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Rosavin Plus:  Freeze Dried and Superior to Spray Dried Form

Rosavin Plus: Freeze Dried and Superior to Spray Dried Form

Freeze Dried vs the Spray Dried Form of Rhodiola Rosea

In testing Swimming Time to Exhaustion in rats, the Freeze-Dried form of Rhodiola rosea root extract produced superior physical strength and endurance performance, compared to the Spray-Dried form of the extract.
Experimental Biology and Medicine, 2005, in press

Seifulla Roshen1
(1) Russian Center for Professional Sport Education and Training, Institute of Immunopathology, Russian Academy of Natural Sciences, Molodogvardeiskaya 46/1, Moscow-121351, Russia

* (1) This work was supported by grants from the Center of Modern Medicine, and Institute of Immunotherapy, Russian Academy of Natural Sciences

Summary

In this study we investigated the effect of freeze-dried (FDE) and spray-dried (SDE) Rhodiola rosea root extracts, standardized to 3% rosavins and 0.8% salidroside, on swimming time to exhaustion, plasma fatty acids and corticosterone, and muscle glycogen concentration in rats. The two experimental groups of animals received 50mg/kg of either FDE or SDE of R. rosea, administered 30 minutes prior to an exhaustive swimming test. The control group received 50mg/kg maltodextrin.

Results revealed quite surprising and dramatic pharmacological differences between the performance enhancing effects of the two types of R. rosea extracts. In the FDE-treated group the swimming time was 15.4±3.2% longer that the SDE-treated group (P < 0.05) and 43±5.5% longer that the control group (p<0.05). Furthermore, after a 30-minute rest, the FDE-treated animals recovered faster and were able to swim for an additional 18.1±3.2 minutes, 27.5% longer than the SDE group’s additional 14.2 ±2.8 minutes, and 74.0% longer than the control group’s additional 10.4 ±3.4 minutes.

Predictably, the Swimming to Exhaustion test predictably increased the plasma corticosterone and free fatty acids levels, and reduced the muscle glycogen concentration in all three groups of animals. Although the administration of both SDE and FDE measurably reduced corticosterone in swimming rats from 289.4 ng/ml (placebo) to 254.1ng/ml and 214.3ng/ml, respectively, FDE performed significantly better than the SDE (P<0.05). Furthermore, FDE-treated animals had higher plasma fatty acid levels and spared more muscle glycogen level compare with SDE-treated and placebo group.

The main processing difference between spray drying and freeze drying the extract is the relatively brief exposure to heat, at temperatures of 160oC+, (320oF) in spray-drying, compared to no heat exposure in freeze drying.

Previous studies have established that the group of rosavins characteristic of extracts of the R. rosea species do, in fact, account for part of the performance-enhancing effects of Rhodiola products. However, since both dried forms of the extract used in this study are standardized to 3% rosavins and 0.8% salidroside, these results clearly suggest that even the relatively brief high temperature exposure of the extract to heat during the spray-drying process is sufficient to degrade or destroy one or more additional key performance-enhancing components in R. rosea extract that have not yet been identified. Consequently, further research is warranted to specifically identify these key additional components.

Introduction

Rhodiola rosea (Crassulaceae) is a phytomedicine traditionally used in Russia and other European and Asian countries (Saratikov and Krasnov 1987, Brown et al. 2002, Abidov et al. 2004). Extensive animal and human research has revealed that the R.rosea root liquid tinctures and standardized extracts helps to alleviate mental and physical disorders (Saratikov and Krasnov 1987; Spasov et al. 2000; Shevtsov et al. 2003). Specifically, R.rosea preparations manifest lipolytic and anabolic activity, and stimulate muscle protein synthesis, creatine phosphate and ATP synthesis in muscle and brain tissue (Adamchuk 1969; (Dambueva 1968; Revina 1969; Saratikov et al. 1971; Salnik 1970; Saratikov and Krasnov, 1987; Abidov et al. 2003).

