Attenuation of Long-Term Rhodiola rosea Supplementation on Exhaustive Swimming-Evoked Oxidative Stress in the Rat

Abstract

Rhodiola rosea improves exercise endurance and fatigue. We hypothesized that ingredients in Rhodiola rosea may increase antioxidant capability against swimming induced oxidative stress. In this study, we have identified the Rhodiola rosea ingredients, p-tyrosol, salidroside, rosin, rosavin and rosarin by high performance liquid chromatography-mass spectrometer and evaluated their O2(-)*, H2O2, and HOCl scavenging activities by a chemiluminescence analyzer. We next explored the effect and mechanism of Rhodiola rosea on 90-min swimming-induced oxidative stress in male Wistar rats fed with three doses of Rhodiola rosea extracts in drinking water (5, 25, 125 mg/day/rat) for 4 weeks. Our results showed that the 4 major ingredients (salidroside, rosin, rosavin and rosarin) from Rhodiola rosea extracts scavenged O2(-)*, H2O2, and HOCl activity in a dose-dependent manner. The ninety-min swimming exercise increased the O2(-)* production in the order: liver > skeletal muscle > blood, indicating that liver is the most sensitive target organ. The level of plasma malonedialdehyde, a lipid peroxidation product, was also increased after exercise. Treatment of 4 weeks of Rhodiola rosea extracts significantly inhibited swimming exercise-enhanced O2(-)* production in the blood, liver and skeletal muscle and plasma malonedialdehyde concentration. The expression in Mn-superoxide dismutase Cu/Zn-superoxide dismutase, and catalase in livers were all enhanced after 4 weeks of Rhodiola rosea supplementation especially at the dose of 125 mg/day/rat. Treatment of Rhodiola rosea extracts for 4 weeks significantly increased swimming performance. In conclusion, treatment of Rhodiola rosea extracts for 4 weeks could reduce swimming-enhanced oxidative stress possibly via the reactive oxygen species scavenging capability and the enhancement of the antioxidant defense mechanisms.

Full text

Corresponding author: Dr. Chiang-Ting Chien, No. 7 Chung-Sun South Road, Department of Medical Research, National Taiwan University
Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC. Tel: +886-2-23123456 ext. 5720, Fax: +886-2-
23947927, E-mail: ctchien@ntuh.gov.tw
Received: April 28, 2008; Revised: February 11, 2009; Accepted: February 13, 2009.
2009 by The Chinese Physiological Society. ISSN : 0304-4920. http://www.cps.org.tw
Chinese Journal of Physiology 52(5): 316-324, 2009
DOI: 10.4077/CJP.2009.AMH029
Attenuation of Long-Term Rhodiola rosea
Supplementation on Exhaustive
Swimming-Evoked Oxidative Stress in the Rat
Shih-Chung Huang1, Fang-Tsai Lee2, Tz-Yin Kuo3, 4, Joan-Hwa Yang3,
and Chiang-Ting Chien4
1Departments of Cardiology and
2Orthopaedic Surgery, Kuang-Tien General Hospital, Taichung
3Department of Food Science, Nutrition, and Nutraceutical Biotechnology,
Shih-Chien University College of Human Ecology, Taipei
and
4Department of Medical Research, National Taiwan University Hospital and National Taiwan
University College of Medicine, Taipei, Taiwan, Republic of China
Abstract
Rhodiola rosea improves exercise endurance and fatigue. We hypothesized that ingredients in
Rhodiola rosea may increase antioxidant capability against swimming induced oxidative stress. In this
study, we have identified the Rhodiola rosea ingredients, p-tyrosol, salidroside, rosin, rosavin and
rosarin by high performance liquid chromatography-mass spectrometer and evaluated their O2–., H2O2,
and HOCl scavenging activities by a chemiluminescence analyzer. We next explored the effect and
mechanism of Rhodiola rosea on 90-min swimming-induced oxidative stress in male Wistar rats fed with
three doses of Rhodiola rosea extracts in drinking water (5, 25, 125 mg/day/rat) for 4 weeks. Our results
showed that the 4 major ingredients (salidroside, rosin, rosavin and rosarin) from Rhodiola rosea
extracts scavenged O2–., H2O2, and HOCl activity in a dose-dependent manner. The ninety-min
swimming exercise increased the O2–. production in the order: liver > skeletal muscle > blood, indicating
that liver is the most sensitive target organ. The level of plasma malonedialdehyde, a lipid peroxidation
product, was also increased after exercise. Treatment of 4 weeks of Rhodiola rosea extracts significantly
inhibited swimming exercise-enhanced O2–. production in the blood, liver and skeletal muscle and
plasma malonedialdehyde concentration. The expression in Mn-superoxide dismutase Cu/Zn-superoxide
dismutase, and catalase in livers were all enhanced after 4 weeks of Rhodiola rosea supplementation
especially at the dose of 125 mg/day/rat. Treatment of Rhodiola rosea extracts for 4 weeks significantly
increased swimming performance. In conclusion, treatment of Rhodiola rosea extracts for 4 weeks could
reduce swimming-enhanced oxidative stress possibly via the reactive oxygen species scavenging capability
and the enhancement of the antioxidant defense mechanisms.
