Poria cocos Ethanol Extract Enhances Pentobarbital- Induced Sleeping Behaviors via GABAA-ergic Systems : 복령 추출물의 GABAA-ergic 시스템을 통한 Pentobarbital 유도 수면 증가 효과

국문 초록 (Abstract)

불면증의 효과적인 치료를 위한 다양한 약물학적 시도를 수행하고 있다. 복령은 pachymic acid (PA)를 주성분으로 수면유도 효과가 있으며 pentobarbital 유도 수면 행동을 증가시킨다. 복령 추출물 및 PA 를 경구투여하고 자발운동을 억제하는데 이는 GABA 수용체에서의 benzodiazepine 결합부위에 관한 상호작용과 관련이 있다는 것을 추정하였다. 이는 중추에서 pentobarbital 과 GABA system 을 통한 작용으로 생각된다. 또한 복령 추출물과 PA 는 1 차 배양 시상하부 신경세포에서 세포 내 연소이온을 증가시켰다. 복령추출물이나 PA 가 GABA 의 생합성을 통하여 GABA 수용체를 활성화 활 수 있다. 따라서, 복령추출물과 PA 가 GAD 의 활성을 신경세포에 8 일간 배양하여
측정한 결과 GAD 를 증가시키는 것을 확인하였다. 더 나아가,
GABAA 수용체의 아형 (알파, 베타, 감마)의 과발현을 조사하였으며, 이들 알파 및 감마 아형은 아형의 단백질이 과발현하였으며, 감바는 다소 억제되었다. 이와 같은 결과를 바탕으로 복령 추출물과 PA 는 GABA 수용체 및 염소이온의 세포내 증가로 인한 억제성을 증가로 수면을 유도하는 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Poria cocos (Hoelen), a Chinese herbal medicine (CHM) has been commonly used for treating insomnia in Asian countries for centuries. However, its claimed benefits and mechanism are not fully understood. The present study aimed to investigate whether the herb ethanol extract (PCE) and its active constituent pachymic acid (PA), possesses hypnotic effects and/or enhances pentobarbital-induced sleeping behaviors. In addition, the mediation of γ-aminobutyric acid (GABA)-ergic systems were defined to understand the possible mechanism. Oral administration of PCE and PA inhibited the locomotion activity in mice. Moreover, both the samples prolonged pentobarbital-induced sleep time, and shorten sleep latency at the hypnotic (40 mg/kg) dose of pentobarbital in mice. Interestingly, it was observed that both PCE and PA increased the number of sleeping mice and total sleeping times, showing synergistic effects similar to muscimol (0.2 mg/kg) at the sub-hypnotic (28 mg/kg) dose of pentobarbital in experimental animals. Notably, the extract and PA raised intracellular chloride levels in primary cultured hypothalamic neuronal cells. PCE and PA increased glutamic acid decarboxylase (GAD65/67) protein levels by western blot. Furthermore, α-, β-, and γ-subunits of GABAA receptors were remarkably over-expressed by PCE treatment. However, the expression studies showed that α-, and β-subunits were remarkably increased by PA, but decreased γ-subunit protein levels in GABAA receptors. To sum up, the data suggest that the herb ethanol extract and its major constituent PA may be useful for the treatment of insomnia via GABAA-ergic mechanisms.

목차 (Table of Contents)

• I. Introduction 1
• 1. Traditional Chinese Medicine 1
• 2. Poria cocos 1
• 3. Insomnia 5
• 4. g-aminobutyric acid 6
• 5. Hypothalamic and cerebellar granule cells 9
• 6. Pentobarbital-induced sleep 10
• 7. Purpose of the study 11
• II. Material and method 13
• 1. Preparation of crude extract 13
• 2. Reagents and chemicals 14
• 3. Animals 14
• 4. Locomotor activity 15
• 5. Pentobarbital-induced sleep 16
• 6. Primary cell culture 17
• 7. Measurement of intracellular Cl influx 18
• 8. Expression of GAD65/67 and GABAA receptors subunits 19
• 9. Statistical analysis 21
• III. Results 22
• 1. Characterization of PCE and PA by HPLC 22
• 2. Suppression of locomotor activity in mice by PCE and PA 24
• 3. Improvement on the onset and duration of sleep in mice by PCE 27
• 4. Improvement on the onset and duration of sleep in mice by PA 30
• 5. Effects of PCE on sleep onset of mice treated by sub-hypnotic dosage of pentobarbital 33
• 6. Effects of PA on sleep onset of mice treated by sub-hypnotic dosage of pentobarbital 35
• 7. Increased intracellular chloride ion influx in primary cultured hypothalamic neuron cells by PCE and PA 37
• 8. Modulation of GAD65/67 expression by PCE and PA 40
• 9. Modulation of GABAA receptors subunits expression by PCE 43
• 10. Modulation of GABAA receptors subunits expression by PA 46
• IV. Disscussion 49
• V. References 57
• VI. Summary in Korean 66
• Acknowledgment 67