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Effect of Rhodiola rosea and Rhodiola crenulata (Crassulaceae) root extracts on ATP content in Muscle Mitochondria

• Abidoff Musa, Center of Modern Medicine, Russian Ministry of Defense Industries, Moscow-121351, Russia.
• Krendal Felix & Grachev Sergey, IM Sechenev Medical Academy, Bolshaya Pirogovskaya, 2/6, Moscow, Russia.
• Seifulla Roshen, Russian Center for Higher Sport Education, Moscow.
• Tim Ziegenfuss, Pinnacle Institute of Health & Human Performance, Wadsworth Medical Center, STE 103 323 High Street, Wadsworth, OH 44281

Abstract

We investigated the effect of oral Rhodiola rosea and Rhodiola crenulata root extract supplementation on swimming time to exhaustion and concentrations of adenosine 5'-triphosphate (ATP) in muscle mitochondria of rats. Animals received either 50mg/kg R.rosea extract (standardized to 3% rosavins and 0.8% salidroside) or 50 mg/kg R.crenulata (standardized to 2% salidroside) 30-minutes prior to completing an exhaustive swimming test conducted over 6-days. R.rosea increased swim time to exhaustion by 24.6% compared to control (0% change) and R.crenulata (4.3% change, P<0.05). There were no statistically significant differences between the control and R.crenulata extract-treated groups, despite the latter animals receiving salidroside in a dosage 2.5 times greater than the R.rosea. The exercise test reduced the content of mitochondrial ATP from 5.46±0.3 to 3.8±0.2 mmol/g protein (31.5±2.2%) in both the control and R.crenulata extract-treated groups, while its level in the R.rosea extract-treated group decreased to 22.02±1.6%, from 5.51±0.4 to 4.85±0.2 mmol/g. In addition, after 24 h resting levels of ATP in the R.rosea-extract treated group had recovered to 5.42±0.3mmol/g, while its content in both control and R.crenulata-extract treated groups were significantly (P<0.05) lower (4.26±0.2 and 4.46±0.3 mmol/g, respectively). These results indicate that R.rosea extract increases the synthesis or re-synthesis of ATP in mitochondria, and stimulates energetic recovery processes after intensive muscular work. Results of this study provide strong evidence that R.rosea and R.crenulata possess significantly different pharmacological properties, and that R.rosea is superior in terms of enhancing physical performance.

Introduction

Rhodiola rosea (Crassulaceae) or “golden root” is a plant indigenous to the high altitude Arctic Regions and is considered a phytomedicine in Russia, Scandinavia and Asia (Saratikov and Krasnov 1987, Brown et al. 2002). Extensive animal and human research has revealed that the R.rosea root extract possesses anti-stress and anti-depression properties, alleviates emotional, mental, and physical disorders (Saratikov and Krasnov 1987; Spasov et al. 2000; Shevtsov et al. 2003), and reduces exhaustion after intensive training workloads (Dambueva 1968; Adamchuk 1969; Salnik 1970, Lapaev, 1982; Azizov and Seifulla 1998). It was also demonstrated that R. rosea root extract increases the level of brain norepinephrine (NE), dopamine (DA), serotonin (5-HT), and has nicotinic cholinergic effects in the central nervous system (Saratikov et al. 1978, Stancheva et al. 1987; Lazarova et al. 1986; Petkov et al. 1986).

Animal studies have revealed that R.rosea stabilizes the ultrastructure of mitochondria during exhaustive swimming (Salnik 1970; Saratikov and Krasnov, 1987), stimulates the release fatty acids from adipose tissue (Dambueva 1968), and enhances creatine phosphate synthesis in muscle and brain, and muscle protein synthesis (Adamchuk 1969; Revina 1969; Saratikov et al. 1971). It has also been reported that professional athletes have used R.rosea to enhance physical performance, promote anabolism in muscle, prolong stamina during peak periods of physical stress, and enhance cardiovascular recovery time after intense training (Fulder 1980; Saratikov et al. 1968; Azizov & Seifulla, 1998; Seifulla, 1999).