Key Words: Rhodiola rosea, exercise, oxidative stress, reactive oxygen species, rat
Introduction
Rhodiola rosea (Golden Root, Roseroot) is a
plant in the Crassulaceae family growing in the
mountainous and arctic regions of North America,
Europe, and Asia. Rhodiola rosea can combat fatigue
316

Rhodiola rosea Supplementation Attenuates Oxidative Stress 317
by its several unique ingredients (5, 10). Among
these, p-tyrosol, salidroside, rosin, rosavin and rosarin
are the major active components of Rhodiola rosea
with adaptogenic characteristics (5) for improvement
of cognitive function (35) and endurance performance
(1, 12, 35), reduction of mental fatigue (10, 36), anti-
diabetic effect (22) and reactive oxygen species (ROS)
production (2, 13, 20, 21).
Physical exercise is characterized by an increase
in O2 uptake and consumption and induced stressors
such as elevations of body temperature, the formation
of reactive oxygen species (ROS), and a decrease in
glycogen (3, 25, 26). In extreme conditions such as
ischemia/reperfusion or exhaustive exercise, the
increased ROS can oxidize macromolecules con-
tributing to abnormal signal transduction or cellular
dysfunction, impairment of both enzymic and non-
enzymic antioxidant defense systems of target tissues
and trigger erythrocyte hemolysis (4) and the cascade
of apoptosis, autophagy and necrosis (7, 8, 26, 30-
33). Exhaustive exercise enhances xanthine oxidase
activities of plasma and skeletal muscle, muscular
myeloperoxidase activity and malondialdehyde
concentrations of plasma and tissues (25). Exhaustive
exercise leads to oxidative damage in the liver in-
cluding rough endoplasmic reticulum fragmentation
and dilatation, glycogen depletion, and mitochondrial
enlargement (34, 37). Therefore, exhaustive exercise-
enhanced oxidative stress may impair liver, kidney,
skeletal muscle and other tissues by different degrees
of ROS production. It has been speculated that in-
creased antioxidant/oxidative damage-repairing
enzyme activities, increased resistance to oxidative
stress and lower levels of oxidative damage may
protect oxidative stress-related cardiovascular, kidney,
liver and neuronal damages (8, 33, 41). The long-
term effects of Rhodiola rosea supplementation on
exhaustive exercise-induced oxidative stress have not
clearly been demonstrated. The purpose of the current
study was to identify the active components in the
Rhodiola rosea extract and to examine the long-term
effect and mechanism of Rhodiola rosea supplemen-
tation on ROS production and oxidized biomarkers in
the liver, skeletal muscle and blood after exhaustive
exercise.
