Sleep and Biological Rhythms 2009; 7: 78–83

ORIGINAL ARTICLE

sbr_391

doi:10.1111/j.1479-8425.2009.00391.x

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Effects of red ginseng extract on sleep architecture and electroencephalogram power spectra in rats Yuan MA,1 Yun B KIM,1 Sang Y NAM,1 Jae-Hoon CHEONG,2 Se H PARK,3 Hae J KIM,3 Jin T HONG4 and Ki-Wan OH4 1

Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, 2College of Pharmacy, Sahmyook University, Seoul, 3Dong-Won Ginseng Co. Ltd., Jincheon, and 4College of Pharmacy, Chungbuk National University, Cheongju, Seoul, Korea

Abstract We evaluated the ability of the ethanol extract of red ginseng (RGE) to regulate sleep architecture. Adult rats were chronically fitted with sleep–wake recording electrodes. Following post-surgical recovery, rats were habituated extensively to freely moving polygraphic recording conditions. Polygraphic signs of undisturbed sleep–wake activities were recorded for 12 h after RGE administration. Ginseng treatment produced more time in non-rapid eye movement (NREM) sleep and total sleep. The total percentage of wakefulness decreased comparably, and the number of sleep–wake cycles was reduced after 10 and 50 mg/kg RGE. RGE (10 mg/kg) administration decreased the power density of cortical electroencephalogram (EEG) d-waves (0.75–4.5 Hz) and increased a-waves (8.0–13.0 Hz) in NREM and rapid eye movement (REM) sleep. It also decreased d-wave power density during wakefulness. Although 100 mg/kg RGE showed little effect on the power densities in NREM and REM sleep, it increased d-wave and decreased q-wave (5.0–9.0 Hz) power densities during wakefulness. Thus, RGE increases spontaneous sleep and NREM sleep. Regulation of sleep architecture by RGE involves decreased d- and q-wave and increased a-wave activities according to cortical EEG. Key words: electroencephalogram (EEG), non-rapid eye movement (NREM), power density, rapid eye movement (REM), red ginseng extract (RGE).

INTRODUCTION The regulation of sleep is a homeostatic process, where control mechanisms are activated to compensate for insufficient and excess sleep.1 In mammals, sleep consists of two major stages, rapid eye movement (REM) sleep and non-REM (NREM) sleep. REM sleep alternates with episodes of NREM sleep, and the spontaneous NREM–REM sleep cycle takes about 12 to 20 min in rats.2 Over the past four decades, most sleep research Correspondence: Dr Ki-Wan Oh, College of Pharmacy, Chungbuk National University, Cheongju. 361-763, Seoul, Korea. Email: [email protected] Accepted for publication 2 March 2009 Published online 6 May 2009.

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has focused on identifying relevant brain structures, neuronal networks, and transmitters involved in the generation and regulation of NREM and REM sleep.3 Little is known about the mechanisms and modulation for the periodic occurrence of NREM–REM sleep. Ginseng, the root of Panax species, has been used as a traditional medicine for thousands of years. People take ginseng to boost energy and sharpen the mind. Ginseng has a complex activity profile that is sometimes difficult to reconcile with neurochemical reports. Ginseng stabilizes and balances physiology, and may help maintain normal sleep and wakefulness.4 Ginseng has been clinically used for the treatment of insomnia.5 Most the benefits of ginseng are direct consequences of ginsenosides, the main active ingredients in ginseng