There are about 20 species of the genus Rhodiola, and the phytochemistry and pharmacological properties of these plants seem to depend entirely upon which species is being used (Komarov, 1939; Saratikov 1974; Kurkin and Zapesochnaya 1986). Principal constituents in R.rosea are cinnamyl alcohol vicyanoside rosavin, rosin, rosarin, (collectively the rosavins) and hydroxyphenylethanol-2-D-glucopyranoside (salidroside, also known as rhodioloside) (Saratikov et al. 1968; Kurkin and Zapesochnaya 1986a,b). The presence of rosavins seems to be specific to R.rosea only (Kurkin and Zapesochnaya, 1996a,b; Dubichev et al. 1991), while the presence of salidroside was shown in all plant species of the genus Rhodiola (Barnaulov et al. 1965; Wang et al. 1992a,b; Kang et al. 1992; Yoshikawa et al. 1996; Linh et a. 2000). In contrast, R. crenulata is medicinal plant in Uzbekistan, China and other Asian countries (Wang et al. 1992b; Cui S et al 2003). It is believed that R.crenulata possesses notifying properties, and that salidroside is responsible for this effect, although it has not yet been clinically evaluated (Xiu 2002).

In this study, we investigated the effect of R. rosea and R. crenulata root extracts on the concentration of ATP in rat muscle mitochondria before and after a swimming test to exhaustion. Results of this study demonstrate that administration of R.rosea root extract increased the time to exhaustion and the content of mitochondrial ATP compared with compare to controls and the R.crenulata extract-treated group.

Materials and Methods

Plant material

Underground parts of R. rosea were collected in Eastern Siberia (Russia), and R. crenulata Fish et Mey root and rhizome was received as a gift from the Uzbekistan Academy of Science. Voucher samples from each original lot were retained in our laboratory. Plant material was collected in the late flowering period, separated from soil residues, and carefully sliced into 1-3cm cuts and dried under continuous airflow chambers at 30oC. When the moisture content was reduced to 5-6%, plant material was milled to 2mm particles size and used in extract preparations.

Powdered plant materials were extracted with an ethanol: water mixture (60:40, v/v) in 100 L extraction vessels at 45-55oC for 12 h with continuous agitation. The crude extracts were separated from cell-wall debris by centrifugation at 2000 rpm, alcohol removed following distillation under reduced pressure and the liquid alcohol-free extract was freeze dried.

HPLC analysis of rosavins and salidroside

The content of rosavins and salidroside were determined using a Waters System HPLC equipped with 996 Photodiode Array Detector, two model 515 Pumps, a Gradient Mixer Kit 051518, a Pump Control Module, a Bus SAT/IN Module, a model 7725I Injector with 20 ml loop, and a Millenium32 Chromatography Manager (Version 3.0). For all separations a RP-C18 analytical column C-18, 3.9 x 150 mm, 5 mm particle size was used (Symmetry, WATO27324, Waters Associates, Inc.). Two mobile solvents were used to develop a binary gradient, phase A: 0.16 M ammonium acetate solution in water (w/v) (pH is adjusted to 5.5 with acetic acid) and, phase B: methanol. The mobile phase flow rate was adjusted to 1.0 ml/min, and UV detection wavelength was set at 2 different wavelengths, 254 for rosavin, rosin and rosarin, and 280 nm for salidroside (Dubichev et al. 1991; Kurkin et al. 1986a; Ramazanov & Bernal Suarez, 1999). After 5 min holding the initial solvent mix, 66:34 (A: B, v/v), a linear ramp up to 60:40 (A: B, v/v) over 17 min was developed, followed by a return to the initial conditions over 5 min, and 5 min equilibration before the next analysis.

The HPLC reference standards of rosavin, rosin, rosarin and salidroside were received as gifts from the Russian Institute of medicinal Plants (VILAR), Moscow. Stock standard solutions were prepared in ethanol: water (85:15, v/v) to a concentration of 1 mg/ml. Four standard solutions containing both components in different concentrations, between 0.01 and 0.3 mg/ml, were injected. The calibration curve for each standard was linear in the described range with correlation coefficients of 0.99.

Animals

Twenty-four adult Sprague-Dawley rats (250±20g) housed in temperature (20±2 °C) and light (08:00h-20.00h) controlled cages were used in this study. Food and water were made available ad libitum. The animals were divided into three groups; control (n=8), the 50mg/kg R.rosea extract-treated group (n=8) and the 50mg/kg R.crenulata extract-treated group (n=8).

Exercise Protocols

A swim test was used to measure the time to exhaustion and to determine the effects of plant extracts on ATP content in muscle mitochondria. The animals were made to swim to exhaustion in a tank filled with water at room temperature. Normally, rats are able to swim for 10–14 hours without rest, but with additional weight attached, exhaustion occurred within 5 hours. The animals were forced to swim for as long as possible over a six day period. They were subjected to two swimming sessions per day until exhaustion, with 30-minutes rest between sessions. Plant extracts (50mg/kg) were administered by oral gavage 30-minutes before each exercise bout. Time to exhaustion was defined as the time between the commencement of exercise and the first occurrence of the animal failing to swim. Immediately after determination of the time to exhaustion, 50% of the rats in each group were sacrificed with an IP injection of sodium pentobarbital (5 mg/100 g body weight) while the other 50% were killed 24 h after the last treatment.