Materials and Methods
Rhodiola rosea and High Performance Liquid
Chromatography-Mass Spectrometry (HPLC-MS)
Dry powders from water extraction of roots of
Rhodiola rosea L. was purchased from Numen Biotech
(Taipei, Taiwan, ROC). In brief, fresh original habitats
of Rhodiola rosea rhizomes from Siberia were thorough-
ly washed with water, shade-dried for 4 weeks, and
powdered using a mixer grinder. A known quantity of
the dried powdered material was soaked in distilled
water for 24 h at 35-42°C and macerated thoroughly
with the help of a mortar and pestle. The mixture
was filtered through Whatman filter paper No. 1, con-
densed using a rotary evaporator and lyophilized.
A model Agilent 1,100 with vacuum degasser,
binary pump, autosampler and thermostatic column
holder was used. The LC separation was performed
using a ZORBAX 300SB-C18 3.5 µm, 1.0 × 150 mm.
The temperature of the column oven was 35°C and the
injection volume was 1 µl. The eluent flow-rate was
0.08 ml/min. The gradient conditions were initially
90% A (aqueous phase with distilled water produced
by Mill-Q, 18.2 MΩ)-5% B (acetonitrile)-5% C
(methanol), changed linearly to 76% A-12% B-12%
C in 16 min. After gradient elution, the column was
washed for 1 min with acetonitrile and equilibrated
for 5 min under the initial conditions leading to a total
time of 25 min for one analysis. All HPLC-MS ex-
periments were performed using a mass spectrometer
(Esquire 3000+, Bruker Daltonik GmbH, Bremen,
Germany) equipped with a positive spray ionization
source in the full-scan mode over the m/z range 100-
600. Nitrogen was used as the drying gas at a flow-
rate of 8 l/min. The dry air temperature was 310°C at
20 psi pressure. For calibration of the HPLC-MS
method with ESI and the eluent system, eight
concentration levels of standard were prepared for
obtaining the calibration curves. We used salidroside,
rosin, rosarin and rosavin for standards (Sigma, St.
Louis, MO, USA). Standard solutions were prepared
in 6% methanol containing 400 ng/ml internal
standard. For all standards, the concentration levels
prepared were 0.5, 2, 5, 20, 50, 200, 500 and 2000
ng/ml. Calibration curves were constructed by plotting
responses of the standard compounds relative to
responses of the internal standard (measured in
triplicate for each concentration) against the con-
centration of standard compounds. Ten mg of the
Rhodiola rosea extracts was dissolved in methanol
and stirred after supersonic vibration for 15 min. The
supernatant was filtered after 0.45 µm and was
analyzed by HPLC-MS. The data were presented in
the Fig. 1. Rhodiola rosea extract contains three
cinnamyl alcohol-vicianosides, rosavin, rosin, and
rosarin, that are specific to this species (14, 15).
Animals and Swimming Model
Male Wistar rats (200-250 g) were housed at the
Experimental Animal Center, National Taiwan
University, at a constant temperature and with a
consistent light cycle (light from 07:00 to 18:00
o’clock). Food and water were provided ad libitum.
All surgical and experimental procedures were

318 Huang, Lee, Kuo, Yang and Chien
approved by the National Taiwan University College
of Medicine and College of Public Health Institu-
tional Animal Care and Use Committee in accordance
with the guidelines of the National Science Council
of Republic of China (NSC 1997).
All the animals were divided into four groups
for oral administration of 0, 5, 25 and 125 mg/day
Rhodiola rosea extracts for 4 weeks. Before the com-
mencement of an experiment, all animals were
familiarized to swimming for 10 min/day for 3 days.
At the indicated time, rats fasted overnight for 8 h
(from 12:00 PM to 8:00 AM) in a 90-min swimming
exercise test were measured (6). The swimming
exercise in free style was carried out in a circular
plastic barrel (diameter, 20 cm; depth, 30 cm) filled
with water maintained at a temperature of 24 ± 1°C.
The rats swam in the circular plastic barrel for 90 min.
After 90 min of swimming challenge, the rats were
killed with an overdose of sodium pentobarbital
intraperitoneally (90 mg/kg body weight) and the
liver, skeletal muscle and blood were removed.
