Nutrition Research and Practice (Nutr Res Pract) 2012;6(6):505-512
pISSN 1976-1457 eISSN 2005-6168
Improvement of andropause symptoms by dandelion and rooibos extract complex
CRS-10 in aging male
Yoo-Hun Noh1,*, Do-Hee Kim1,2,*, Joon Yub Kim1, Jiae Park1,2, Ok Hyeon Kim1, Daeseok Han3,
Won-Yong Kim4, Sung-Su Kim1, Moo-Yeol Lee5, Seok-Hyun Heo7, Misook Kim6, Won Bok Lee1,
Yoonhwa Jeong6§ and Soon Chul Myung8§
1Department of Anatomy and Cell Biology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea
2BioGrand Biomedical Research Center, BioGrand Inc., Gyeonggi 427-800, Korea
3Korea Food Research Institute, Gyeonggi 463-746, Korea
4Department of Microbiology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea
5Department of Physiology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea
6Department of Food Science and Nutrition, College of Natural Science, Dankook University, 152 Jukjeon-ro, Suji-gu, Yongin,
Gyeonggi 448-701, Korea
7Korea Health Supplements Institute, Gyeonggi 463-400, Korea
8Department of Urology, College of Medicine, Chung-Ang University, 221 Heukseok-dong, Dongjak-gu, Seoul 156-756, Korea
Many aging male suffer various andropause symptoms including loss of physical and mental activities. This study evaluated the putative alleviative
effects of CRS-10 dandelion and rooibos extract complex (CRS-10) on the symptoms of andropause. The survival rate of TM3 Leydig cells (TM3
cells) treated with CRS-10 was measured based on typical physiological stress. After daily intake of CRS-10 for 4 weeks, the level of testosterone,
physical activity and both the number and activity of sperm in older rats (18 weeks) were measured. Furthermore, thirty males were surveyed with
AMS (Aging Males' Symptoms) questionnaire after intake of 400 mg of CRS-10. Overall, CRS-10 protected TM3 cells from serum restriction and
oxidative stress via activation of ERK and Akt pathways. The level of testosterone and activation of spermatogenesis in rats were significantly
enhanced. In addition, physical locomotion was markedly improved. Daily intake of 400 mg of CRS-10 improved the quality of life among agingmale
respondents, according to a clinical survey using the AMS. The results indicate the potential of CRS-10 as a safe and efficacious natural substance
for reducing or alleviating andropause symptoms.
Key Words: Leydig cell, testosterone, CRS-10, spermatogenesis, andropause
Leydig cells in the interstitium of testis are the primary source
of testosterone, which stimulates the differentiation of the male
phenotype and spermatogenesis in the testes [1,2]. Loss of Leydig
cells significantly contributes to the age-related decline of serum
testosterone [3-5]. Substantial losses in serum testosterone may
result in the inactivation of spermatogenesis, loss of muscle
strength, as well as declines in muscle performance and physical
function, which can detract from the quality of life (QoL) of
aging males [6-8]. A predominant proportion of aging and older
men have reduced levels of serum testosterone, which is a major
reason for andropause symptoms .
Several recent studies have shown that the restoration of
testosterone level improves andropause symptoms, such as loss
of skeletal muscle, depressed mood, increased fat mass, sexual
dysfunction and osteopenia [9,10]. However, injection of artificial
testosterone can simultaneously induce many side effects inclu-
ding prostate cancer, benign prostatic hyperplasia and cardio-
vascular events [11,12]. Therefore, identification and refinement
of natural substances, which can protect Leydig cells and support
the continued production of serum testosterone, is very crucial
for reducing the symptoms of andropause.
Dandelions are composed of a variety of compounds including
This work was supported by a grant from the Korea Healthcare Technology R&D Project (A085138), Ministry for Health, Welfare & Family Affairs, Republic of Korea.
*These authors contributed equally to this work.