Mitochondrial isolation

Muscle mitochondria were isolated following the method described by Tonkonogi and Sahlin (1999). Muscle specimens were disintegrated with scissors, homogenized in the presence of proteinase and resuspended in an ice-cold isolation medium containing 70mM sucrose, 220mM mannitol, 5mM HEPES (pH 7.2), 0.5mM EDTA. The homogenate was centrifuged at 600 X g for 10 minutes. Supernatant was decanted, re-diluted with the isolation medium, and centrifuged again at 12,000 X g for 10 minutes to obtain the mitochondrial fraction. The final mitochondrial pellet was resuspended in a buffer (pH 7.40) consisting of 225mM mannitol, 75mM sucrose, 10mM Tris, and 0.1mM EDTA. An aliquot of the suspension and muscle were taken for measurements of protein content and citrate synthase activity as a mitochondrial marker to estimate the percentage of mitochondria freed from the muscle (Tonkonogi et al. 1997). The protein concentration was determined by the method of Bradford using nitrogen-calibrated bovine serum albumin as the standard (Marshall and Williams, 1992).

ATP analysis

The content of ATP in muscle and isolated mitochondria was determined using the bioluminescence method described by Drew and Leeuwenburgh (2003) with a commercial ATP kit. This assay is based on the reaction of ATP with firefly luciferase and its substrate luciferin. Measurements of ATP content were made immediately after isolation of the mitochondria to improve the accuracy of the measurements and reduce any inherent errors associated with the luminescent decay or reduced viability of the isolated mitochondria (Drew and Leeuwenburgh, 2003). All mitochondrial samples were performed in triplicate and an average of these results was used in the quantification of ATP content.

Chemicals

The ATP determination kit (A-22066) by Molecular Probes (Eugene, OR) contains D-luciferin, luciferase (40 III of a 5 mg/ml solution in 25 mM Tris-acetase, pH 7.8, 0.2 M ammonium sulfate, 15% (v/v) glycerol and 30% (v/v) ethylene glycol), dithiothreitol (DTT), adenosine 5'-triphosphate (ATP), and a Reaction Buffer (10 ml of 500 mM Tricine buffer, pH 7.8, 100 mM MgSO4, 2mM EDTA and 2 mM sodium azide). The reagents and reaction mixture were combined according to the protocol provided by the manufacturer.

Statistical analysis

All values reported are means ± SE. Differences between means were tested for statistical significance by single-factor analysis of variance (ANOVA) with a repeated-measure design. P< 0.05 was considered as an indicator of significant differences.

Results and discussion

Rhodiola rosea (Crassulaceae) root extract is a phytomedicine used to reduce chronic stress and promote mental and physical performance. The phytoactive compounds responsible for its pharmacological effects are a complex of cinnamyl alcohol vicyanosides rosavin, rosin, rosarin, (the rosavins) and hydroxyphenylethanol-2-D-glucopyranoside (salidroside). Rhodiola crenulata also belongs to the genus Rhodiola and is used as general tonic. It has been speculated that R.crenulata possesses pharmacological properties similar to R.rosea, even though it does not contain the complex of rosavins.

Figure 1 shows the HPLC chromatograms of R.rosea and R.crenulata root extracts used in this study. These results indicate that the HPLC fingerprint of these two plant extracts is very different. R.rosea root extract contains the complex rosavins and salidroside, while R.crenulata root extract contain salidroside only. This is in agreement with previous data obtained by other laboratories (Kurkin and Zapesochnaya 1986a; Dubichev et al. 1991). Using corresponding HPLC reference standards, the content of rosavins in R.rosea root extract was calculated as 3.02% dry weight and salidroside 0.89% dry weight. In contrast, the content of salidroside in the R.crenulata root extract was calculate as 2.05% dry weight. Therefore, the ratio of rosavins to salidroside in true R.rosea root extract is about 3:1, which is in agreement with Kurkin and Zapesochnaya (1996a,b).