Although there are approximately 20 species of the genus Rhodiola (Komarov, 1939), the phytochemistry and pharmacological properties of the phytomedicine seem to depend entirely upon which species is being used to produce the extract (Abidov et al. 2003; Kurkin and Zapesochnaya 1986). Recently, we demonstrated that the administration of 50mg/kg of extract of Rhodiola rosea root, standardized to 3% rosavins and 0.8% salidroside, increased the swimming time to exhaustion in rats by 24%, and stimulated ATP synthesis in muscle mitochondria, compared to controls administered 50mg/lg of maltodextrin. In contrast, 50mg/kg of the alternative extract of R. crenulata root, standardized to 2% salidroside, failed to show any significant physical performance effect compared to identical controls (Abidov et al. 2003). Since R.crenulata root doesn’t contain rosavins, results of this study suggested that the presence of rosavins is responsible for superior biological activity of R.rosea over R. crenulata (Saratikov et al. 1968; Kurkin and Zapesochnaya 1986a,b; Abidov et al. 2003).

The two most practical, cost-effective industrial methods of drying extract such as Rhodiola are spray-drying and freeze-drying. Both methods can be used to produce dried extracts of Rhodiola on a commercial scale, although spray drying is typically preferred because it is more economical. Both forms can be standardized to 3% rosavins and 0.8% salidroside. To date, no controlled studies have been conducted to compare how the two methods of drying might affect the pharmacological properties of the basic extract when equally standardized to these same marker compounds, which was the purpose of our study.

Materials and Methods

Plant material

The Rhodiola rosea root and rhizomes were collected in Eastern Siberia (Russia) in the late flowering period, separated from soil residues, carefully sliced into 1-3cm cuts and dried under continuous airflow chambers at 30oC to moisture content less 9%. Then plant material was milled to 2-3 mm particles size, placed into extraction vessels and extracted three times with warm water at 40-45oC for 4 h each time with continuous agitation. The crude extract was separated from plant debris by filtration, following continuous flow centrifugation at 10oC and 2000 rpm, and obtained clear liquid extract was either freeze-dried or spray-dried. The freeze-dried extract was in golden-yellow color with strong rose odor that is specific to R. rosea root. The dark brown color spray dried extract lacked specific to R. rosea odor of rose flower. The upper temperature in the Spray-dried was settled to 180oC and at temperature at the bottom of tower was 80oC.

HPLC analysis of rosavins and salidroside

The content of rosavins and salidroside were determined as described previously (Ganzera et al. 2001) 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 and phosphate buffer/ Acetonitrile gradient were used (Symmetry, WATO27324, Waters Associates, Inc.). The mobile phase flow rate was adjusted to 0.62 ml/min, and UV detection wavelength was set at wavelength 205nm (Ganzera et al. 2001).

The HPLC reference standard of rosavin and salidroside were received as gifts from the Russian Institute of Medicinal Plants, 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. The content of rosavins and salidroside in the FDE was 3.42% and 0.93% dry weight, and 3.54% and 0.98% dry weight in SDE, respectively. The HPLC fingerprints of used extracts are provided on Figure 1.

Animals

Thirty adult Sprague-Dawley rats (220±10g) 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 received 50mg/kg malto dextrin (Control, n=10), the 50mg/kg freeze-dried extract-treated group (FDE, n=10) and the 50mg/kg spray-dried extract-treated group (SPE, n=10). The extracts of R. rosea were administered by oral gavage 30-minutes before swim test.

The care and treatment of experimental animals conformed to the Center of Modern Medicine guidelines for the ethical treatment of laboratory animals. To avoid circadian variations in physical activity, experiments were carried out from 11.00 am to 17.00 o’clock.

Exercise Protocols

A swim test to exhaustion was used to evaluate the effects of R. rosea extracts on physical straight and on the recovery from intensive muscular workload. The animals were forced to swim for as long as possible over a six-day adaptation period. After this adaptation period, loads equivalent to 5% of their own body weight were attached to the tail of the animals, and they were subjected to swimming session until exhaustion. The exhaustion was defined as the time of first occurrence of the animal failing to swim. The recovery of animals after first exhaustion was evaluated as the time in minutes that rats were able to swim again after exhaustion and 30 minutes rest.

Serum corticosterone

A serum level of corticosterone was measured with radioimmunoassay kits (ICN Biomedicals, Inc. Costa Mesa, CA) according to the manufacturer’s instruction, with 125I-corticosterone with approximate detection limit 10ng/ml.

