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Chinese Medicine, 2012, 3, 215-222
http://dx.doi.org/10.4236/cm.2012.34031 Published Online December 2012 (http://www.SciRP.org/journal/cm)
In Vitro Characterization of the Efficacy and Safety
Profile of a Proprietary Ajuga turkestanica Extract
José M. Zubeldia1*, Aarón Hernández-Santana1, Miguel Jiménez-del-Rio1,
Verónica Pérez-López1, Rubén Pérez-Machín2, José Manuel García-Castellano3
1Polinat S. L. Taibique 4, Polígono Industrial Las Majoreras, Las Palmas, Spain
2Molecular Oncology Group (G-OncoMol) Research Unit, University Hospital of Gran Canaria,
Canary Health and Research Foundation Barranco de la Ballena, Las Palmas, Spain
3Department of Orthopaedic Surgery, Complejo Hospitalario Universitario Insular, Las Palmas, Spain
Email: *jose@polinat.com
Received September 12, 2012; revised October 18, 2012; accepted November 3, 2012
ABSTRACT
Ajuga turkestanica, an herbaceous flowering species in the mint family, has been traditionally used in Turkey and Uz-
bekistan for heart disease, muscle aches and stomach problems. Due to its high levels of phytoecdysteroids (particularly
the characteristic C-11-hydroxylated Turkesterone), anabolic properties have also been reported. The aim of our study
was to screen for early signs of efficacy and safety of a proprietary Ajuga turkestanica extract (ATE) using in vitro
models. C2C12 mouse myotube cell line was used to study potential effects on viability and gene modulation. Cell vi-
ability was evaluated with different concentrations [0.2 - 200 ppm (mg/L)] of ATE. Gene modulation was assessed by
quantitative polymerase chain reaction (qRT-PCR) after 6 h incubation (ATE vs. the androgenic anabolic steroid me-
thandrostenolone). Total androgenic activity was measured using the A-SCREEN bioassay. Ultra-high performance
liquid chromatography analysis showed good correlation between the phytochemical profile of the native plant and our
ATE. C2C12 mouse myotube cells treated with ATE experienced no significant loss of viability (concentrations 0.2 - 200
ppm, 1 - 24 hs, p > 0.05). qRT-PCR array analysis showed significant (p < 0.05) down regulation of Caspase-3 (2-fold)
and Myostatin (4-fold). The extract showed no androgenic activity within the dose range used. Our results indicate the
potential for an ATE to support muscle mass without typical androgenic side effects of synthetic anabolic drugs.
Keywords: Ecdysteroids; Ajuga turkestanica; Turkesterone; Caspase 3; Myostatin; Androgenic Activity;
Sarcopenia
1. Introduction
The genus Ajuga (Labiatae) is comprised of more than
40 species widely distributed in temperate regions of
both hemispheres and contains at least three classes of po-
tentially bioactive compounds: clerodane diterpenes, phy-
toecdysteroids and iridoid glycosides. Ajuga turkesta-
nica (Regel) Briq is a perennial herb growing mainly in
Central Asia known as a rich source of bioactive sub-
stances and used by local people to treat heart diseases,
muscle and stomach aches [1]. With regards to phytoec-
dysteroids several bioactive compounds have been iso-
lated including turkesterone, 20-hydroxyecdysone (20-
HE), cyasterone, cyasterone 22-acetate, ajugalactone, aju-
gasterone B, α-ecdysone and ecdysone 2,3-monoaceto-
nide [2,3]. A characteristic feature of Ajuga turkesta-
nica is the presence of the C11-hydroxylated turkeste-
rone, which has not been observed in other species of the
same genus [4].
Ecdysteroids are polyhydroxylated ketosteroids with
long carbon side chains. These steroid hormones control
moulting and reproduction in arthropods, but their role in
plants is less well known as they do not elicit any of the
classical plant hormone responses [5]. Plants may use
ecdysteroids as a chemical defense against insects by
disrupting their hormonal balance and moulting process
[6]. The discovery of these steroid molecules in 1966 in
several plant species led to their availability in large
amounts for pharmacologic studies in search of safer
more specific insecticides. While showing no signs of
toxicity, ecdysteroids had other possible beneficial ef-
fects that could support their use in folk medicine such as
immunomodulation, antiarrythmic, hepatoprotective, or
antidiabetes effects [7-10].