Changes of Lucigenin- and Luminol-Enhanced
Chemiluminescence Counts (CL)
The antioxidant activities of 5 major ingredients
(tyrosol, salidroside, rosin, rosarin and rosavin) and
Rhodiola rosea extracts on xanthine (0.75 mg kg-1,
dissolved in 0.01 N NaOH) and xanthine oxidase (24.
8 mU kg-1) enhanced O2–., 0.03% H2O2 induced H2O2
Fig. 1. A. Structures of four compounds. B. Standards and Rhodiola rosea extract extracted chromatograms from HPLC-MS experi-
ment (full-scan mode) with positive ESI and eluent system.
HO
A
B
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
OO
OO
OO
OO
O
OH
OH
OH
OH OH
OH
OH
OH
Rosin
Rosavin
Rosarin
Salidroside
Standard
Salidroside
Sample (0.1 mg/ml)
Salidroside
(0.4 µg/ml) Rosin
Rosarin
Rosarin
Rosin
Rosavin
Rosavin
EIC 379 +Al
EIC 323 +Al
EIC 451 +Al
EIC 451 +Al
EIC 319 +Al
EIC 323 +Al
Time [min]
18161412108642
0
2
4
0
0.0
0.5
1.0
2
4
0
2
4
0
0
1
2
4
k10
4
k10
4
k10
4
k10
4
k10
4
k10
4
Irdene

Rhodiola rosea Supplementation Attenuates Oxidative Stress 319
activity, and % HOCl induced HOCl activity were
evaluated.
ROS levels were measured using a CL analyzing
system (CLD-110, Tohoku Electronic Industrial,
Sendai, Japan) as previously described (7). The
system contained a photon detector (Model CLD-
110), a CL counter (Model CLC-10), a water circulator
(Model CH-200) and a 32-bit IBM personal computer
system. A cooler circulator was connected to the
model CLD-110 photon detector to keep the tem-
perature at 5°C. Under these conditions, radiant
energy as low as 10-15 W could be detected.
CL was measured in an completely dark chamber
of the CL analyzing system. We demonstrated that
using the CL-emitting substance lucigenin (N,N’-
dimethyldiacridinium, Sigma, St. Louis, MO, USA)
for O2–. or luminol (5-amino-2,3-dihydro-1,4-
phthalazinedione, Sigma, St. Louis, MO, USA) for
H2O2 or HOCl to enhance the CL counts provided
similar data to those reported in our previous in vivo
study (7). The lucigenin-enhanced CL method
provides a reliable assay for superoxide. After 100 s,
1.0 ml of 0.1 mM lucigenin in PBS (pH = 7.4) was
mixed with the tested sample. CL in the tested sample
was measured continuously for a total of 600 s. The
assay was performed in duplicate for each sample,
and the results are expressed as CL counts (10 s)-1.
The total amount of CL in 600 s was calculated by
integrating the area under the curve. The means ±
S.E.M. CL level for each sample was calculated.
We used 1 ml blood samples and 0.2 g homoge-
nized liver and leg skeletal muscle to measure
ROS levels. The analysis of lipid peroxidation,
malondialdehyde (MDA), concentrations of plasma
samples was assessed colorimetrically at 586 nm
using a commercial kit (Calbiochem 437634;
Calbiochem-Novabiochem, La Jolla, CA, USA) as
previously described (40). Concentration was ex-
pressed in µM in plasma and in µmol/mg protein in
tissue samples.
Effect of Rhodiola rosea Extract on MnSOD, Cu/Zn
SOD, Catalase Protein Expression
Protein concentration was determined by a
BioRad Protein Assay (BioRad Laboratories,
Hercules, CA, USA). Ten g of protein was electro-
phoresed as described below. The expression of
MnSOD, Cu/Zn SOD and catalase in liver tissues was
evaluated by western immunoblotting and densito-
metry as described (41). Briefly, total proteins were
homogenized with a prechilled mortar and pestle
in an extraction buffer of 10 mM Tris-HCl (pH 7.6),
140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride,
1% Nonidet P-40, 0.5% deoxycholate, 2% β-
mercaptoethanol, 10 µg/ml pepstatin A and 10 µg/ml
aprotinin. The mixtures were homogenized com-
pletely by vortexing and kept at 4°C for 30 min. The
homogenate was centrifuged at 12,000 g for 12 min at
4°C, the supernatant was collected, and protein
concentrations were determined by the BioRad Pro-
tein Assay (BioRad Laboratoriess Hercules, CA,
USA).