§Corresponding Author: Yoonhwa Jeong, Tel. 82-31-8005-3176, Fax. 82-31-8005-4054, Email. email@example.com
Sun Chul Myung, Tel. 82-2-820-1808, Fax. 82-2-6294-1406, Email. firstname.lastname@example.org
Received: September 18, 2012, Revised: October 14, 2012, Accepted: October 28, 2012
ⓒ2012 The Korean Nutrition Society and the Korean Society of Community Nutrition
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Improvement of andropause symptoms by CRS-10
sesquiterpene lactones and phenylpropanoids, which appear to
possess a broad-spectrum of anti-bacterial, anti-fungal, anti-viral,
anti-diabetic, anti-inflammatory, hepatoprotective, diuretic and
tumor apoptosis-inducing properties [13-16]. Rooibos, which is
produced in leaves of Aspalathus linearis, contains flavonol,
quercetin, flavones (orientin and soorientin), luteolin dihydro-
chalcone, aspalathin, isoquercitrin and rutin [17-19]. Rooibos has
been implicated as being beneficial in the reduction of inflam-
mation, hyperglycemia oxidative stress and liver damage [20-22].
The effects, if any, of dandelion extract and rooibos on the
reduction of andropause symptoms of aging male have not been
In this study, we assessed the prowess of extracts from
dandelion (ED) or rooibos (ER), individually and the complex
of ED and ER (CRS-10) could protect TM3 Leydig cells (TM3
cells) and increase serum testosterone in aging male rats. Also,
the clinical availability of CRS-10, which reduces andropause
symptoms, was evaluated with aging male.
Materials and Methods
Preparation of CRS-10
Dried dandelion was extracted twice with hot water and
filtered, followed by concentration and drying processes.
Fermented leaves of Rooibos (Aspalathus linearis) were extracted
twice with hot water and filtered, followed by concentration and
spray drying processes. The dandelion and rooibos extracts were
mixed in order to generate CRS-10.
TM3 cells, an immature mouse Leydig cell line (ATCC, USA),
were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/
F-12 medium (GIBCO®, Life Technologies Co., USA), supple-
mented with glutamine, antibiotics and 10% fetal bovine serum
(FBS; GIBCO®) at 37℃. Cells (75,000 cells/1.0 ml medium/well)
were seeded in 24-well cluster dishes (Corning, USA) for binding
and competition assays, unless otherwise specified.
Cell viability assay
The cells were plated on 96-well plates (Corning) in 10%
FBS/DMEM/F-12 and incubated for 24 h. To create oxidative
cellular stress, hydrogen peroxide (H2O2) was treated for two
hours. To make low serum stress, the cells were incubated in
1% FBS/DMEM/F-12 for 24 h. Subsequently, Alamar Blue
(Invitrogen, USA) was aseptically added to the cells from either
the oxidative stress condition or from the low serum. The cells
were incubated for 3 h and the absorbance of the cells was
measured at a wavelength of 570 nm using an ELISA Reader
(Molecular Devices, USA). The background absorbance was
measured at 600 nm and was then subtracted. The cell viability
was defined as [(test sample count - blank count) ÷ (untreated
control count - blank count)] × 100.
After treatment, total cellular protein was obtained by placing
cell in a lysis buffer consisting of 0.1% sodium dodecyl sulfate
(SDS) in phosphate buffered saline (PBS), followed by a brief
sonication. Protein concentration was determined by a BCA
protein assay (Pierce Chemical Co., USA). Thirty micrograms
of total cellular protein were separated by 12% SDS-polyacry-
lamide gel electrophoresis (SDS-PAGE) and then transferred to
nitrocellulose membranes. Blots were probed with an antibody
specific for the following proteins: β-actin (1:5,000 dilution;
Assay Designs, USA); phosphate-extracellular signal regulated
kinase (p-ERK) and total ERK (1:1,000 dilution; Cell Signaling
Technology, USA), and phosphate-Akt (p-Akt) and total Akt
(1:500 dilution; Santa Cruz Biotechnology, USA). Detection of
bound antibody on each blot was assessed with horseradish
peroxidase-conjugated secondary antibody visualized by enhanced
chemiluminescence (ECL; Western blot detection kit; Amersham
Pharmacia Biotech, USA).