Ecdysteroids are structurally different from mammal-
*Corresponding author.
C
opyright © 2012 SciRes. CM
J. M. ZUBELDIA ET AL.
216
ian steroids, and they are not expected to bind to verte-
brate steroid receptors. However, anabolic effects have
been reported in vertebrates: increased growth in mice,
rats, sheep, or pigs, and increased physical performance
without training in rats with increased synthesis of myo-
fibrillar proteins [11].
The potential for any substance to increase protein
synthesis in muscle by-passing secondary effects com-
mon with steroid synthetic drugs may be an attractive
approach for important health issues such as sarcopenia,
a condition in which subjects have progressive general-
ized loss of skeletal muscle mass and function. It has
been associated with adverse outcomes such as falls,
mobility limitations, incident disability, and fractures in
the elderly [12]. It is also associated with insulin resis-
tance in both non-obese and obese individuals and ab-
normal blood glucose levels in obese individuals, espe-
cially in those younger than 60 years of age [13]. Finally,
sarcopenia plays a key role in the development of ca-
chexia, a syndrome occurring at terminal stages of cancer,
chronic heart or kidney failure, or AIDS [14]. Proposed
treatments include testosterone supplementation, which
would require close monitoring of androgenic side ef-
fects such as prostate hypertrophy [15].
The aim of our study was to initially screen a proprie-
tary Ajuga turkestanica extract (ATE), rich in ecdyster-
oids for early signs of efficacy (increase/protection of
muscle mass) and safety (lack of androgenic activity).
2. Materials and Methods
2.1. Chemicals and Materials
HPLC grade acetonitrile and methanol were purchased
from Merck (Spain). Water was purified and deionized
by a Milli-Q ultrapure water system. Turkesterone and 20-
hydroxyecdysone (20-HE) reference standards were ob-
tained from Chromadex (Irvine, USA). Methandroste-
nolone, a commercially available synthetic anabolic ster-
oid, and 17β-Estradiol (E2) were purchased from Sig-
ma-Aldrich (Spain). Methyltrienolone (R1881), a non-
me- tabolizable synthetic anabolic steroid, was provided
from Perkin Elmer (Spain).
2.2. Plant Extraction
Ajuga turkestanica was collected in Uzbekistan. The dri-
ed whole plant (1 kg) was extracted in a percolator at
room temperature using 10 L of 85% ethanol in water for
2 hs. The liquid fraction was removed and the whole pro-
cess was repeated. The two liquid fractions (20 L) were
combined and ethanol was removed in vacuo before
freeze-drying (Telstar Cryodos benchtop freezedrier).
Samples were vacuum sealed in plastic bags and stored at
room temperature inside a dessicator.
2.3. Chromatography
Ajuga turkestanica whole dried plant (finely ground) or
powdered extract (0.1 g) was diluted in methanol (25 ml)
and sonicated for 15 min at room temperature. The solu-
tion was filtered through a 0.2 μm syringe filter (Micron
Analytical, Spain) before analysis by ultra high perfor-
mance liquid chromatography (UPLC). UPLC analysis
was performed on a Waters Acquity H-Class UPLC sys-
tem coupled to a photodiode array detector (PDA). Se-
paration was carried out on an Acquity C18 BEH column
(Waters, 100 × 2.1 mm, 1.7 µm). The mobile phase con-
sisted of ultrapure water (A) and acetonitrile (B). The
following linear gradient was used: 0 - 5 min, 10% - 50%
B; 5 - 6 min, 50% - 100% B, 6 - 8 min, 100% B. Each
run was followed by an equilibration period of 2 min.