The polyclonal anti-MnSOD (Stressgen
Bioreagents Limited, Victoria, Canada) rabbit anti-
human Cu/Zn SOD (Stress Marq Biosciences Inc.,
Victoria, Canada) and catalase (Chemicon Interna-
tional Inc., Temecular, CA, USA) antibodies, and the
monoclonal mouse anti-mouse β-actin (Sigma, Saint
Louis, MI, USA) were used at 1:1000 dilutions. All
of these antibodies cross-reacted with the respective
rat antigens (29).
Statistical Analysis
All values were expressed as means ± standard
error mean (SEM). Differences within groups were
evaluated by paired t-test. One-way analysis of
variance was used for establishing differences among
groups. Intergroup comparisons were made by
Duncan’s multiple-range test. A chi-square test was
performed in the hepatic antioxidant expression.
Differences were regarded as significant if P < 0.05
was attained.
Results
Ingredient Analysis of Rhodiola rosea Extract
In the present study, the structures of four
standard components including salidroside, rosin,
rosarin and rosavin are demonstrated in Fig. 1A. The
original diagram obtained from the four standard
components (upper panel) and the samples of Rhodiola
rosea extract (lower panel) was analyzed by HPLC-
MS (Fig. 1B). Rhodiola rosea ingredient was analyzed
by HPLC-MS as shown in Table 1. By the use of our
techniques, Rhodiola rosea extract was shown to
contain four major components including salidroside,
rosin, rosarin and rosavin. Among these four com-
ponents, the salidroside content was the highest (13128
ppm). Our data verified that the Rhodiola rosea
extract in this study contains approximately 1.3%
salidroside, 0.4% rosin, 0.4% rosarin and 1% rosavin,
but did not contain p-tyrosol in our HPLC-MS analysis.
Rhodiola rosea extract supplementation was provided
to the rat in drinking water in a dose-dependent
manner. Table 1 shows the level of the four com-
ponents in the three dosages of Rhodiola rosea extract
supplementation. The salidroside, rosin, rosarin and
rosavin contents were dose-dependently increased
with the Rhodiola rosea extract dose.

320 Huang, Lee, Kuo, Yang and Chien
Antioxidant Activity of the Rhodiola rosea Extract
We first compared the antioxidant activities of
O2–., H2O2, HOCl of five ingredients, p-tyrosol,
salidroside, rosin, rosavin and rosarin, of the Rhodiola
rosea extract in 2, 20 and 200 µg/ml. As shown in Fig.
2, p-tyrosol, salidroside, rosin, rosavin and rosarin
significantly (P < 0.05) inhibited xanthine- and
xanthine oxidase-induced O2–. levels at 2, 20 and
200 µg/ml. The inhibited O2–. ability was most
prominent in p-tyrosol and rosavin at 200 µg/ml.
Also, p-tyrosol, salidroside, rosin, rosavin and rosarin
significantly (P < 0.05) inhibited H2O2 and HOCl
activities (Fig. 2) at 2, 20 and 200 µg/ml. The in-
hibited H2O2 ability was most prominent in p-tyrosol
and rosin at 200 µg/ml. The inhibited HOCl ability
was most prominent in p-tyrosol at 200 µg/ml.
The second part of study was to explore the
antioxidant activity in different dosages of Rhodiola
rosea extract with serial dilutions at 0, 0.2, 1, 5, 25
and 125 mg/mL. Rhodiola rosea extract significantly
reduced xanthine- and xanthine oxidase-induced O2–.,
H2O2, and HOCl activities in a dose-dependent manner
(Fig. 3).
