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

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 Ð. 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 Ð. extracts scavenged O2 , H2O2, and HOCl activity in a dose-dependent manner. The ninety-min Ð. 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 Ð. 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 plant in the Crassulaceae family growing in the mountainous and arctic regions of North America, Rhodiola rosea (Golden Root, Roseroot) is a Europe, and Asia. Rhodiola rosea can combat fatigue

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: [email protected] 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 316 Rhodiola rosea Supplementation Attenuates Oxidative Stress 317 by its several unique ingredients (5, 10). Among powdered using a mixer grinder. A known quantity of these, p-tyrosol, salidroside, rosin, rosavin and rosarin the dried powdered material was soaked in distilled are the major active components of Rhodiola rosea water for 24 h at 35-42°C and macerated thoroughly with adaptogenic characteristics (5) for improvement with the help of a mortar and pestle. The mixture of cognitive function (35) and endurance performance was filtered through Whatman filter paper No. 1, con- (1, 12, 35), reduction of mental fatigue (10, 36), anti- densed using a rotary evaporator and lyophilized. diabetic effect (22) and reactive oxygen species (ROS) A model Agilent 1,100 with vacuum degasser, production (2, 13, 20, 21). binary pump, autosampler and thermostatic column Physical exercise is characterized by an increase holder was used. The LC separation was performed in O2 uptake and consumption and induced stressors using a ZORBAX 300SB-C18 3.5 µm, 1.0 × 150 mm. such as elevations of body temperature, the formation The temperature of the column oven was 35°C and the of reactive oxygen species (ROS), and a decrease in injection volume was 1 µl. The eluent flow-rate was glycogen (3, 25, 26). In extreme conditions such as 0.08 ml/min. The gradient conditions were initially ischemia/reperfusion or exhaustive exercise, the 90% A (aqueous phase with distilled water produced increased ROS can oxidize macromolecules con- by Mill-Q, 18.2 MΩ)-5% B (acetonitrile)-5% C tributing to abnormal signal transduction or cellular (methanol), changed linearly to 76% A-12% B-12% dysfunction, impairment of both enzymic and non- C in 16 min. After gradient elution, the column was enzymic antioxidant defense systems of target tissues washed for 1 min with acetonitrile and equilibrated and trigger erythrocyte hemolysis (4) and the cascade for 5 min under the initial conditions leading to a total of apoptosis, autophagy and necrosis (7, 8, 26, 30- time of 25 min for one analysis. All HPLC-MS ex- 33). Exhaustive exercise enhances xanthine oxidase periments were performed using a mass spectrometer activities of plasma and skeletal muscle, muscular (Esquire 3000+, Bruker Daltonik GmbH, Bremen, myeloperoxidase activity and malondialdehyde Germany) equipped with a positive spray ionization concentrations of plasma and tissues (25). Exhaustive source in the full-scan mode over the m/z range 100- exercise leads to oxidative damage in the liver in- 600. Nitrogen was used as the drying gas at a flow- cluding rough endoplasmic reticulum fragmentation rate of 8 l/min. The dry air temperature was 310°C at and dilatation, glycogen depletion, and mitochondrial 20 psi pressure. For calibration of the HPLC-MS enlargement (34, 37). Therefore, exhaustive exercise- method with ESI and the eluent system, eight enhanced oxidative stress may impair liver, kidney, concentration levels of standard were prepared for skeletal muscle and other tissues by different degrees obtaining the calibration curves. We used salidroside, of ROS production. It has been speculated that in- rosin, rosarin and rosavin for standards (Sigma, St. creased antioxidant/oxidative damage-repairing Louis, MO, USA). Standard solutions were prepared enzyme activities, increased resistance to oxidative in 6% methanol containing 400 ng/ml internal stress and lower levels of oxidative damage may standard. For all standards, the concentration levels protect oxidative stress-related cardiovascular, kidney, prepared were 0.5, 2, 5, 20, 50, 200, 500 and 2000 liver and neuronal damages (8, 33, 41). The long- ng/ml. Calibration curves were constructed by plotting term effects of Rhodiola rosea supplementation on responses of the standard compounds relative to exhaustive exercise-induced oxidative stress have not responses of the internal standard (measured in clearly been demonstrated. The purpose of the current triplicate for each concentration) against the con- study was to identify the active components in the centration of standard compounds. Ten mg of the Rhodiola rosea extract and to examine the long-term Rhodiola rosea extracts was dissolved in methanol effect and mechanism of Rhodiola rosea supplemen- and stirred after supersonic vibration for 15 min. The tation on ROS production and oxidized biomarkers in supernatant was filtered after 0.45 µm and was the liver, skeletal muscle and blood after exhaustive analyzed by HPLC-MS. The data were presented in exercise. the Fig. 1. Rhodiola rosea extract contains three cinnamyl alcohol-vicianosides, rosavin, rosin, and Materials and Methods rosarin, that are specific to this species (14, 15).