The flow rate was 0.5 ml/min and the injection volume 1
µL. The column temperature was 50˚C and the detec-
tion wavelength was set to 245 nm.
2.4. Gene Expression Study
A mouse skeletal muscle cell line, C2C12 (American Type
Culture Collection, UK), was cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) with high glucose
(Thermo Fisher Scientific, Spain) supplemented with
10% fetal bovine serum (Lonza Group, Switzerland), 2
mM glutamine, 100 units/ml penicillin and 100 μg/ml
streptomycin. Cells between passages 3 and 10 were
seeded at a density of 10,000 cells per cm2. Cells were
grown for 48 hs until they reached 80% - 90% conflu-
ence. To induce myogenic differentiation, the medium
was replaced with differentiation medium, DMEM sup-
plemented with 2% horse serum (PAA Laboratories,
Austria) [11,16]. After 10 days the myoblasts had fused
into multinucleated myotubes. Cells were maintained at
37˚C in a humidified 5% CO2 incubator and medium was
changed every other day.
Cell viability after ATE treatment was determined us-
ing the Presto Blue cell viability kit (Invitrogen, Spain)
following the manufacturer’s instructions. C2C12 cells
were plated and differentiated to myotubes into 96-well
plates. After differentiation, the culture medium was re-
placed with DMEM containing various concentrations of
ATE (0.2 - 200 ppm) for 1, 3, 6 and 24 hours. Before
Presto Blue kit reagents were added, the medium was
removed and cells were washed with PBS. The cells
were incubated with Presto Blue for 20 min at 37˚C. Flu-
orescence was measured on a MX3005P Q-PCR Sys-
tem (Agilent Technologies, Spain) using a Cy3 filter set
on plate read mode.
For RNA extraction, C2C12 cells were plated and dif-
ferentiated to myotubes into 12-wells plates. After dif-
ferentiation, cells were incubated with 20 ppm ATE
(approx. 1 µM total ecdysteroids) or 1 µM methandros-
Copyright © 2012 SciRes. CM
J. M. ZUBELDIA ET AL. 217
tenolone for 6 h. RNA was then extracted using an All
Prep RNA/Protein Kit (Qiagen, Spain). Total RNA was
quantified using a fluorometric method with Quant-iT kit
(Invitrogen, Spain). RNA was stored at −80˚C until fur-
ther use. cDNA was reverse-transcribed from the RNA
extract using RT2 First Stand cDNA kit and we used a
RT2 Profiler PCR Array to analyze a panel of 84 genes
involved in skeletal muscle development and disease
(Qiagen, Spain). Quantitative real-time RT-PCR was
carried out using a SYBR-Green/ROX detection in a
MX3005P Q-PCR System. Samples were heated at 95˚C
for 10 min, followed by a second stage composed of 15
sec at 95˚C, 1 min at 60˚C which was repeated 40 times
and third stage for dissociation curve composed of 1 min
at 95˚C, 30 sec at 55˚C and 30 sec at 95˚C.
To analyze the PCR-array data, an MS-Excel sheet
with macros was downloaded from the manufacturer’s
website (http://www.sabiosciences.com). This program
calculated relative gene expression and statistical sig-
nificance.
2.5. Androgenic Study
MCF-7-AR1 cells were kindly provided by Nicolas Olea
from Granada University, Spain. The MCF-AR1 cells
result from stably transfected MCF-7 human breast can-
cer cell with a full human AR 27. The MCF-7-AR1 cell
line was grown routinely in a humidified atmosphere of
5% CO2 at 37˚C in Dulbecco’s modified Eagle’s Me-
dium (DMEM) without phenol red containing 10% fetal
bovine serum (FBS) supplemented with 2 mM glutamine,
100 units/ml penicillin, 100 μg/ml streptomycin, 15 mM
HEPES and 4.2 mM sodium bicarbonate (FBS-DMEM)
(Lonza Group, Switzerland). Cells become proliferative
quiescent when transferred into the same Culture me-
dium but supplemented with 10% charcoal-dextran-treated
FBS (CD-FBS, steroid free) (Thermo Fisher Scientific,
Spain) instead of FBS. The CD-FBS-DMEM medium
was used as experimental medium. MCF-7-AR1 cells
proliferate maximally in experimental medium plus 100
pM E2, and respond to androgens by decreasing their
proliferation rate in a dose-dependent fashion.
