1. RAPID COMMUNICATION
Salicylic Acid Treatment Increases the Levels of Triterpene
Glycosides in Black Cohosh (Actaea Racemosa) Rhizomes
Annette De Capite1,2
& Tyler Lancaster1
& David Puthoff1
Received: 1 July 2015 /Revised: 4 November 2015 /Accepted: 16 November 2015
# Springer Science+Business Media New York 2015
Abstract Black cohosh (Actaea racemosa) serves as the host
plant for the Appalachian azure butterfly, Celastrina
neglectamajor. Overharvesting of Black cohosh for the die-
tary supplement industry may result in its extirpation, and may
also cause the elimination of the dependent butterfly. One way
to increase or maintain the number of host plants in forested
environments would be to reduce the number harvested, for
example by increasing the levels of the desired metabolites in
Black cohosh rhizomes. The secondary metabolites actein and
deoxyactein are triterpene glycosides and are among the com-
pounds associated with the putative activity of Black cohosh
extracts. Acetein and deoxyacetein are used to standardize
Black cohosh supplements. To gain an understanding of
mechanisms that may control actein and deoxyactein accumu-
lation, Black cohosh rhizomes were treated with exogenous
salicylic acid, jasmonic acid, or ethylene, or were mechanical-
ly wounded. Salicylic acid treatment significantly increased
the levels of actein and deoxyactein in the rhizome of Black
cohosh, suggesting that the synthesis of triterpene glycosides
is controlled in part by salicylic acid. Using salicylic acid or
related chemicals to increase the levels of actein and
deoxyactein in rhizomes may help supply the supplement in-
dustry and, simultaneously, help conserve Black cohosh and
species dependent upon it.
Keywords Jasmonic acid . Ethylene . Wounding . Actein .
Deoxyactein . Secondary metabolite
Introduction
Black cohosh (Actaea racemosa) is a medicinal plant native to
the U.S. that is found mainly in shaded environments within
wooded areas of Appalachia. Most of the Black cohosh for the
supplement industry is collected from wild populations al-
though harvesting the roots and rhizomes of the plant dimin-
ishes the Black cohosh population. Black cohosh provides a
vital niche for other species within the same environment. For
example, the caterpillars of the Appalachian azure butterfly
(Celastrina neglectamajor) feed exclusively on the buds of
A. racemosa (NatureServe 2015). This butterfly is found in
only 20 sites within all of Indiana (Shuey 2005). and is ranked
as vulnerable-secure (S3-S4) in Kentucky by NatureServe
(2015). In addition, this species is listed as S2 (imperiled) in
Maryland and S3 (vulnerable) in PA and WV (NatureServe
2015). Loss of wild Black cohosh plants due to harvesting
could contribute to further pressures on the Appalachian azure
butterfly.
In addition to risks to associated insects, collection of wild
A. racemosa is risky because of the likelihood of confusion
between several Actaeeae species including the threatened
(A. podacarpa) and poisonous (A. pachypoda) members.
Actaea podacarpa has few characteristics that distinguish it
from A. racemose including only a small difference in the fruit
and flowers (Ramsey 1987). Limiting harvest of Black cohosh
will not only protect Black cohosh’s wild genetic diversity and
the diversity of closely-related, difficult to distinguish species
but also the ecology of species dependent upon this plant.
Black cohosh contains two secondary metabolites, actein
and deoxyactein, which belong to the terpenoid class
* David Puthoff
dpputhoff@frostburg.edu
1
Department of Biology, Frostburg State University, 101 Braddock
Rd, Frostburg, MD 21532, USA
2
Present address: Department of Microbiology and Plant Biology,
Oklahoma University, 770 Van Vleet Oval, Norman, OK 73019,
USA
J Chem Ecol
DOI 10.1007/s10886-015-0655-x

2. (Nagarajan 2002). These compounds have been used to stan-
dardize nutritional supplemental extracts from Black cohosh
(Ganzera et al. 2000). While most of the research involving
actein and deoxyactein has been focused on potential pharma-
cological activity in humans, our research was aimed at deter-
mining how Black cohosh regulates production of these sec-
ondary metabolites in order to develop methods to increase
production of bioactive extracts and to better understand the
natural history of these compounds.
Given that most secondary plant metabolites are used to
defend plants against attackers (Howe and Jander 2008). we
hypothesized that plant-defense related hormones (jasmonic
acid, salicylic acid, ethylene) or the process of wounding
would cause an increase in the levels of actein and
deoxyactein in Black cohosh rhizome.