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root. Ginsenosides affect metabolism, including increasing vitality, boosting mental efficiency, and promoting greater stamina and endurance, thereby preventing tiredness and the debilitating effects of old age. Ginsenosides work as adaptogen to help the body adapt to biological stress in a natural way. Korea red ginseng extract (RGE) is 100% organically grown and contains a richer blend of minerals, vitamins, essential oils, amino acids, and enzymes than other ginsengs. Due to the location of the Korean Peninsula (latitude 36–38N), Korea’s soil and climate are the most suitable for the cultivation of ginseng. Korea ginseng roots, which take 6 years to mature, contain more active elements than any other ginseng root. The soil where Korea ginseng is cultivated requires special care, demanding an interval of at least 10 years between harvests.6 Together with its health-promoting effects, ginseng enhances brain activity and promotes psychological stability by stimulating and tranquilizing the mind. Korea ginseng extract contains three times more ginsenosides than any other ginseng, making it the most effective of all ginsengs.6 Thus, Korea RGE is an unparalleled natural source of nutrients for a healthy body and mind. However, sleep disorders are the most commonly experienced adverse effects of ginseng.7 To assess the role of ginseng in regulating sleep–wake fluctuations and sleep architecture and to determine the possible mechanisms of NREM–REM sleep modulation, we examined its effect on the amount of total sleep and wakefulness, and selectively investigated power density changes in recorded electroencephalograms (EEG) of specific sleep–wake stages in freely moving rats.

METHODS Materials Six-year-old red ginseng roots were purchased from a Korea ginseng market. RGE (100 g) was extracted three times with 1000 ml 70% ethanol for 4 h under reflux at room temperature. This extract was subjected to filtration and concentration. Finally, the ethanol extract of RGE was freeze-dried, followed by evaporation of the remaining supernatant, and used as the test sample.

Animals Experiments were performed on 32 adult male Wistar rats (Samtako, Osan, Korea) weighing between 250 and 350 g. The rats were housed individually with food and

© 2009 The Authors Journal compilation © 2009 Japanese Society of Sleep Research

water provided ad libitum under an artificial 12-h light/ dark cycle (lights on at 07.00 hours) and at a constant temperature (22 ⫾ 2°C). The rats were housed in the departmental holding room for 1 week before testing. All rats were maintained in accordance with the National Institute of Toxicological Research and the Korea Food and Drug Administration guidelines for the care and use of laboratory animals.

Surgery The animals were divided into four groups (control; RGE 10, 50, and 100 mg/kg groups) with eight rats in each. Each rat was implanted with a transmitter (TA11CTA-F40; Data Sciences International, St. Paul, MN, USA) for recording EEG and activity via telemetry as described previously.8 The body of transmitter was implanted subcutaneously off the midline and posterior to the scapula. It was attached to the skin with three sutures for stabilization. The transmitters were led subcutaneously to the skull and the bare ends placed in contact with the dura through holes in the skull (A: 2.0 [Bregma], L: 1.5; P: 7.0 [Bregma], L: 1.5 contra-lateral). The electrodes were anchored to the skull with screws and dental cement. All surgical procedures were performed stereotaxically under aseptic conditions. Surgical anesthesia was achieved with pentobarbital (50 mg/ kg, i.p.).

Data collection Following 7 days of post-surgical recovery, telemetric recording of cortical EEG and activity were conducted using procedures similar to previous reports.8 For the EEG signal, the gain of transmitters was set at -0.5/+0.5 volts per/units ¥ 2, and raw signals generated from the transmitter were in the range of 0.5–20.0 Hz. The signals were processed by a Data Sciences analog converter and routed to an AD converter (Eagle PC30, Data Sciences International, St. Paul, MN, USA) housed in a PC-class computer. The AD converter digitized the EEG and activity signals at 128 Hz. The digitized data were transferred to the computer and displayed graphically by the program on the computer monitor. An online fast Fourier transformation (FFT) was performed on EEG data every 2 s (256 samples) after a Hanning window treatment. The FFT analyses generated power density values from 0.0 to 20.0 Hz at a resolution of 0.5 Hz. The FFT data were further averaged in the range of 0 to 20 Hz every 10 s. The sleep data and FFT results were

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saved to the hard disk every 10 s for additional off-line analyses. Movement of the animal in relation to the telemetry receiver generated transistor-transistor logic (TTL) pulses that were collected and counted as a measure of activity. Oral administration of RGE was performed 10 min before EEG recording, Recording began at 07.00 hours, and 12-h EEG and activity were recorded in each rat.