To test for the potential cytotoxicity of AE on MC7-
AR1, cells were trypsinized, counted and plated into
96-wells plates (NUNC) at seed density of 4000 cell per
well in FBS-DMEM. After 24 hs to allow attachment,
cells were treated with FBS-DMEM alone and with
FBS-DMEM in the presence of a range of ATE concen-
trations. After incubation for five days, the FBS-DMEM
was gently aspirated and the cells were trypsinized. Try-
pan blue exclusion test was used to count viable cells and
non-viable cells. The method stains selectively non-vi-
able cells. Briefly, a suitable cell suspension was given
into a tube and 0.4% w/v of trypan blue stain was added.
After mixing, solution was incubated 5 mins at room
temperature. Cells were finally counted in a TC10 auto-
mated cell counter (BioRad Laboratories, Spain).
The A-Screen bioassay compares the cell number of
similar inocula of MCF-7-AR1 cells growing in CD-
FBS-DMEN in the absence of any estrogen and andro-
gens (negative control), in the presence of 100 pM of E2
(estrogen control) and 100 pM of E2 plus a range of ATE
concentrations. MCF-7-AR1 were trypsinized and seeded
into 96-wells plate (Nunclon delta) at concentration of
6000 cell/well in experimental medium. After 24 hours to
allow attachment, experimental medium with 100 pM of
E2 containing the various dilutions ATE (0.1 to 100 PPM)
was added into each well. Positive (100 pM E2) and
negative control (CD-FBS-DMEM), as well as plant ex-
tract doses were tested six-fold. In addition, an androgen
reference curve made with R1881 or methandrostenolone
was set up as positive control in every experiment. After
120 hs, the assay was finished by gently removing the
experimental medium and the addition of ice-cold 10%
Thricloroacetic acid (wt/vol). The plates were left on ice
for 30 mins, then rinsed gently 3 times with water and
allowed to air dry. Cells were then stained with 0.4%
sulforhodamine B (SRB) in 1% (vol/col) acetic acid for
20 mins. The bound dye was solubilized with Tris-base
pH 10.6. After short shaking, absorbance was read in a
MW plate reader (Biotek) at 492 nm subtracting the
background measurement at 620 nm. It has been estab-
lished previously that there is a direct linear relationship
between cell number and the absorbance values of the
Tris-SRB solution. Experimental readings were in the
lineal range of the standard curve (Data not shown).
3. Results
The most abundant ecdysteroids in the A. turkestanica
extract were quantified with UPLC-PDA. Figure 1 shows
the chemical structures and the chromatographic separa-
tion of the main bioactive compounds typically found in
Ajuga turkestanica plant extracts and the powder extract
used in this study. The ATE powder contained approxi-
mately 0.69% (w/w) turkesterone and 1.30% (w/w) 20-
HE. No significant difference was observed between the
Ajuga turkestanica plant and the corresponding powder
extract, indicating that the phytochemical profile of the
main bioactive compounds does not change during the
extraction and preparation process. Preservation of the
native constituents in the plant is essential prior to any
biological testing.
To evaluate potential cytotoxicity, myotubes were
treated with ATE powder at concentrations from 0.2 to
200 ppm (mg/L) for up to 24 hs and cell viability was
evaluated using the Presto Blue cell viability kit. This
reagent kit is a resazurin-based assay, where resazurin is
converted to the fluorescent product resorufin by metab-
olically active cells and measured quantitatively [17].
Copyright © 2012 SciRes. CM
J. M. ZUBELDIA ET AL.
Copyright © 2012 SciRes. CM
218
This transformation of non-fluorescent resazurin to fluo-
rescent resorufin is the basis for the use of this fluoro-
metric indicator for the determination of cell viability.