18-week-old male Sprague-Dawley rats were purchased from
SAMTAKO (Gyeonggi, Korea). The animals were used in the
experiments after 1 week of acclimation. The rats were housed
in an air-conditioned room that was controlled for temperature
(23±3℃) and humidity (55%) with a 12 h dark/12 h light cycle
(lights on from 8:00 until 20:00), with unrestricted access to
water and food. Ten rats were orally administrated with 40 mg/kg
flour daily for 4 weeks, as the control (CTL) group. Another
10 rats were orally administrated with 40 mg/kg CRS-10 daily
for 4 weeks, as the experimental group.
Measurement of serum testosterone level
Blood samples were collected from 18-22-week-old male rats
(between 14:00 and 16:00) and then the amount of serum testos-
terone was measured by a standard coated-tube radioimmu-
noassay kit (Diagnostic Systems Laboratories, USA).
The rats were placed on a standard accelerating rotarod device
(Harvard Apparatus, USA). Following a 30-day training session
at a fixed speed, the rats were tested twice on the rotarod at
an accelerating speed of 40 rpm over a duration of 5 min or
until the animals fell off. The time on the bar and the speed
attained on the rotarod prior to the experiment were recorded
and the better score of the two trials was used for analysis.
Yoo-Hun Noh et al.
Fig. 1. Protective effect of CRS-10 against low serum-stress. The cell viability was determined biochemically, using Alamar Blue assay, at 24 h after replacement with
1% FBS-containing medium (A). The photographs represent the optical phase contrast microscopic morphology (B). The values are mean ±SE of four separate independent
experiments. The difference from the cells incubated with 10% serum was statistically significant *P < 0.05.
Swimming retention test
A swimming retention test evaluating the physical function of
the rats was performed, and the results were recorded throughout
the 4 weeks. The swimming exercise was performed in a plastic
barrel filled with water with the temperature controlled at 32-3
6℃. After 30 days, the rats were subjected to the swimming
exercise daily for 4 weeks. This exercise always took place at
noon. The swimming times was recorded weekly. Swimming
times, definedas the time from the start of swimming until the
time when the rats were exhausted and sank below the surface
of the water for 10 sec, were measured. After completing the
measurement of the swimming time, the rats were immediately
rescued from the water to avoid drowning.
Measurement of the number of sperm and its activity
Two cauda epididymis from 18-22 weeks-old male rats were
harvested at the same time each day (between 14:00 and 16:00).
The sperm-containing fluid was squeezed out of the cauda, which
was later cut into pieces. The sperm fluid and pieces of cauda
were suspended in HEPES buffer (GIBCO®). Suspensions of
spermatozoa were fixed in 10% formalin. A hemocytometer was
used to determine the number of spermatozoa. To measure the
activity of the sperm, the sperm were incubated in M199 medium
(GIBCO®) containing 0.5% BSA for 5 min. Mobilesperm were
Thirty men over 40 years of age and a matching placebo group
were recruited at Chung-Ang University Medical Center, Seoul,
Korea. Males were excluded from the study if they were
presented with any of the following conditions: metabolic
diseases, prostatic hyperplasia, development of clinical prostate
cancer, urethral stricture, neurogenic bladder, cardiovascular
events, thyroid disease during the past year or use of medications
containing sex hormones.
Demographics and other clinical dates were obtained through
the Aging Males' Symptoms (AMS) questionnaire. The study
protocol was approved by the Institutional Review Boards at the
participating hospitals (Chung-Ang University Medical Center).
Volunteers were randomly distributed into one of three groups
by a pharmacist who was not aware of any clinical information
of the subjects. Group 1 (n = 10) was the placebo group. Group
2 (n = 10) was treated with 200 mg of CRS-10 daily and group
3 (n = 10) was treated with 400 mg of CRS-10 daily. During
the study period, the investigator, rater and the study participants
were double-blinded. AMS data were compiled before the
CRS-10 regimen and after the 4-week regimen.
Student’s t-test was used in order to compare the demographic
variables between the CRS-10 and placebo groups. One-way
ANOVA was used for comparisons either between CRS-10 and
the control group or between the before and after administration
of CRS-10.All statistical analyses were performed using SPSS
11.0 (SPSS, USA) with statistical significance defined at an alpha
level < 0.05.