To explore the effect of 90-min exhaustive swim-
ming exercise on blood and tissue ROS production,
we investigated lucigenin-dependent O2–. chemilu-
minescence counts in the blood and in homogenized
liver and skeletal muscle. As shown in Fig. 4, after
90-min swimming, the level of lucigenin-dependent
O2–. chemiluminescence counts was significantly (P <
0.05) increased in blood, liver and skeletal muscle.
The enhancement in O2–. production was in an order of
liver > skeletal muscle > blood. The plasma level of
malonedialdehyde, a lipid peroxidation product, was
also significantly (P < 0.05) increased.
Long-term Rhodiola rosea extract supplemen-
tation at different dosages for 4 weeks significantly
decreased the swimming exercise-enhanced O2–.
chemiluminescence counts in blood, liver and skeletal
Table 1. Aqueous concentrations of Rhodiola rosea extracts as determined by HPLC-MS
Salidroside (µg) Rosin (µg) Rosarin (µg) Rosavin (µg)
R0 (0 mg) 0 ±00±00±00±0
R5 (5 mg) 71 ±520±221±352±6
R25 (25 mg) 334 ±39 87 ±10 85 ±9 247 ±32
R125 (125 mg) 1,645 ±169 416 ±61 382 ±42 1,187 ±155
Fig. 2. Different dosages of five standards displaying O2–., H2O2
and HOCl scavenging activities in a dose-dependent
manner. The sample concentration was 2-200 µg/ml.
Each data point was tested for four times. *P < 0.05
when compared to the control value.
% of O
2
-. inhibition
0
20
40
60
80
100
120
140 2 µg
20 µg
200 µg
Control salidroside tyrosol rosarin rosin rosavin
*
**
*
*
*
*
**
*
*
*
*
% of H
2
O
2
inhibition
0
20
40
60
80
100
Control salidroside tyrosol rosarin rosin rosavin
*
*
*
**
*
*
*
*
*
*
*
*
*
*
% of HOCl inhibition
0
20
40
60
80
100
*
*
*
*
*
*
*
*
*
*
**
*
**
Control salidroside tyrosol rosarin rosin rosavin
Fig. 3. The antioxidant activity against O2–., H2O2 and HOCl
in different dosages of Rhodiola rosea extract with
serial dilutions at 0 (R0), 0.2 (R0.2), 1 (R1), 5 (R5), 25
(R25) and 125 (R125) mg/ml. *P < 0.05 when com-
pared to the control value.
% of ROS inhibition
0
2
4
6
8
60
70
80
90
100 O
2
-.
H
2
O
2
HOCl
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Control R0.2 R1 R5 R25 R125
(mg/mL)

Rhodiola rosea Supplementation Attenuates Oxidative Stress 321
muscle. The increased plasma malonedialdehyde
level induced by swimming was also depressed by
long-term Rhodiola rosea extract supplementation
(Fig. 4).
Chronic Rhodiola rosea Treatment Enhanced Hepatic
Antioxidant Enzymes Expression
We explored whether 4 weeks of Rhodiola rosea
extract supplementation affected expression of
antioxidant enzymes in the rat liver. As shown in Fig.
5, Mn SOD, Cu/Zn SOD and catalase were all ex-
pressed in the liver of R0 group. After 90 min of
swimming exercise, the expression of Cu/Zn SOD,
but not Mn SOD and catalase, was significantly
decreased in the R0 group. Four weeks of administra-
tion of the Rhodiola rosea extract at 25 and 125 mg
enhanced hepatic Mn SOD and Cu/Zn SOD expression
before the exhaustive swimming exercise. Catalase
expression was mildly enhanced at the dosage of
125 mg of Rhodiola rosea extract for four weeks.
Four weeks of Rhodiola rosea extract supplemen-
tation attenuated the depression of Cu/Zn SOD after
exhaustive swimming.
Rhodiola rosea Extract Supplementation Increased
Exercise Performance
After 4 weeks of Rhodiola rosea extract supple-
mentation at different dosages, 5% weight-loaded
swimming was used to evaluate the exercise perfor-
mance by the swimming time to fatigue. Treatment
of four weeks of the Rhodiola rosea extract at 5, 25
and 125 mg significantly (P < 0.05) increased the
swimming performance by 18.8%, 46.8% and 59.3%
(n = 5 each).