Serum fatty acids

For estimation of the basal plasma levels of corticosterone, the animals were kept undisturbed the night before the experiment. Next morning blood samples (50/µL) taken from the tail of animals (baseline) and 45 minutes after swimming test completed. Samples were centrifuged and stored at 20°C until measurement of free fatty acids (FFA). Serum content of FFA was determined using a commercially available enzymatic colorimetric kit (Wako Chemicals USA, Inc., Richmond, VA, USA).

Skeletal muscle glycogen

Muscle glycogen was measured by a phenol-sulfuric acid colorimetric assay as described by Lo et al. (1970) in modification (Anthony et al. 1999. A standard curve ranging from 0 to 100 mg/L was prepared using a glycogen stock solution (Bovine Liver Glycogen/L, Sigma, St. Louis, MO) in distilled water.

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

The effect of Rhodiola rosea SDE and FDE on swimming time to exhaustion.

The effects of Rhodiola rosea FDE and SDE on the swimming time to exhaustion in rats were investigated in this study. Results of our study revealed two extracts obtained from the same plant possess surprisingly different pharmacological effects.

In animals received 50 mg/kg FDE exhaustion occurred more slowly and the time of swimming was prolonged by 24±2.5% (P<0.05) compared to SDE-treated group and 43±5.5% compared to control group (Fig. 2) (P < 0.05). Surprisingly, animals treated with 50mg/kg SDE the swimming time to exhaustion increased only to 15.4±3.2% (P<0.05) compared to control animals, indicating that the FDE possesses significantly superior endurance properties.

The next we examined how both types of extracts would affect on the recovery capacity exhausted animals after 30-minutes rest. Results of this study revealed that the FDE-treated group was able to swim again after 30 minutes rest for another 18.1±3.2 minutes, while their counterparts from SDE -treated and control groups for 14.2 ±2.8 minutes and 10.4 ±3.4 minutes, respectively (Fig. 3).

The effect of Rhodiola rosea SDE and FDE on muscle glycogen and serum fatty acids concentration:

The baseline concentrations of muscle glycogen and plasma FFA in resting animals were 5.3 ±03mg/g tissue and 0.56±0.4mmol/L, respectively (Fig. 4 and 5). The Swimming to Exhaustion test predictably reduced the muscle glycogen concentration and increased the plasma FFA levels in all three groups of animals. However, FDE-treated animals spared 20±3% more muscle glycogen during exhaustion swimming exercise than those in SDE group and 55±7% more than in control group. Furthermore, the administration of R. rosea extracts caused a marked increase in serum FFA, although the effect of the FDE was significantly stronger than in those SDE-treated and control group. For instance, the FDE-treated group had 11±2.6% higher level of plasma FFA compare with the SDE group and 30±3.2% higher than in those from control group. These findings suggest that the increase in serum FFA was advantageous to the mobilization and utilization of fat for the enhancement of swimming capacity in animals, which might preserve usage of muscle glycogen.

The effect of Rhodiola rosea FDE and SDE on serum corticosterone

The swimming test to exhaustion induced a robust secretion of corticosterone (Fig. 6). To determine whether the R. rosea extracts will influence the increased secretion of corticosterone, the FDE and SDE were administered 30 minutes before swim test. Pretreatment of animals with both FDE and SPE reduced swim -induced raise plasma in corticosterone secretion (Fig. 6), again the effect inhibition of exercise induced corticosterone release was significantly stronger in FDE-treated animals in that in those in SPE-treated and control groups. The concentration of plasma corticosterone increased from 120.5±10ng/ml to 289.4±9ng/ml (237±20% increase) in control group, while in animals received SDE and FDE the corticosterone level increased to 254.1±8ng/ml 212±11% increase) and 214.3±11ng/ml (177±12% increase), respectively (P<0.05). Therefore, these results indicate that the FDE was effective to 55±7% compared to control and 35±6% compare to SDE in its inhibition of the exercise induced corticosterone increase. The mechanism of the reduction of swim induced corticosterone secretion is not clear, but we propose that reduction of swim induced corticosterone concentration might be related to anti-stress and anti-depressant properties of Rhodiola rosea extract. Certainly, further investigations are required to elucidate the mechanisms underlying the anti-cortisone effects of Rhodiola rosea extract