CRS-10 enhanced survival of TM3 cells
To evaluate the effects of ED, ER and CRS-10, TM3 cells
were incubated with either 10 or 50 µg/ml of natural extracts
in low serum (1%) medium. At the low serum medium, TM3
cells displayed a generally decreased proliferation rate and
induction of apoptotic cell death. However, treatment of CRS-10
increased the survival rate of TM3 cells (Fig. 1A). Treatment
of CRS-10 also increased cell viability of TM3 cells by upto
38%, while statistically significant increased cell viability was
Improvement of andropause symptoms by CRS-10
Fig. 2. Protective effects of CRS-10 against H2O2. TM3 cells were treated with
40 μM of H2O2 for 2 h. The cells were pretreated with either CRS-10 or dandelion
or rooibos 2 h before H2O2 treatment. The cell viability was determined biochemically
using the Alamar Blue assay. Open bar is the cell viability of non-treated TM3 cells
in 10% FBS-medium. The difference from the cells treated with H2O2 alone (black
bar) was statistically significant *P < 0.05.
Fig. 3. Enhanced activation of ERK and Akt by CRS-10 may protect TM3 cells against H2O2. After 24 h of treatment, CRS-10 treatment enhanced the activation of
ERK and Akt (A). The cells were pretreated with either 10 μM PD98059 (ERK inhibitor) or 10 μM LY294002 (Akt inhibitor) 2 h before CRS-10 and H2O2 co-treatments.
The cell viability was determined biochemically using the Alamar Blue assay. The difference from the cells treated with H2O2 alone was statistically significant *P < 0.05 (B).
ERK, Extracellular signal-regulated kinases.
not detected from the treatment of either ED or ER. Moreover,
the cells in CRS-10 treated media were healthy without apoptotic
features, whereas some control cells displayed apoptotic morpho-
logical features, such as floating and loss of cellular shape (Fig.
1B). These results were consistent with the ability of CRS-10,
which are necessary to maintain TM3 cells in a viable and healthy
state when under physiological stress.
CRS-10 protected TM3 cells against H2O2-induced cellular stress
To determine whether CRS-10 could protect TM3 cells from
H2O2-induced damage, the Alamar Blue assay was examined. As
expected, 2 h incubation with H2O2 significantly decreased cell
viabilityby 40% compared to the cultured cell without H2O2 (Fig.
2). Cell viabilities were increased to 138% and 148% by the
treatment with CRS-10 concentrations of 10 µg/ml and 50 µg/ml,
respectively, indicating a dose-dependent protection (Fig. 2).
Even though the cell viabilities were increased by 50 µg/ml ED
(approximately 120%) and by 10 and 50 µg/ml ER (both
approximately 128%), the improvement was lower compared to
CRS-10. A dose-dependent effect was not evident using ED (Fig.
2). Thus, CRS-10 was much more effective in protecting TM3
cells than were ED or ER, even at a lower concentration.
The next experiment assessed whether CRS-10 could protect
TM3 cells from H2O2-induced injury by regulating ERK and Akt
phosphorylation. Phosphorylation of both kinases is instrumental
in general cellular defense against various stresses in human cells.
As shown in the Western blot analyses, the phosphorylation of
ERK and Akt were significantly increased by 50 µg/ml of
CRS-10 (Fig. 3A). Cell viability in the CRS-10-treated group
was reduced to approximately 125% and 120%, respectively, by
treatment with the ERK inhibitor PD98059 or the Akt inhibitor
LY294002 (Fig. 3B). These results indicated that CRS-10 was
capable of effectively protecting against H2O2-induced injury
through asimultaneous activation of ERK and Akt.
CRS-10 improved serum testosterone level, capacity for locomo-
tion and spermatogenesis in aged male rats
We further studied the effect of CRS-10 on serum testosterone
level, capacity for locomotion and spermatogenesis in aging male
rats (18 weeks). Ten milligrams of CRS-10 was feed each day
for 4 weeks. CRS-10 significantly up-regulated serum testos-
terone levels from approximately 3.52 ng/ml to 5.04 ng/ml (Fig.