Rhodiola rosea and High Performance Liquid Animals and Swimming Model Chromatography-Mass Spectrometry (HPLC-MS) Male Wistar rats (200-250 g) were housed at the Dry powders from water extraction of roots of Experimental Animal Center, National Taiwan Rhodiola rosea L. was purchased from Numen Biotech University, at a constant temperature and with a (Taipei, Taiwan, ROC). In brief, fresh original habitats consistent light cycle (light from 07:00 to 18:00 of Rhodiola rosea rhizomes from Siberia were thorough- o’clock). Food and water were provided ad libitum. ly washed with water, shade-dried for 4 weeks, and All surgical and experimental procedures were 318 Huang, Lee, Kuo, Yang and Chien

A HO HO O HO O O HO HO O OH HO OH OH

Salidroside Rosin HO OH O O O OH HO HO OH O HO O O HO O OH HO O HO OH

Rosarin Rosavin B Irdene EIC 379 +Al k104 1 Standard (0.4 µg/ml) Rosin

0 k104 EIC 323 +Al 4 Salidroside 2

0 k104 EIC 451 +Al Rosavin 4 Rosarin 2

0 k104 EIC 319 +Al 1.0 Sample (0.1 mg/ml) Rosin 0.5

0.0 k104 Salidroside EIC 323 +Al 4

0 k104 EIC 451 +Al Rosavin 4

2 Rosarin

0 2 4 6 8 10 12 14 16 18 Time [min]

Fig. 1. A. Structures of four compounds. B. Standards and Rhodiola rosea extract extracted chromatograms from HPLC-MS experiment (full-scan mode) with positive ESI and eluent system.