Methods and Materials
Rhizome Collection and Treatments Rhizomes of Black
cohosh for the wounding experiments were harvested from
the Frostburg State University campus. For all other treat-
ments, rhizomes were purchased from Catoctin Mountain
Botanicals (Jefferson, MD, USA). Collected rhizomes gener-
ally ranged from 20 to 50 g while those that were purchased
were generally smaller (~15–25 g). Immediately after harvest
or purchase, rhizomes were divided into two random groups
and the above-ground tissues were removed with a scalpel.
Each rhizome then was sliced cross-wise (down the vertical
axis, perpendicular to the soil line and parallel to the length of
the rhizome) in half with a razor blade. For the jasmonic acid,
salicylic acid, and ethylene treatments, including controls, one
half of the rhizome was submerged with the selected com-
pound dissolved in 50 mM phosphate buffer (pH 7.2) while
the other half was incubated in buffer alone for 24 h. In gen-
eral, 5–15 cm2
of exposed surface area were treated. Jasmonic
acid was applied as a 10 μM solution, ethylene as a 29 ppm
solution of ethephon (Sigma), and salicylic acid as a 100 μM
solution. In order to wound rhizomes, they were squeezed to
almost the breaking point with an electrician’s pliers every
1 cm. After treatment, rhizomes were removed from treat-
ment, rinsed briefly, blotted dry, and allowed to air dry for 3
d at room temperature.
Rhizome Processing and Extraction The treated rhizomes
were sliced longitudinally and immediately dried at 50 °C for
48 h followed by storage at room temperature for up to 1 d.
Rhizomes were ground for 2–5 min, depending on rhizome size,
in a Bel-Art Products Micro-mill (Peaquannock, NJ, USA).
Ground samples were stored at −80 °C until extraction. One half
gram of dried rhizome material was extracted × 3 with 3 ml of
methanol with sonication followed by 20 s vortexing. The super-
natants were combined, and methanol was added to bring the
volume to 10 ml. Each 10 ml extract was filtered through a
0.45 μm filter (Fisher) and stored at -80 °C.
Preparation of Actein and Deoxyactein Standards
Standards of actein and 23-epi-26-deoxyactein (deoxyactein)
(Chromadex, Irvine, CA, USA) were prepared as stock solu-
tions (0.2 mg/ml) in methanol and were stored at −20 or
−80 °C. Serial dilutions were performed to obtain mixtures
containing each compound at the following concentrations:
0.1 mg/ml, 0.05 mg/ml, and 0.025 mg/ml. The standards were
utilized to establish a standard curve for analysis of actein and
deoxyactein concentration in rhizome extracts using HPTLC.
High Performance Thin Layer Chromatography (HPTLC)
Silica gel HPTLC plates (20 × 20 cm) (EMD Millipore, Merk,
Darmstadt, Germany) were dried at 40 °C for at least 24 h.
Actein and deoxyactein standards and plant extracts were
spotted 1.5 cm above the bottom of plate with 0.5 cm between
each spot. Each standard spot consisted of either 10 or 5 μl to
ensure that the standard curve amounts bracketed the plant
samples. Each plant extract consisted of 4 μl. Spotted plates
were dried for 15 min at room temperature and then one hr at
40 °C before development. The mobile phase consisted of
ethyl formate: toluene: formic acid (30:50:20). The develop-
ment chamber was equilibrated with the mobile phase using
Whatman paper for 30 min prior to loading plates inside. After
the solvent front had reached 0.5 cm from the top of the plate,
plates were removed from the development chamber and dried
at room temperature for 15 min then transferred to 40 °C for
15 min. Plates were immersed in enough Anisaldehyde re-
agent (5 % sulfuric acid, 5 % acetic acid, 90 % methanol) to
cover the plate (~20 ml) for 1 s, allowed to dry, and then were
transferred to a hot plate (100 °C) for 1 min. Plates were
examined under visible light and digitally photographed im-
mediately after staining. The intensity of each spot from the
standard curve and the plant extracts were quantified using
spot densitometry (AlphaEaseFC ver3.2.3 AlphaInnotech
(now ProteinSimple), San Leandro, CA, USA). A Student’s
t-test was used to compare the treated and control halves of the
rhizomes.
Results
In order to elucidate treatments that alter the levels of actein and
deoxyactein in rhizomes, Black cohosh rhizomes were either
wounded or treated with exogenous jasmonic acid, salicylic acid,
or ethylene. The rhizomes used for these studies were collected
from wild populations, and thus represent an unknown genetic
diversity. In addition, environmental conditions (e.g., soil types,
light levels, pathogens/pests) may have varied among the rhi-
zomes so that each rhizome was split and one-half was used
for treatment and the other half as the control.