We did not see any significant loss of cell viability for
the range of concentrations and treatment period used in
this study (Figure 2(a), p > 0.05).
Our ATE showed a 2-fold downregulation of cas-
pase-3 in myotubes while the androgenic anabolic steroid
methandrostenolone downregulated caspase-3 but to a
lesser extent compared to the ATE (˂2-fold). Myotubes
treated for 6 hs with ATE showed a 4-fold down-regula-
tion of Myostatin. Methandrostenolone treatment also down-
regulated myostatin to a lesser extent (˂2-fold) (Figure
2(b), p < 0.05).
Since androgenic activity is ultimately based on cell
number end-point, it was necessary to establish whether
any decrease in proliferation could be associated with a
cytotoxic effect of the extract rather than AR agonist ac-
tion before the A-Screen was performed. Trypan blue
dye exclusion assay was used to examine ATE-mediated
cytotoxicity (expressed as non-viable cells) and to assess
cell viability upon exposure to ATE in complete medium
(FBS-DMEM). ATE treatment reduced both total cell
number and viability of MCF7-AR1 cells in a dose-de-
pendent manner (Figure 3). The results show that ATE
inhibits MCF7-AR1 cell viability at very high doses (200
and 600 ppm) by inducing both a cytotoxic cell response
and reducing the number of viable cells. Based on the
above, the concentration of ATE used in the A-Screen
assay was limited to a range of 0.1 - 100 ppm.
Results of the A-Screen test are shown in Figure 4.
The synthetic androgen methyltrienolone (R1881) was
used as the reference compound (Ca, positive control)
and a dose-response curve showed that R1881 inhibited
Figure 1. Chemical structures of the main bioactive compounds in Ajuga turkestanica: (1) turkersterone; and (2) 20-HE.
UPLC chromatograms of (a) Ajuga turkestanica whole plant; and (b) the corresponding freeze-dried powder extract (ATE)
measured at 245 nm.
Figure 2. (a) Effect of AE treatment on C2C12 muscle cell viability. Cells were cultured with media containing different con-
centrations of ATE (0.2 - 200 ppm) for 24 hs (n = 3). Results are expressed in fluorescence units (FU) and percentage of vi-
ability, calculated using the following equation: (FU treated/FU control) × 100; (b) Effect of ATE and methandrostenolone on
Caspase-3 (Casp-3) and myostatin (Mstn) gene expression levels in C2C12 muscle cells. Test groups were treated with 20 ppm
of ATE for 6 hours (n = 3). *p ˂ 0.05.
J. M. ZUBELDIA ET AL. 219
Figure 3. Effect of ATE on cell viability of MCF-7AR1 de-
termined by trypan blue dye exclusion assay. Cells were
grown for 5 days with FBS-DMEM in the presence of a
range of AE concentrations (0.1 - 600 ppm). C-: FBS-
DMEM only. Results are showed as fraction of viable and
nonviable cells and are expressed as percentage of the nega-
tive control total (viable and non viable) cell number.
cell proliferation at very low concentrations (Figure
4(a)), IC50 = 20 pM). Methandrostenolone also inhibited
cell proliferation at higher concentrations (Figure 4(b),
IC50 = 350 pM). The addition of ATE at a range of con-
centrations (0.1 - 100 ppm) to the culture media in the
presence of E2 did not show a significant proliferative
inhibition compared with the control (MCF7-AR1 cells
plus E2) at doses up to 100 ppm (Figure 4(c)).
4. Discussion
The C2C12 mouse cell line is a well-established in vitro
model for skeletal muscle studies [18]. C2C12 myoblasts
may be readily differentiated into multinucleated myo-
tubes under controlled conditions, and these cells behave
in many ways like skeletal muscle fibers, contracting
when stimulated and expressing characteristic muscle
proteins [19,20]. In this context, C2C12 myotubes were
used to study cell viability and changes in modulation of
genes associated with muscle skeletal, muscle develop-
ment and disease upon treatment with our ATE.