approved by the National Taiwan University College with water maintained at a temperature of 24 ± 1°C. of Medicine and College of Public Health Institu- The rats swam in the circular plastic barrel for 90 min. tional Animal Care and Use Committee in accordance After 90 min of swimming challenge, the rats were with the guidelines of the National Science Council killed with an overdose of sodium pentobarbital of Republic of China (NSC 1997). intraperitoneally (90 mg/kg body weight) and the All the animals were divided into four groups liver, skeletal muscle and blood were removed. for oral administration of 0, 5, 25 and 125 mg/day Rhodiola rosea extracts for 4 weeks. Before the com- Changes of Lucigenin- and Luminol-Enhanced mencement of an experiment, all animals were Chemiluminescence Counts (CL) familiarized to swimming for 10 min/day for 3 days. At the indicated time, rats fasted overnight for 8 h The antioxidant activities of 5 major ingredients (from 12:00 PM to 8:00 AM) in a 90-min swimming (tyrosol, salidroside, rosin, rosarin and rosavin) and exercise test were measured (6). The swimming Rhodiola rosea extracts on xanthine (0.75 mg kg-1, exercise in free style was carried out in a circular dissolved in 0.01 N NaOH) and xanthine oxidase (24. -1 Ð. plastic barrel (diameter, 20 cm; depth, 30 cm) filled 8 mU kg ) enhanced O2 , 0.03% H2O2 induced H2O2 Rhodiola rosea Supplementation Attenuates Oxidative Stress 319 activity, and % HOCl induced HOCl activity were aprotinin. The mixtures were homogenized com- evaluated. pletely by vortexing and kept at 4°C for 30 min. The ROS levels were measured using a CL analyzing homogenate was centrifuged at 12,000 g for 12 min at system (CLD-110, Tohoku Electronic Industrial, 4°C, the supernatant was collected, and protein Sendai, Japan) as previously described (7). The concentrations were determined by the BioRad Pro- system contained a photon detector (Model CLD- tein Assay (BioRad Laboratoriess Hercules, CA, 110), a CL counter (Model CLC-10), a water circulator USA). (Model CH-200) and a 32-bit IBM personal computer The polyclonal anti-MnSOD (Stressgen system. A cooler circulator was connected to the Bioreagents Limited, Victoria, Canada) rabbit anti- model CLD-110 photon detector to keep the tem- human Cu/Zn SOD (Stress Marq Biosciences Inc., perature at 5°C. Under these conditions, radiant Victoria, Canada) and catalase (Chemicon Interna- energy as low as 10-15 W could be detected. tional Inc., Temecular, CA, USA) antibodies, and the CL was measured in an completely dark chamber monoclonal mouse anti-mouse β-actin (Sigma, Saint of the CL analyzing system. We demonstrated that Louis, MI, USA) were used at 1:1000 dilutions. All using the CL-emitting substance lucigenin (N,N’- of these antibodies cross-reacted with the respective dimethyldiacridinium, Sigma, St. Louis, MO, USA) rat antigens (29). Ð. for O2 or luminol (5-amino-2,3-dihydro-1,4- phthalazinedione, Sigma, St. Louis, MO, USA) for Statistical Analysis H2O2 or HOCl to enhance the CL counts provided similar data to those reported in our previous in vivo All values were expressed as means ± standard study (7). The lucigenin-enhanced CL method error mean (SEM). Differences within groups were provides a reliable assay for superoxide. After 100 s, evaluated by paired t-test. One-way analysis of 1.0 ml of 0.1 mM lucigenin in PBS (pH = 7.4) was variance was used for establishing differences among mixed with the tested sample. CL in the tested sample groups. Intergroup comparisons were made by was measured continuously for a total of 600 s. The Duncan’s multiple-range test. A chi-square test was assay was performed in duplicate for each sample, performed in the hepatic antioxidant expression. and the results are expressed as CL counts (10 s)-1. Differences were regarded as significant if P < 0.05 The total amount of CL in 600 s was calculated by was attained. integrating the area under the curve. The means ± S.E.M. CL level for each sample was calculated. Results We used 1 ml blood samples and 0.2 g homogenized liver and leg skeletal muscle to measure Ingredient Analysis of Rhodiola rosea Extract ROS levels. The analysis of lipid peroxidation, malondialdehyde (MDA), concentrations of plasma In the present study, the structures of four samples was assessed colorimetrically at 586 nm standard components including salidroside, rosin, using a commercial kit (Calbiochem 437634; rosarin and rosavin are demonstrated in Fig. 1A. The Calbiochem-Novabiochem, La Jolla, CA, USA) as original diagram obtained from the four standard previously described (40). Concentration was ex- components (upper panel) and the samples of Rhodiola pressed in µM in plasma and in µmol/mg protein in rosea extract (lower panel) was analyzed by HPLC- tissue samples. MS (Fig. 1B). Rhodiola rosea ingredient was analyzed by HPLC-MS as shown in Table 1. By the use of our Effect of Rhodiola rosea Extract on MnSOD, Cu/Zn techniques, Rhodiola rosea extract was shown to SOD, Catalase Protein Expression contain four major components including salidroside, rosin, rosarin and rosavin. Among these four com- Protein concentration was determined by a ponents, the salidroside content was the highest (13128 BioRad Protein Assay (BioRad Laboratories, ppm). Our data verified that the Rhodiola rosea Hercules, CA, USA). Ten g of protein was electro- extract in this study contains approximately 1.3% phoresed as described below. The expression of salidroside, 0.4% rosin, 0.4% rosarin and 1% rosavin, MnSOD, Cu/Zn SOD and catalase in liver tissues was but did not contain p-tyrosol in our HPLC-MS analysis. evaluated by western immunoblotting and densito- Rhodiola rosea extract supplementation was provided metry as described (41). Briefly, total proteins were to the rat in drinking water in a dose-dependent homogenized with a prechilled mortar and pestle manner. Table 1 shows the level of the four com- in an extraction buffer of 10 mM Tris-HCl (pH 7.6), ponents in the three dosages of Rhodiola rosea extract 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, supplementation. The salidroside, rosin, rosarin and 1% Nonidet P-40, 0.5% deoxycholate, 2% β- rosavin contents were dose-dependently increased mercaptoethanol, 10 µg/ml pepstatin A and 10 µg/ml with the Rhodiola rosea extract dose. 320 Huang, Lee, Kuo, Yang and Chien