J Chem Ecol

3. Wounded Black cohosh rhizomes did not have significant-
ly different levels of either actein or deoxyactein compared to
untreated controls (Fig. 1a). There was an overall higher level
of deoxyactein as compared to actein in extracts of unwound-
ed and wounded rhizomes. Treatment of rhizomes with
jasmonic acid did not result in significant differences in the
levels of actein and or deoxyactein compared to untreated
controls (Fig. 1b). Likewise, when comparing rhizomes that
were either treated or not with ethylene, there were no signif-
icant differences (Fig. 1c). As seen with the rhizomes from the
other experiments in this study, the level of deoxyactein was
higher overall as compared to actein.
In contrast to the other treatments, salicylic acid treated
rhizomes did have significantly more actein and deoxyactein
compared to the untreated controls (P = 0.026, 0.009 for
actein and deoxyactein, respectively) (Fig. 1d). Levels of
deoxyactein rose from just under 3 mg/g dry rhizome
to 4 mg/g dry weight. Actein levels rose from just over
2 mg/g dry weight to just under 5 mg/g dry weight.
The increases between salicylic acid-treated and control
rhizomes were 2.2- and 1.4-fold for actein and deoxyactein,
respectively. Unlike untreated plants or plants from other
treatments, in the salicylic acid-treated rhizomes, the
amount of actein and deoxyactein were approximately
equal (Fig. 1d).
Discussion
It has been noted that conservation of plant species in general
simultaneously and substantially conserves insect diversity
(Panzer and Schwartz 1998). This is of particular importance
for species, such as the Appalachian azure butterfly, that de-
pend upon only one plant for their survival. Because there is a
great demand for its rhizomes, most of which are harvested
from wild populations, Black cohosh is in a unique category. It
is highly sought after from the wild, while at the same time its
harvest permanently removes individuals from wild popula-
tions. While previous research has shown that Black cohosh
can recover from low levels of harvesting, intensely harvested
populations do not recover after one year (Small et al. 2011).
Overharvesting could lead to extirpation of not only Black
cohosh, but also the insects that rely on this host.
One mechanism to relieve some of this harvest pressure is
to harvest fewer rhizomes and identify new ways to produce
more active compounds from those rhizomes. Many plants
have been manipulated or put under different stresses to obtain
desirable ingredients (Schilmiler et al. 2008; Sharma et al.
2011). Given that most specialized metabolites are produced
by plants to defend from attack, this study examined the use of
plant-defense hormones to elicit production of actein and
deoxyactein. While it is not known how the treatments
0
0.5
1
1.5
2
2.5
3
3.5
deoxyactein actein
mg/gdryweight
control
JA-treated
B
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
deoxyactein actein
mg/gdryweight
control
wounded
A
*
*
0
0.5
1
1.5
2
2.5
deoxyactein actein
mg/gdryweight
control
Eth-treated
C
0
1
2
3
4
5
6
7
deoxyactein actein
mg/gdryweight
control
SA treated
**
D
Fig. 1 For each of the sections of
the figure above, Black cohosh
rhizomes were split in half with
one half treated with indicated
hormone as described in Methods
and Materials with the other half
rhizome serving as its control;
wounded (a), treated with
jasmonic acid (JA) (b), treated
with ethylene (c), or treated with
salicylic acid (d). One half gram
of each rhizome then was
extracted, and actein and
deoxyactein were quantified
accordingly. Asterisk indicates a
significant difference (P-value
<0.05) using a Student’s t-test.
Error bars represent standard error
of the mean
J Chem Ecol

4. employed here may alter/reflect endogenous salicylic acid,
jasmonic acid, or ethylene levels, the treatments have been
shown to elicit biomarkers similar to those produced
when a plant is experiencing a genuine pathogen attack
(Chao et al. 1999). Only exogenous treatment with
salicylic acid caused a significant change (increase) in
the levels of actein and deoxyactein. Wounding rhi-
zomes or treating them with exogenous jasmonic acid
or ethylene did not cause any change in levels of the
monitored compounds. Previous research demonstrated that
other medicinal plants (e.g., Mitragyna speciosa), have a se-
ries of salicylic acid inducible genes (Jumali et al. 2011). The
data presented here provide preliminary support of a similar
inducible network of genes that could function in plant de-
fenses in Black cohosh.
Having the ability to increase the levels of needed com-
pounds traditionally collected from wild plants will reduce
the need to harvest more individuals. For Black cohosh, re-
ducing the number of plants removed by harvesting could
protect both the plant and dependent species such as the
Appalachian azure. The research presented here provides
one chemical treatment that could be used to increase valuable
compounds in Black cohosh tissues to achieve the long term
goal of conserving the genetic diversity of Black cohosh, its
close relatives, and species dependent upon it.
Acknowledgments The authors would like to gratefully acknowledge
the Appalachian Center for Ethnobotanical Studies for funding this re-
search project. Also acknowledged are helpful comments of the anony-
mous reviewers.
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