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Time spent (h)

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Data analysis The time spent (min) in NREM, REM, and total sleep (NREM + REM), and the number of sleep–wake cycles were processed to obtain 12-h periods for each rat. We further calculated the time of each recording spent in each sleep–wake state (wake, NREM, REM). The absolute EEG power during wakefulness, NREM, and REM were calculated in 0.5 Hz bins from 0.5 to 20 Hz for the entire 12-h recording. Afterwards, EEG power density in three selected frequency bands for wakefulness, NREM, and REM (d-wave [0.75–4.5 Hz], q-wave [5.0– 9.0 Hz], and a-wave [8.0–13.0 Hz]) were evaluated.

Statistical analysis All statistical analyses were conducted using SigmaStat software (SPSS, Chicago, IL, USA). One-way ANOVA with post-hoc Dunnett’s tests was used.

RESULTS Effects of RGE on sleep architecture RGE did not change REM sleep during the 12-h recording, but did change the amount of wakefulness, NREM, and total sleep. RGE (10 and 50 mg/kg) induced a significant decrease in total time awake and an increase

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Determination of behavioral states and analysis in EEG power

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The amount of time in wakefulness, NREM sleep, and REM sleep were determined from digitized data in 10-s epochs using the animal sleep analysis software SleepSign 2.1 (Kissei Comtec, Matsumoto, Japan). Briefly, the software identifies wakefulness as a high-frequency, lowamplitude EEG and NREM by the presence of spindles interspersed with slow waves. EEG power during REM is significantly reduced to lower-frequency d-waves (0.75–4.0 Hz) and increased q-wave activity (5.0– 9.0 Hz, peak at 7.5 Hz).

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Figure 1 Effects of red ginseng extract (RGE) on sleep architecture. The data represent the mean ⫾ SE of time spent in the sleep–wake state (wake, sleep [total sleep], NREM [nonrapid eye movement] sleep, REM sleep) and number of sleep/wake changes. (a) Time spent in sleep–wake state; (b) number of sleep/wake changes. *P < 0.05, significantly different from control.

in NREM and total sleep. Similarly, RGE 100 mg/kg decreased total wake time and increased total sleep time, and tended to increase NREM and REM sleep (P > 0.05) (Fig. 1A). RGE (10 and 50 mg/kg but not 100 mg/kg) reduced the number of sleep–wake cycles during the 12-h recording time (Fig. 1B).

Effects of RGE on EEG power density during NREM sleep During NREM sleep, the rats had reduced d-wave, increased a-wave, and similar q-wave power after RGE

© 2009 The Authors Journal compilation © 2009 Japanese Society of Sleep Research

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(10 mg/kg) administration. RGE (50 mg/kg) administration increased the power density of a-waves only, and KRG (100 mg/kg) did not change EEG power density (Fig. 2).

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Effects of RGE on EEG power density during REM sleep

Effects of RGE on EEG power density during wake time RGE (10, 50, and 100 mg/kg) did not change EEG a-wave power density (P > 0.05). Low and high doses of RGE showed different effects on d- and q-wave power densities: RGE 10 mg/kg significantly decreased d-wave power density without influencing q-wave power density (P > 0.05), RGE 50 mg/kg did not change d- or q-wave power densities (P > 0.05), and RGE 100 mg/kg increased d-wave but decreased q-wave power densities during wakefulness (Fig. 2).

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20 10 0 δ-wave Percentage of power density (%) during each stage

During REM sleep, RGE 10 mg/kg significantly decreased d-wave, and increased a-wave power density, without influencing q-wave power density (P > 0.05). RGE (50 and 100 mg/kg) did not induce changes in EEG power density (P > 0.05) (see Fig. 2).