Evaluation of the molecular pathways involved in the
putative anabolic effect of the ecdysteroids present in the
ATE suggests two possible pathways. During muscle
wasting caspase-3 activation and the ubiquitin protea-
some system (UPS) act synergistically to increase the
degradation of muscle proteins. Activation of the former
is required to convert actomyosin and myofibrils into
substrates of the UPS. Caspase-3 cleaves specific 19 S
proteasome subunits in C2C12 muscle cells with a cell-
specific activity. Caspase-3 cleaves different subunits in
myoblasts and myotubes hence intervening in cell dif-
(a)
(b)
(c)
Figure 4. Androgenic activity bioassay (A-Screen): Dose-
response curve to (a) methyltrienolone (R1881) and (b)
methandrostenolone by MCF7-AR1 cells in the presence E2.
(c) Proliferative response of E2 treated MCF7-AR1 cells to
ATE in a range of concentrations (0.1 - 100 ppm). C-, non-
treated; C+, E2; Ca, E2 plus R1881.
ferentiation or muscle wasting [21]. Recently, Bhatnagar
and colleagues have shown that adding a caspase-3 in-
hibitory peptide to myotube cultures resulted in inhibit-
tion of tumor necrosis factor-like weak inducer of apop-
tosis (TWEAK) induced loss of myosin heavy chain and
myotube diameter [22]. Our ATE showed a 2-fold down-
regulation of caspase-3 in myotubes supporting its potential
to protect muscle form wasting, as opposed to methan-
Copyright © 2012 SciRes. CM
J. M. ZUBELDIA ET AL.
220
drostenolone which downregulated caspase-3 to a lesser
extent (˂2-fold).
Myostatin is mostly expressed in skeletal muscle and
normally functions as a negative regulator of muscle
growth. Upon the binding to activin type IIB receptor,
this extracellular cytokine initiates several different sig-
naling cascades resulting in the down-regulation of the
important myogenesis genes. Muscle size is regulated via
a complex interplay of myostatin signaling with the insu-
lin-like growth factor 1/phosphatidylinositol 3-kinase/
Akt pathway responsible for increase in protein synthesis
in muscle 14. Myostatin blockage or its natural absence
leads to a significant increase in muscle mass [23]. Myo-
tubes treated for 6 h with ATE showed a 4-fold down-
regulation of myostatin, supporting the putative anabolic
effects of the plant. Methandrostenolone treatment also
downregulated myostatin but to a lesser extent (˂2-fold).
These results are also compatible with other investiga-
tions. Gorelick-Feldman et al. studied the mechanism of
action of ecdysteroids in murine C2C12 myotubes in
which a 95% ethanol-based extraction preparation pro-
voked a 15% increase in protein synthesis.
However, when myotubes were pretreated with a Pho-
sphatidyl Inositol 3-kinase (PI3K) inhibitor, the effect on
protein synthesis was significantly reduced indicating the
involvement of this particular molecular pathway [11].
Knockout of the myostatin gene has been associated with
the up-regulation of proteins involved in glycolitic shift
of muscle and down regulation of proteins involved in
oxidative energy metabolism. Specifically, investigators
have found increased expression of genes belonging to
the PI3K pathway in myostatin-null mice as opposed to
the wild type [24]. Hence, down-regulation of myostatin
by our ATE in C2C12 myotubes goes in accordance with
previous published results.
Testosterone and other androgenic steroid drugs have
been used in the past to increase or maintain muscle mass.
These drugs act via the androgen receptors and as such
have shown significant side effects that must be carefully
evaluated before initiating any therapy. There is an in-
creased risk for prostate hyperplasia and cancer in men,
virilization in women, and cardiac hypertrophy and athe-
rosclerosis for both [25]. Safer approaches have been
evaluated as well. A randomized, double-blind, placebo
controlled, multicenter trial was conducted to evaluate
the safety and efficacy of a novel selective androgen re-
ceptor modulator (SARM). A total of 120 healthy men
and postmenopausal women were evenly randomized to
take placebo or 0.1, 0.3, 1 or 3 mg of SARM for 12
weeks. The incidence of adverse events was similar
amongst groups with no serious events reported. Spe-
cifically, the novel compound did not have effects on
sebum production or hair growth in women while elicit-
ing a dose-dependent increase in total lean body mass,
highlighting the benefits of dissociating the anabolic and
androgenic activities when a therapeutic effect is sought
[26].
