chemical constituents and antimicrobial effects of A.
borealis, based on gas chromatography and biological assays?
These efforts will provide a foundation for genetic and biomedical
research in the coming years at Black Hills State University as well as
resources for student course instruction and scholarly endeavors. Because
most of the aforementioned research activities remain in progress during
August, this poster will focus on preliminary findings related to medicinal
chemistry and environmental distribution of Achillea.
Long-term goals:
- Develop a Staphylococcus aureus bioassay for further investigation of
yarrow extracts.
- Expand the antifungal bioassay to include a species of filamentous
fungi.
- Determine specific compounds in Achillea and isolate relevant findings.
- Investigate environmental and genetic contributions of phenotypic
differences among Achillea populations.
At each site, vegetation and soil sampling
was conducted in ~2,500 m2 plots that
circumscribed focal populations of
Achillea. Presence-absence inventories of
vascular plant communities were
performed three times throughout the
growing season. Soil collections were
collected and Achillea leaf measurements
were conducted in early July. Stem
measurements were made in late July.
Fig. 1 - Distribution of study sites and
phenotypic characteristics of Achillea
populations. Lower panel: Site elevation and
latitude on a north-south transect from the
North Dakota border to the southern Black
Hills. Upper panel: Variation in plant size and
architecture in populations (mean ± 95%
confidence interval) that includes order-of-
magnitude differences in leaf area.
Leaf dimensions differ significantly
across sites (ANOVA, F = 159.586,
P<0.001). Multivariate analysis of
presence-absence vegetation data
matrix (Fig. 2) suggest variation in
plant community composition is
strongly associated with elevation.
Fig. 2 – Plant species and community assemblages
in study sites along elevational transect. Top
panel: Ordination (cluster analysis) of vegetation
data suggests community differences between
high elevation (>5,000') and low elevation
(<4,500') areas. Below: Notable plant species
encountered.
Bear Mountain
Crooks Tower
Potato Springs
Flag Mountain
Cement Ridge (Upper)
Odakota Mountain
Big Hill
Cement Ridge (Lower)
Eagle Cliffs
Upper Tinton
Ditch Creek
Iron Creek
Iron Lake
Belle Fourche Reservoir
Benonite
Four Mile Ridge
Pine Slope
Deerfield Lake
Ft. Meade
E. Camp Crook
The Castles
E. Short Pine Hills
Higgins Gulch
Lookout Mountain
McKenna
Based on the factors explained
above, we deduced the best sites
for collection to be Upper Tinton
and Deerfield Lake. These sites were
chosen for their similar elevations
but their drastically different
ecotypes – Upper Tinton is a
forested riparian zone, whereas
Deerfield Lake is a short grass
prairie. Material weight (Table 1)
was measured after leaves were
collected and completely dried.
Table 1 – Comparison of the two collection sites.
Wild yarrow has long been recognized as a medicinally significant plant.
Native Americans reportedly have been using Achillea for hundreds of years
as a pain reliever, a diuretic, a digestive aid, a stimulant, and to reduce fever.
Additionally, the mythical Greek hero Achilles, whom the genus is named
after, reportedly carried leaves of Achillea to battle.
Specimens of wild yarrow found at Flag
Mountain (above) and in Harding County (left).
(Above) Plant material from Deerfield Lake
soaking in hexanes for ~24 hours.
(Below) Material after pouring off solvent.
(Above) Extract on high vacuum for ~12 hours.
(Below) Completed and labeled extracts.
Cultures of two Candida species – Albicans
and Krusei.
Top panel: 96-well plate prepared for
experimentation and analysis.
Bottom panel: plate template used.
(Left) Field team surveying Lower Cement Ridge. (Center) Field team collecting yarrow specimens
from Upper Tinton. (Right) Yarrow leaves left in trays to dry in the sun.