Discussion
In the present study, we described a HPLC-MS
technique to identify and quantify 4 major components,
salidroside, rosin, rosarin and rosavin, in the Rhodiola
rosea extract. The application of HPLC for deter-
mining hydrophilic extracts from Rhodiola rosea and
Rodiola quadrifida has led to the identification of
cinnamic alcohol, chlorogenic acid, rhodiooctanoside,
rosiridin, rosavin and the phenolic compounds
salidroside, rhodiolin and viridoside (20, 24, 38).
Fig. 4. Antioxidant effects by different dosages of Rhodiola
rosea extract against the O2–. level in blood, liver and
muscle, and plasma MDA in the rats subjected to 90-min
swimming exercise that significantly increased the
oxidative stress. The animals were fed Rhodiola rosea
extract at 0 (R0), 5 (R5), 25 (R25) and 125 (R125) mg/ml
in the drinking water for 4 weeks. *P < 0.05 when
compared to the control value.
Fig. 5. Effects of different dosages of Rhodiola rosea extract
supplement and swimming test on MnSOD, Cu/Zn SOD
and catalase expression in the rat livers. R0, no Rhodiola
rosea extract supplement; R5, 5 mg/ml; R25, 25 mg/ml;
R125, 125 mg/ml. *P < 0.05 when compared to the
control value of R0 group.
Plasma MDA
(nmol/mL)
0.0
0.2
0.4
0.6
0.8
*
*
Blood O
2
-.
(counts/10 s)
0
100
200
300
400
500
Control n = 8 each
Swimming n = 8 each
*
*
Liver O
2
-.
(counts/10 s)
0
500
1000
1500
2000
2500
3000
*
*
Muscle O
2
-.
(counts/10 s)
0
200
400
600
800
1000
1200
*
*
R0 R5 R25 R125
**
*
*
*
*
Mn SOD
Cu/Zn SOD
Catalase
β-actin
R5R0 R125R25
Control Swim Control Swim Control Swim Control Swim
1.4
1.2
1.0
0.8
0.6
1.8
1.5
1.2
0.9
0.6
1.6
1.4
1.2
1.0
0.8
MnSOD/β-actin
ratio
CuZnSOD/β-actin
ratio
Catalase/β-actin
ratio

322 Huang, Lee, Kuo, Yang and Chien
Our technique did not detect p-tyrosol in our Rhodiola
rosea extract.
It is frequently stated, but poorly demonstrated,
that exercise could have adverse effects related to
inflammatory response, ROS production and accu-
mulation of oxidative damage in several organs (3,
11, 16, 19, 33). Unlike the skeletal muscle, liver con-
tains high levels of xanthine dehydrogenase; during
exercise, xanthine dehydrogenase is converted to
xanthine oxidase generating ROS and oxidative
damage (31). In the present study, we found that 90-
min exhaustive swimming exercise increased O2–.
production in an order liver > skeletal muscle > blood
indicating that liver is the most sensitive target organ.
This result is agreement with a previous report with
nuclear 8-hydroxydeoxyguanosine as the oxidative
stress; the report showed that the nuclear 8-hydrox-
ydeoxyguanosine content increased in liver, not in
skeletal muscle and brain (33). The increased blood
ROS after exhaustive exercise may contribute to the
enhanced level of plasma malonedialdehyde, a lipid
peroxidation product, found in our analysis. Increased
blood ROS including O2–., H2O2 or HOCl may induce
oxidation of phospholipid bilayers in the erythrocytes,
increase phosphotidylcholine hydroperoxide and
malonedialdehyde accumulation in the erythrocyte
membrane and consequently contributes to hemoly-
sis (13, 17, 18).
Therefore, an increased activity in the antioxi-
dant defense mechanism or a decrease in oxidative
stress may protect organs against oxidative damages.