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

2 µg 140 µ 100 -. 20 g O2 120 200 µg 90 * H2O2 100 * 80 HOCl 80 * * inhibition * * * * 70

-. *

2 * * 60 * 60 40 * * * 8

% of O 20 * 6 0 * Control salidroside tyrosol rosarin rosin rosavin % of ROS inhibition 4 * * * * 2 * * 100 * 0 * * * * * Control R0.2 R1 R5 R25 R125 80 * * (mg/mL) 60 * * * inhibition 2 * Ð.

O Fig. 3. The antioxidant activity against O2 , H2O2 and HOCl 2 40 * * * in different dosages of Rhodiola rosea extract with * 20 * * * serial dilutions at 0 (R0), 0.2 (R0.2), 1 (R1), 5 (R5), 25

% of H * (R25) and 125 (R125) mg/ml. *P < 0.05 when com- 0 Control salidroside tyrosol rosarin rosin rosavin pared to the control value.

100 80 activities (Fig. 2) at 2, 20 and 200 µg/ml. The in- * 60 hibited H2O2 ability was most prominent in p-tyrosol and rosin at 200 µg/ml. The inhibited HOCl ability * * 40 * * * * was most prominent in p-tyrosol at 200 µg/ml. * * * 20 * * The second part of study was to explore the * * % of HOCl inhibition * Rhodiola 0 antioxidant activity in different dosages of Control salidroside tyrosol rosarin rosin rosavin rosea extract with serial dilutions at 0, 0.2, 1, 5, 25 Ð. and 125 mg/mL. Rhodiola rosea extract significantly Fig. 2. Different dosages of five standards displaying O2 , H2O2 Ð. and HOCl scavenging activities in a dose-dependent reduced xanthine- and xanthine oxidase-induced O2 , manner. The sample concentration was 2-200 µg/ml. H2O2, and HOCl activities in a dose-dependent manner Each data point was tested for four times. *P < 0.05 (Fig. 3). when compared to the control value. To explore the effect of 90-min exhaustive swimming exercise on blood and tissue ROS production, Ð. we investigated lucigenin-dependent O2 chemilu- Antioxidant Activity of the Rhodiola rosea Extract minescence counts in the blood and in homogenized liver and skeletal muscle. As shown in Fig. 4, after We first compared the antioxidant activities of 90-min swimming, the level of lucigenin-dependent Ð. Ð. O2 , H2O2, HOCl of five ingredients, p-tyrosol, O2 chemiluminescence counts was significantly (P < salidroside, rosin, rosavin and rosarin, of the Rhodiola 0.05) increased in blood, liver and skeletal muscle. Ð. rosea extract in 2, 20 and 200 µg/ml. As shown in Fig. The enhancement in O2 production was in an order of 2, p-tyrosol, salidroside, rosin, rosavin and rosarin liver > skeletal muscle > blood. The plasma level of significantly (P < 0.05) inhibited xanthine- and malonedialdehyde, a lipid peroxidation product, was Ð. xanthine oxidase-induced O2 levels at 2, 20 and also significantly (P < 0.05) increased. Ð. 200 µg/ml. The inhibited O2 ability was most Long-term Rhodiola rosea extract supplemen- prominent in p-tyrosol and rosavin at 200 µg/ml. tation at different dosages for 4 weeks significantly Ð. Also, p-tyrosol, salidroside, rosin, rosavin and rosarin decreased the swimming exercise-enhanced O2 significantly (P < 0.05) inhibited H2O2 and HOCl chemiluminescence counts in blood, liver and skeletal Rhodiola rosea Supplementation Attenuates Oxidative Stress 321

500 Mn SOD * 400 Control n = 8 each -. * 2 Swimming n = 8 each 1.4 * -actin 1.2

300 β 1.0

200 ratio 0.8 * 0.6 Blood O 100 (counts/10 s) MnSOD/ 0 Cu/Zn SOD

0.8 * -actin 1.8 * β 1.5 * 0.6 1.2 ratio 0.9 * 0.6 0.4 * CuZnSOD/ Catalase (nmol/mL) 0.2 Plasma MDA 0.0 1.6 -actin