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DISCUSSION RGE increased total and NREM sleep and decreased wakefulness and the number of sleep–wake cycles. Panax ginseng improves general physical and mental wellbeing, with sleep-stabilizing and balancing effects. Our results indicate that ginseng improves sleep, especially NREM sleep.9,10 Total sleep amount and frequency depends on the activity patterns of cortical EEG waves.10 NREM sleep, or slow wave sleep, is deep sleep and is defined by an oscillation of the EEG in the d-wave frequency range (<2 Hz in humans, 0.75 to 4.0 Hz range in mice).11 EEG activities in the d-wave frequency represent widespread synchronized firing patterns of cortical and thalamocortical neurons.11 Sleep deprivation, or forced wakefulness, induces a large increase in delta power.12 This mechanism of sleep has an essential role for rest and maintenance of neural function, and the regulation of d-wave activity in sleep is linked with synaptic potentiation and downscaling.12 Local increases in d-wave activity in sleep after a motor learning task correlate

© 2009 The Authors Journal compilation © 2009 Japanese Society of Sleep Research

**

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Figure 2 Effects of red ginseng extract (RGE) on electroencephalogram (EEG) power density during sleep and wake stages. EEG power density in d-wave, q-wave, and a-wave were evaluated. The data represent the mean ⫾ SE of EEG power densities in three selected frequency bands in the non-rapid eye movement (NREM), REM, and wakefulness stages. *P < 0.05; **P < 0.01, significantly different from control.

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with improved performance in humans.12 The power of d-wave activity is a reliable parameter for assessing sleep depth and regulation.13 d-Wave activity relates to prior waking and is a reliable indicator of time spent awake.14 The intensity of d-wave activity in cortical EEG is the single most important process for the regulation of NREM sleep, declining over the course of a daily sleep period, and increasing in recovery sleep after a period of prolonged wakefulness.15,16 Furthermore, d-wave activity is reduced in NREM sleep after a nap and/or excess sleep.16 However, in our results, RGE increased NREM sleep with decreased d-wave activity. Therefore, RGE modulates sleep architecture like a nap or excess sleep. Unlike NREM sleep, the EEG correlation for REM sleep regulation remains poorly understood. In humans, the EEG a-wave activity (frequency range of 8–13 Hz) might be a marker of REM sleep regulation.17 However, in rats, a-wave activity may not be involved in REM sleep, but d-wave activity instead.18 Preliminary analyses on the dose-response relationship of RGE on sleep indicated effective doses. RGE (10 mg/kg) decreased d-wave and increased a-wave activity in both NREM and REM sleep, and significantly increased NREM and total sleep. RGE (50 mg/kg) increased the power density of a-wave activity on NREM sleep, and also increased NREM and total sleep without changing REM sleep. RGE (100 mg/kg) did not change EEG power densities of d-, q-, or a-waves and only increased total sleep. Thus, low-dose (10 mg/kg) and high-dose (100 mg/kg) RGE affected d-wave and q-wave power densities differently. Lower doses of RGE are more effective at modulating sleep and specifically affected NREM sleep. Oral administration of RGE powder before transient forebrain ischemia prevented the occurrence of ischemia-induced learning disabilities and hippocampal neuron loss.19 Ginsenosides, such as Rb1, like 21aminosteroids, are centrally acting neuro-protective agents as potent as peptide growth factors.20 They protect neurons against lethal ischemic damage by scavenging neurotoxic hydroxyl radicals that are overproduced in cases of brain ischemia and reperfusion.20 Ginsenosides modulate ligand binding to gaminobutyric acid (GABA)A and GABAB receptors.21 Ginsenosides also inhibit the development of reverse tolerance and psychological dependence induced by methamphetamine and cocaine.22 Ginseng simultaneously inhibits analgesic tolerance and morphine dependence, and inhibits hyperactivity, reverse tolerance, and conditioned place preference induced by psychostimulants.23

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RGE administration in humans causes vasodilatation and slight body temperature elevation. Thus, ginsenosides may also protect neurons by modulating temperature and/or blood flow before and after ischemic insult.24 RGE contains three times more ginsenosides than any other ginseng.6 The ginsenosides in RGE modulated sleep behavior by modulating neuronal function. In summary, RGE increases spontaneous sleep and NREM sleep and decreases the number of sleep-wake cycles. Regulation of sleep architecture by RGE involves decreased d- and increased a-wave activities in cortical EEG. RGE could contain multiple active compounds that modulate sleep, but further study is required to clarify this possibility.

ACKNOWLEDGMENT This work was supported by a 2008 IndustryUniversity-Institute joint research and development project funded by the Small and Medium Business Administration.

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