Table 1 summarizes the effect of R. rosea and R.crenulata root extracts on time to exhaustion for the swim test. The mean time to exhaustion was 34.2±2.5, 35.5±3.1 and 44.9±2.1 minutes in control, R.crenulata and R.rosea extract-treated groups, respectively. The mean time to exhaustion of animals in R.crenulata extract-treated group was no different from that in control group (p>0.05). These results indicate that R.crenulata root extract does not enhance physical performance compared with R.rosea extract, despite on fact that R.crenulata root extract used in this study contain 2.5 times higher salidroside content. Therefore, the complex of rosavins and or its combination with salidroside might be responsible for the observed effects in this study, although the role of other yet to be identified yet compounds cannot be excluded.

More evidence supporting distinct pharmacological differences between these two plants of the genus Rhodiola can be gleaned from their effects on mitochondrial ATP content. Table 2 shows that the swim exercise significantly reduced the content of ATP in muscle mitochondria from 5.38±0.3 mmol/g protein to 3.86±0.4 mmol/g in the control group and from 5.48±0.5 to 3.81±0.5 mmol/g protein in the R.crenulata extract-treated group. However, the reduction in ATP content was substantially lower in the R.rosea extract treated-group, from 5.41±0.4 to 4.85±0.3 mmol/g protein (P<0.05). These results indicate that R.rosea extract appears to enhance the synthesis/re-synthesis of mitochondrial ATP in exercised muscle.

The current findings agree with previous reports demonstrating that R.rosea root extract can enhance mitochondrial levels of high energy phosphates. Revina investigated the effect of R.rosea extract on the level of brain ATP and CP in rats under experimental conditions similar to described above (Revina 1969). The results of that study revealed that administration of R. rosea (1:1 liquid tincture in 40% ethanol) before exhaustion swimming exercise extract increased the level of both ATP and CP in the brain at the same time that these decreased in the control group (Revina 1969; Saratikov and Krasnov 1987). Results of this study suggest that stimulation of synthesis of ATP and CP in brain might contribute to endurance or stamina during physical exertion, simultaneously reducing the recovery time after intensive physical workloads. Similar results have been reported by other researchers (Adamchuk 1969; Adamchuk and Salnik 1970; Saratikov and Krasnov 1987; Salnik, 1970).

The anabolic properties of R. rosea root extract were first studied in mice (Salnik et al. 1968; Adamchuk 1969; Saratikov et al. 1971). Concentrations of RNA and DNA, activity of aminoacyl-t-RNA synthetase and ATP in muscle were analyzed before and after exhaustive swimming exercise. Results indicated that the activity of aminoacyl-t-RNA-synthetase, and the concentration of RNA and ATP were significantly higher in muscle of animals received R.rosea extract. In addition to indicating a potential anabolic effect from R. rosea root extract, data from Adamchuk and other authors suggest that the anabolic activity of whole extract of R.rosea root is superior to that of isolated individual compound salidroside.

Intramuscular lactic acidosis and ammonia are other metabolic factors that can cause fatigue during resistance exercise and reduce the rate of synthesis of ATP and CP in mitochondria (Seifulla 1999; Lambert and Flynn, 2002). High levels of lactic acid that accumulate during intensive muscular exercise impair the respiratory function of skeletal muscle mitochondria and induce reorganization of the ultrastructure of the mitochondria. Notably, at least one study has reported that mice receiving R. rosea have reduced muscle ammonia concentrations. Since ammonia metabolism is fundamental to muscle function, this effect may explain how R. rosea accelerates muscle recovery after intensive muscular workloads (Saratikov and Krasnov 1987).

Research on the effects of R. rosea on athletes and others performing maximum physical and mental workloads has confirmed the same pattern of improved performance demonstrated in animal studies (Revina 1969; Saratikov and Krasnov 1987). Human test subjects taking R. rosea showed more robust pulse rates, greater back muscle strength and hand endurance under static tension, better coordination, and reduced recovery times. Extensive experiments on skiers and other athletes have reliably demonstrated the significant and unique value of R. rosea tincture for increasing stamina and accelerating recovery from physical exertion (Saratikov and Krasnov 1987). Based on the multitude of Russian studies beginning with 35 years ago, Soviet scientists and trainers have recommend R. rosea with increased frequency in many arenas of athletic performance to improve speed and strength, stamina, energy reserves, and short recovery time between competitions (Lapaev 1982; Saratikov et al. 1968; Saratikov and Krasnov 1987; Seifulla 1999).

In summary, these results indicate that R.rosea root extract possesses pharmacological properties unique to this plant species. Aside from the positive effects on mitochondrial ATP synthesis we observed that animals treated with R.rosea root extract increase the time to exhaustion and reduce their recovery time after intense exercise. Our results also suggest that the complex of rosavins might be responsible for the beneficial effects observed in this study, however, the role of other yet to be identified compounds in R.rosea cannot be ruled out.

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