4A). The improved rate was about 43% compared with the
baseline (Fig. 4B). However, no significant increase was detected
in the control group (Fig. 4A).
Testosterone enhances physical locomotion capacity . To
evaluate the effect of CRS-10 on the adaptation process of rat
skeletal muscles to long-term physical strength, Rotarod and
Yoo-Hun Noh et al.
Fig. 4. Increased serum testosterone level by CRS-10. Testosterone level in the serum represents the mean ±SE. The serum testosterone levels before and after
administration of CRS-10 were compared using one-way ANOVA (A). The solid bar shows the percentage of serum testosterone level of CRS-10 and placebo groups compared
with the baseline (B). Significant differences are denoted by * vs. before, P < 0.05 and †vs. control, P < 0.05.
Fig. 5. Positive role of CRS-10 in the enhancement of physical activity. To evaluate the enhancement of physical function by CRS-10, Rotarod and swimming retention
test were performed. The rats were trained with rotarod and swimming exercise 30 min a day for 30 days. Running time on the wheel is presented as the mean±SE
(A). Retention time, swimming time without stop for 10 sec, was determined weekly (B). Significant differences are denoted by * vs. before, P < 0.05 and †vs. control, P
Testis wt2) (mg)
309.52 ± 14.7 312.42 ± 21.7 318.75± 11.54 311.00 ± 8.93
1.55 ± 0.541.59 ± 0.63 1.58 ± 0.311.64 ± 0.51
1)Mean±SD (n = 10)
Table 1. Demographic characteristics of older male rats between study groups
swimming retention tests were performed. Control and CRS-
10-treated rats were trained three times a day for 4 weeks. The
latency time on the rotarod was increased from about 200 sec
to 250 sec by treatment of CRS-10, representing a 25% improve-
ment. The controls showed no increased time (Fig. 5A). Further,
the CRS-10 treated rats showed a significantly increased swimming
time with practice, while the control rats did not (Fig. 5B).
Testosterone has an important role during spermatogenesis in
the number and activity of the sperm. To evaluate the effects
of CRS-10 on spermatogenesis, the number and activity of the
sperm was determined in control and CRS-10-treated rats. The
sperm number per gram of epididymis was significantly increased
from approximately 51.6 × 106 to approximately 60.8 × 106 by
CRS-10 treatment (Fig. 6A), representing a 20% improvement
rate (Fig. 6C). However, no significant change in the sperm
number was detected in the control rats (Fig. 6A).
The activity of sperms was represented as a portion of active
sperms 4 h after sperm acquisition. The portion of active sperm
was markedly increased from 50% to 71% by the treatment of
CRS-10 (Fig. 6B), representing over a 40% improvement (Fig.
6C). However, no significant variation was detected in the control
(Fig. 6B). The control group displayed no differences in both
the number and activity of sperms. No significant changes of
body and testis were detected in the CRS-10 and control groups
Intake of 400 mg CRS-10 improved QoL in aging men
The dose-dependent effects of CRS-10 on the QoL of aging
male were assessed using the AMS clinical questionnaire. Ten
normal men were treated with 400 mg of CRS-10/day and 10
other men were treated with 200 mg/day for 4 weeks. Also, 10
normal men, as the placebo group, were treated with 400 mg
of dextran for 4 weeks. All groups then completed the AMS
Improvement of andropause symptoms by CRS-10
Fig. 6. Enhanced spermatogenesis and sperm activity by CRS-10. Number of sperm before and after administration of CRS-10 was compared using one-way ANOVA
(A). Activated sperms by CRS-10, which can maintain movement after 5 min of medium incubation, were counted (B). These improvements by CRS-10 were presented
as the percentage compared with the baseline (C). Significant differences are denoted by * P < 0.05.