The anabolic effect of ecdysteroids is connected with
the acceleration of translocation processes instead of the
induction of new RNA synthesis. Ecdysteroids are not
likely to act as the classical steroids, via cytoplasmic
receptor and regulation of gene transcriptional activity.
In fact, an androgen dependent development is a prereq-
uisite before the action of ecdysteroids in rats. Using
radioligand assays Báthori et al. found that none of 11
tested ecdysteroids (including turkesterone) bound sig-
nificantly to estrogenic, glucocorticoid or androgenic
receptors [27]. Ecdysteroids display significant structural
differences from anabolic-androgenic steroid hormones,
which may explain the different mechanisms of their
anabolic action.
MCF7-AR1 is a human cancer-derived cell line which
has been genetically engineered to over express the AR
[28]. The A-Screen cell bioassay, developed to measured
anti-androgenic activity using MCF7-AR1 cell number
as the end point, is used to identify androgenic chemicals
among environmental pollutants and it has proved to be
very sensitive and reproducible assay for detecting an-
drogenic activity [29]. This assay measures androgen‐
dependent inhibition of proliferation of the androgen
receptor (AR)‐positive human mammary carcinoma cell
line, MCF7‐AR1. This cell line has been stably trans-
fected with a full human AR and expresses approxima-
tely five times more AR than wild‐type cells. MCF7‐
AR1 cells retain the capacity to proliferate in response to
estrogen treatment (E2). Androgens inhibit estrogen‐in-
duced proliferation and cells arrest in G0/G1 phase in a
dose-dependent manner [28].
The A-Screen bioassay compares the cell number of
similar inocula of MCF-7-AR1 cells growing in media in
the absence of any estrogen and androgens (C-, negative
control), in the presence of E2 (C+, estrogen control) and
in the presence of E2 in combination with different con-
centrations of the suspected androgen (Figure 4). An-
drogenic activity of a test compound results in the in-
hibit-tion of cell proliferation compared to the E2 control.
As expected both androgens (R1881 and Methandros-
tenolone) inhibited cell proliferation. However, adding
ATE at a non-cytotoxic concentrations (0.1 - 100 ppm) to
the media did not show a significant proliferative inhibi-
tion compared with the control (MCF7-AR1 cells plus
E2) (Figure 4(c)). Therefore and based on these results,
it was established that our ATE does not show andro-
genic activity within the dose range used.
In conclusion, we have shown the feasibility of ob-
taining a standardized Ajuga turkestanica extract that
retains the main bioactives in a ratio similar to that of the
root material. Preservation of this natural ratio is essen-
Copyright © 2012 SciRes. CM
J. M. ZUBELDIA ET AL. 221
tial to maintain the synergistic effect of the different phy-
toactive compounds. Biological activity of the high con-
tent in ecdysteroids (and particularly turkesterone) of the
ATE was demonstrated by real time qRT-PCR. Cas-
pase-3 and Myostatin were both significantly down re-
gulated, supporting the results by others which indicate
ecdysteroids may protect against muscle waste. Results
of the A-screen assay showed with high sensitivity the
lack of androgenic activity of the ATE, a desired trait in
developing alternative approaches for managing sarco-
penia in humans. We believe further clinical work is
warranted.
5. Conflict of Interests
José M Zubeldia, Aarón Hernández-Santana, Miguel Ji-
ménez del Rio, and Verónica Pérez work for Polinat SL,
the company which has developed and manufactures the
Ajuga turkestanica extract. Jose Manuel García Castel-
lano and Rubén Pérez Machin have no disclosures. This
work was funded by Polinat SL.
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