β 1.4 * 1.2 3000 * ratio 1.0 2500 0.8 -. Catalase/ 2 2000 β-actin 1500 1000

Liver O Control Swim Control Swim Control Swim Control Swim

(counts/10 s) * 500 R0R5 R25 R125 0 Fig. 5. Effects of different dosages of Rhodiola rosea extract 1200 supplement and swimming test on MnSOD, Cu/Zn SOD * -. 1000

2 and catalase expression in the rat livers. R0, no Rhodiola 800 rosea extract supplement; R5, 5 mg/ml; R25, 25 mg/ml; 600 R125, 125 mg/ml. *P < 0.05 when compared to the 400 control value of R0 group. Muscle O * (counts/10 s) 200 0 R0 R5 R25 R125 expression was mildly enhanced at the dosage of Fig. 4. Antioxidant effects by different dosages of Rhodiola Ð. 125 mg of Rhodiola rosea extract for four weeks. rosea extract against the O2 level in blood, liver and muscle, and plasma MDA in the rats subjected to 90-min Four weeks of Rhodiola rosea extract supplemen- swimming exercise that significantly increased the tation attenuated the depression of Cu/Zn SOD after oxidative stress. The animals were fed Rhodiola rosea exhaustive swimming. extract at 0 (R0), 5 (R5), 25 (R25) and 125 (R125) mg/ml in the drinking water for 4 weeks. *P < 0.05 when Rhodiola rosea Extract Supplementation Increased compared to the control value. Exercise Performance

After 4 weeks of Rhodiola rosea extract supple- muscle. The increased plasma malonedialdehyde mentation at different dosages, 5% weight-loaded level induced by swimming was also depressed by swimming was used to evaluate the exercise perfor- long-term Rhodiola rosea extract supplementation mance by the swimming time to fatigue. Treatment (Fig. 4). of four weeks of the Rhodiola rosea extract at 5, 25 and 125 mg significantly (P < 0.05) increased the Chronic Rhodiola rosea Treatment Enhanced Hepatic swimming performance by 18.8%, 46.8% and 59.3% Antioxidant Enzymes Expression (n = 5 each).

We explored whether 4 weeks of Rhodiola rosea Discussion extract supplementation affected expression of antioxidant enzymes in the rat liver. As shown in Fig. In the present study, we described a HPLC-MS 5, Mn SOD, Cu/Zn SOD and catalase were all ex- technique to identify and quantify 4 major components, pressed in the liver of R0 group. After 90 min of salidroside, rosin, rosarin and rosavin, in the Rhodiola swimming exercise, the expression of Cu/Zn SOD, rosea extract. The application of HPLC for deter- but not Mn SOD and catalase, was significantly mining hydrophilic extracts from Rhodiola rosea and decreased in the R0 group. Four weeks of administra- Rodiola quadrifida has led to the identification of tion of the Rhodiola rosea extract at 25 and 125 mg cinnamic alcohol, chlorogenic acid, rhodiooctanoside, enhanced hepatic Mn SOD and Cu/Zn SOD expression rosiridin, rosavin and the phenolic compounds before the exhaustive swimming exercise. Catalase salidroside, rhodiolin and viridoside (20, 24, 38). 322 Huang, Lee, Kuo, Yang and Chien