Discussion

Results of this study provide convincing evidence that the FDE R.rosea possess significantly superior pharmacological effects when compared to the SDE, although both phytomedicines were standardized to 3% rosavins and 0.8% salidroside. The FDE was much more efficient in its reducing exhaustion time and in the recovery processes after exhausting workload than spray-dried extract, which indicates that in addition to rosavins, the freeze dried extract contain other constituents that contributes to its superior pharmacological effects. The spray drying process, which includes treatment of liquid, extracts droplets with 300-400oF may cause significant impact on phytochemical profile in plant extracts. Although HPLC chromatograms of both extracts seems looks not very different each from other, but based on pharmacological effects on animals body it is become clear that there some constituents in freeze dried R.rosea extract that together with rosavins contributes to its superior pharmacological. Yet, these potential compounds most likely are heat-not stable and degrade during spray drying process.

The complex of rosavins with other essential compounds might be responsible for the observed effects in this study. For example highly biological active terpenes and aroma volatiles been isolated and extensively characterized in R. rosea (Rohloff 2002). The dried rhizomes contained rosiridin and rosiridol, essential oil with the main chemical classes are monoterpene hydrocarbons, monoterpene alcohols and straight chain aliphatic alcohols. n-Decanolm geraniol and 1,4-p-menthadien-7-ol were the most abundant volatiles detected in the essential oil, and a total of 86 compounds were identified in both the SD and HS-SPME samples. Geraniol was identified as the most important rose-like odour compound besides geranyl formate, geranyl acetate, benzyl alcohol and phenylethyl alcohol (Rohloff 2002). At this moment it is not clear how drying method affects these compounds. Furthermore, we don’t know whether these compounds are responsible factors that contributes to superior pharmacological activity of freeze dried extract over spray-dried phytomedicine.

The results of our study provide strong evidence that besides analysis of rosavins and salidroside content, the method of drying which apparently affects the pharmacological properties of R. rosea preparations, should be strongly considered.

Our research on the effects of R. rosea on animals performing maximum physical workloads has confirmed the same pattern of improved performance demonstrated in animal studies by other authors (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, our results indicate that the freeze-dried R. rosea root extract possess pharmacological properties unique to this plant species. Our results as well as of other researchers also suggest that the complex of rosavins and other heat-degradable compounds might be responsible for the beneficial effects observed in this study.

On the other hand, Rhodiola rosea liquid tincture ingestion produces an ergogenic effect during prolonged endurance exercise. According to Saratikov and Krasnov (1987), the R.rosea liquid tincture elevated the catecholamine concentration that stimulated fat metabolism, either by the increase of adipose tissue and/or muscle triglycerides lipolysis. Plasma FFA were elevated in FDE and SDE treated groups and also in some other previously reported studies ().The enhanced fat metabolism may subsequently reduce muscle glycogenolysis during exercise and may delay exhaustion in prolonged endurance exercise (Essig et al. 1980 ). A significant increase in the swimming capacity was observed in FDE treated group, when the serum FFA concentration was significantly increased compare with SDE and control animals. We suggest that the FDE-induced increase in serum FFA may also increase their utilization in the muscle; therefore this may be advantageous to increase endurance in mice. Increased fat oxidation prolongs exercise performance in mice.

The increased serum FFA may inhibit the muscle glycolysis during the early period of exercise and stimulate FFA uptake by muscles, suggesting that the spared glycogen is available during the later stage of exercise, resulting in a prolonged time to exhaustion. The present findings showed that swimming capacity is increased significantly in FDE treated group compare with control and SDE, indicating the possible involvement some other constituents in FDE that those compounds are missing in the SDE.

The present findings suggest that FDE may be used as a nutritional aid to enhance the exercise capacity in humans. Detailed studies on phytochemical characteristics of FDE and SPE are underway. Elevated plasma FFA may results in the sparing of muscle glycogen due to enhancement of fatty acid utilization (Kim et al. 1997 ).

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Figure 1. Typical HPLC fingerprint of Siberian Rhodiola rosea root extract

Figure 2. Effect of the freeze-dried and spray dried Rhodiola rosea root extract (50mg/kg body weight) on the swimming time to exhaustion, and the recovery after 30-minutes rest.

Fig 2. The effect of FDE and SDE Rhodiola rosea on swimming capacity in rats after exhaustion swimming exercise and 30 minutes rest . Values are means ± SEM, n = 10 or 11. * Significantly different from placebo, P < 0.05.

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