Exhaustive exercise on the treadmill resulted in
significant increases in lipid peroxidation of skeletal
muscle, liver and kidney inrats, and this was prevented
by superoxide dismutase derivatives (30, 31). Previous
studies have indicated that Rhodiola rosea extracts
containing specific ingredients may have beneficial
effects in enhancing exercise performance (1, 12, 23,
35) and in reducing ROS levels (2, 13, 21), but the
major active component has not clearly been demon-
strated. It has been reported that some Rhodiola
species did not have antioxidant effects on hypoxemia
and oxidative stress (39). The chemical composition
and physiological properties of Rhodiola species are
to a degree species-dependent although overlaps in
constituents and physiological properties do exist in
many Rhodiola species (20, 27, 28). Rhodiola rosea
contains a range of biologically active substances
including organic acids, flavonoids, tannins and phe-
nolic glycosides. The stimulating and adaptogenic
properties of Rhodiola rosea were originally attributed
to two compounds isolated from its roots identified as
p-tyrosol and the phenolic glycoside salidroside found
in all studied species of Rhodiola (9). However, other
active glycosides, including rosavin, rosin and rosarin,
have not been found in the Rhodiola species examined
(20). Because of this variation within the Rhodiola
genus, verification of Rhodiola rosea by HPLC is
dependent on the content of the rosavin, rosin and
rosarin rather than salidroside and p-tyrosol (9, 14,
15, 20). Based on a comparative analysis, the most
uniquely active chemical constituents are rosavin
(the most active), rosin, rosarin, salidroside and its
aglycon, p-tyrosol. In our data and in a previous
study, p-tyrosol, salidroside, rosavin, rosin, and rosarin
are all antioxidant substances (13, 20, 21, 42, 43),
especially in p-tyrosol. In the present study, we have
clearly identified that Rhodiola rosea extracts used
in our study contained rosavin, rosin and rosarin, but
did not contain p-tyrosol as analyzed by our HPLC-
MS technique. A previous study has indicated that
Rhodiola rosea roots contain 1.3 to 11.1 mg/g salidro-
side and 0.3 to 2.2 mg/g p-tyrosol (20). Our HPLC-
MS data show that 13 mg/g salidroside, 3.3 mg/g
rosin, 3.1 mg/g rosarin and 9.5 mg/g rosavin are
found in our Rhodiola rosea extracts. We therefore
suggest that high contents of rosin, rosarin and ro-
savin may exert a more efficient potential than p-
tyrosol (not detected or too low in our Rhodiola
rosea extract) in the reduction of swimming-induced
oxidative stress.
Our results also showed that four weeks of
Rhodiola rosea extract supplementation can up-
regulate Mn SOD and Cu/Zn SOD protein expression
in the rat liver. Although we did not know the detailed
mechanisms involving Rhodiola rosea-enhanced
antioxidant protein expression, direct scavenging ROS
activity and enhancement of several antioxidant
proteins of Rhodiola rosea extract may have provided
hepatic protection against exhaustive exercise- induced
oxidative stress in the liver. In the present study, we
have clearly indicated that p-tyrosol, salidroside, rosin,
rosarin and rosavin or the Rhodiola rosea extract
can significantly and dose-dependently decreased
O2–., H2O2 and HOCl activity in vitro. In addition,
chronic Rhodiola rosea extract supplement signifi-
cantly and dose-dependently reduced swimming
exercise-enhanced plasma malonedialdehyde con-
centrations and O2–. levels in the liver, skeletal muscle
and blood. In the present study, we evaluated all the
responses after Rhodiola rosea extract supplemen-
tation. We did not determine the responses after
terminating the supplementation. However, we suggest
that upregulation of several antioxidant proteins in the
liver may persist for several days until these proteins
are degraded. This potential effect requires further
investigation for the mode and kinetics of possible
medicinal applications of the Rhodiola rosea extract.
Acknowledgment
This work was supported in part by the National

Rhodiola rosea Supplementation Attenuates Oxidative Stress 323
Science Council of the Republic of China (NSC96-
2320-B002-007) to Dr. Chien CT and in part by the
Kuan-Tien General Hospital Research Funds to Dr.
Lee FT.
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