Our technique did not detect p-tyrosol in our Rhodiola (20). Because of this variation within the Rhodiola rosea extract. genus, verification of Rhodiola rosea by HPLC is It is frequently stated, but poorly demonstrated, dependent on the content of the rosavin, rosin and that exercise could have adverse effects related to rosarin rather than salidroside and p-tyrosol (9, 14, inflammatory response, ROS production and accu- 15, 20). Based on a comparative analysis, the most mulation of oxidative damage in several organs (3, uniquely active chemical constituents are rosavin 11, 16, 19, 33). Unlike the skeletal muscle, liver con- (the most active), rosin, rosarin, salidroside and its tains high levels of xanthine dehydrogenase; during aglycon, p-tyrosol. In our data and in a previous exercise, xanthine dehydrogenase is converted to study, p-tyrosol, salidroside, rosavin, rosin, and rosarin xanthine oxidase generating ROS and oxidative are all antioxidant substances (13, 20, 21, 42, 43), damage (31). In the present study, we found that 90- especially in p-tyrosol. In the present study, we have Ð. min exhaustive swimming exercise increased O2 clearly identified that Rhodiola rosea extracts used production in an order liver > skeletal muscle > blood in our study contained rosavin, rosin and rosarin, but indicating that liver is the most sensitive target organ. did not contain p-tyrosol as analyzed by our HPLC- This result is agreement with a previous report with MS technique. A previous study has indicated that nuclear 8-hydroxydeoxyguanosine as the oxidative Rhodiola rosea roots contain 1.3 to 11.1 mg/g salidro- stress; the report showed that the nuclear 8-hydrox- side and 0.3 to 2.2 mg/g p-tyrosol (20). Our HPLC- ydeoxyguanosine content increased in liver, not in MS data show that 13 mg/g salidroside, 3.3 mg/g skeletal muscle and brain (33). The increased blood rosin, 3.1 mg/g rosarin and 9.5 mg/g rosavin are ROS after exhaustive exercise may contribute to the found in our Rhodiola rosea extracts. We therefore enhanced level of plasma malonedialdehyde, a lipid suggest that high contents of rosin, rosarin and ro- peroxidation product, found in our analysis. Increased savin may exert a more efficient potential than p- Ð. blood ROS including O2 , H2O2 or HOCl may induce tyrosol (not detected or too low in our Rhodiola oxidation of phospholipid bilayers in the erythrocytes, rosea extract) in the reduction of swimming-induced increase phosphotidylcholine hydroperoxide and oxidative stress. malonedialdehyde accumulation in the erythrocyte Our results also showed that four weeks of membrane and consequently contributes to hemoly- Rhodiola rosea extract supplementation can up- sis (13, 17, 18). regulate Mn SOD and Cu/Zn SOD protein expression Therefore, an increased activity in the antioxi- in the rat liver. Although we did not know the detailed dant defense mechanism or a decrease in oxidative mechanisms involving Rhodiola rosea-enhanced stress may protect organs against oxidative damages. antioxidant protein expression, direct scavenging ROS Exhaustive exercise on the treadmill resulted in activity and enhancement of several antioxidant significant increases in lipid peroxidation of skeletal proteins of Rhodiola rosea extract may have provided muscle, liver and kidney inrats, and this was prevented hepatic protection against exhaustive exercise- induced by superoxide dismutase derivatives (30, 31). Previous oxidative stress in the liver. In the present study, we studies have indicated that Rhodiola rosea extracts have clearly indicated that p-tyrosol, salidroside, rosin, containing specific ingredients may have beneficial rosarin and rosavin or the Rhodiola rosea extract effects in enhancing exercise performance (1, 12, 23, can significantly and dose-dependently decreased Ð. 35) and in reducing ROS levels (2, 13, 21), but the O2 , H2O2 and HOCl activity in vitro. In addition, major active component has not clearly been demon- chronic Rhodiola rosea extract supplement signifi- strated. It has been reported that some Rhodiola cantly and dose-dependently reduced swimming species did not have antioxidant effects on hypoxemia exercise-enhanced plasma malonedialdehyde con- Ð. and oxidative stress (39). The chemical composition centrations and O2 levels in the liver, skeletal muscle and physiological properties of Rhodiola species are and blood. In the present study, we evaluated all the to a degree species-dependent although overlaps in responses after Rhodiola rosea extract supplemen- constituents and physiological properties do exist in tation. We did not determine the responses after many Rhodiola species (20, 27, 28). Rhodiola rosea terminating the supplementation. However, we suggest contains a range of biologically active substances that upregulation of several antioxidant proteins in the including organic acids, flavonoids, tannins and phe- liver may persist for several days until these proteins nolic glycosides. The stimulating and adaptogenic are degraded. This potential effect requires further properties of Rhodiola rosea were originally attributed investigation for the mode and kinetics of possible to two compounds isolated from its roots identified as medicinal applications of the Rhodiola rosea extract. p-tyrosol and the phenolic glycoside salidroside found in all studied species of Rhodiola (9). However, other Acknowledgment active glycosides, including rosavin, rosin and rosarin, have not been found in the Rhodiola species examined This work was supported in part by the National Rhodiola rosea Supplementation Attenuates Oxidative Stress 323

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