Fig. 7. Improved AMS score by 400 mg/day CRS-10. AMS questionnaire was completed at 0 and 4 weeks. Scores of the AMS answersare presented as mean±SE
(A). The scores before and after administration of CRS-10 were compared using one-way ANOVA. The solid bar shows the percentage of the AMS score of CRS-10 and
placebo groups compared with the baseline (B). Significant differences are denoted by * (P < 0.05). AMS, Aging Males' Symptoms.
questionnaire in a double-blind manner. The men treated with
400 mg of CRS-10 reported significantly fewer negative answers
to QoL-related questions. A negative answer score of about 39
prior to CRS-10 treatment was reduced to about 30 following
the treatment regimen (Fig. 7A), representing a 20% improvement
rate (Fig. 7B). The group receiving 200 mg of CRS-10 and the
placebo group did not display significant differences before and
after treatment, although the number of negative answers was
slightly reduced in the 200 mg CRS-10 treated group (Fig. 7A).
Safe natural substances capable of reducing andropause
symptoms of men are essential, due to the fact that an increasing
proportion of men will become affected as the general life span
is prolonged. The present study screened for the efficacy and
safety of the combined extract of dandelions and rooibos
Leydig cells of males are continuously exposed to various
Yoo-Hun Noh et al.
harmful stimulations, particularly from mid-age onward. These
cells sustain gradual and progressive damage, along with
age-related loss of physiological functions. As a result, their
capacity to produce testosterone declines. Andropause syndrome
in aging male is mainly due to testosterone reduction. Conse-
quences include loss of skeletal muscle, decrease of locomotive
capacity, depression, oligozoospermia and inactivation of sperm
[6,23-26]. The present results indicate that CRS-10 may play a
significant positive role in the health of TM3 cells, and thus
result in the sustained production of testosterone.
Aged men suffer from accumulated physiological stresses.
Through the results of this study, it has been revealed that
CRS-10 increased TM3 cells viability in stressed conditions (e.g.,
low serum medium) and effectively protected TM3 cells against
physiological oxidative stress. It has been known that testosterone
exerts various positive roles including protection of the body
from the stress and reduction of andropause symptoms in aging
males. The testosterone is produced in Leydig cells, similar to
TM3 cells in the testis. Hence, the protective effect of CRS-10
on the reproductive cells and organ might be crucial for the health
and anti-aging of men. CRS-10 treatment could maintain the
health of the cells by the activation of the ERK and Akt pathways.
The present results are consistent with previous reports,
suggesting the involvement of the pathways in the protection of
cells from various insults, which is critical for maintaining a
functional population of adult Leydig cells as well as for fertility
The collective data supports the suggestion that the increase
of testosterone in the serum of CRS-10 treated rats might be
engendered by the protective and activating roles of CRS-10 on
Leydig cells, since the cells produce testosterone in the testis
Interestingly, the testosterone level of CRS-10 treated rats was
increased, together with asignificant enhancement of physical
locomotion activities. Several previous studies showed that
testosterone improves skeletal muscle and strength in a dose-
dependent manner, and thus improves physical performance
[7,9,32]. Increased testosterone production in the presence of
CRS-10 implicated in the improved physical capacity of the
treated rats in the present study.
Also, CRS-10 simultaneously increased spermatogenesis and
sperm activity. The serum testosterone level is linked with the
production of active sperm. Testosterone improves the efficacy
of germ cell differentiation and active maturation [33,34].
Recently, testosterone has been used in the clinical treatment of
andropause patients with oligozoospermia and maturation arrest
of sperms, despite its various side effects [10,11,35,36]. Hence,
consistent with previous studies, it can be suggested that the
enhanced activity and health of Leydig cells together with the
enhanced serum testosterone levels by CRS-10 were the bases
of improved spermatogenesis and activation of sperm. Further-
more, CRS-10 was markedly effective in the clinical study with
aging male, as judged using the AMS questionnaire, which is
an established means of diagnosing andropause symptoms of men
[37-39]. Dose dependent effects of CRS-10 were evident, which
related to the specific positive effects on andropause symptoms.
The effective clinical dose was 400 mg/day.
In conclusion, CRS-10 effectively improved andropause synd-
romes clinically and also enhanced physical performance as well
as activation of spermatogenesis. These positive effects may have
occurred through protection and activation of Leydig cells,
together with induced generation of endogenous testosterone.
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