Sleep-inducing activity of water extract and 4-Methyl-2,6-di-tert-butylphenol from blue mussel Mytilus edulis
- Alternative Title
- Mytilus edulis 열수추출물 및 4-Methyl-2,6-di-tert-butylphenol의 수면증진 효과
- Abstract
- Recently, a great deal of interest has been paid by the consumers towards bioactive compounds from natural resources as functional ingredients in nutraceuticals, cosmeceuticals, and pharmaceuticals. Natural sleep aids, which contain specific constituents of foodsand herbal plants, have recently become popular as alternatives sedative–hypnotics to improve sleep quality without side effects. Herbal plants have been use in sleep deprivation treatment for more than 2000 years in China. However, although a large number of studies on hypnotic effects of plant constituents have been performed, fishery products have not been recognized as a potential source of natural hypnotics. Among of them, shellfish have long been a part of the daily diet and have been used in traditional medicine. Blue mussel (ME, Mytilus edulis) is the popular shellfish that is used widely as daily diet. In addition, blue mussel’s peptide was reported that have diverse biological activities, such as antioxidant, antibacterial, anticoagulant, and angiotensin I converting enzyme inhibition.
ME extract was prepared by hot water extraction and filtration though an ultrafiltration (UF) membrane with molecular weight cut off (MWCO) 1 kDa. ME extract was fractionated with n-hexane, ethylacetate, n-butanol, and distilled water. The highest sleep-promoting fraction was chosen for compound isolation. By using chromatography, we isolated 4-Methyl-2,6-di-tert-butylphenol (BHT) from blue mussel water extract. The structure of the above isolated compound was determined by 1H, 13C, HMQC, HMBC, and MS spectroscopic data.
The hypnotic effect of ME extract and BHT were evaluated by using pentobarbital-induced sleep test, electroencephalogram (EEG) and electromyogram (EMG) in mouse model. ME extract, and BHT reduced sleep latency and induced sleep period in mice. ME extract also induced rate of sleep onset and sleep duration in sub-hypnotic dose test. sub-hypnotic dose test result confirmed that ME extract has sleep-induced effect in mice. EEG and electromyogram EMG results showed that ME extract reduced sleep latency, induced non-rapid eye movement sleep (NREMS) without change in rapid eye movement sleep (REMS). Delta activity of ME extract administrated groups did not have significant change compare with vehicle group. This result demonstrated that ME extract induces sleep similar to physiological condition. Furthermore, the hypnotic effect was tested with administration of flumazenil, a GABA receptor antagonist. The result showed that flumazenil, a GABAA receptor inhibitor, inhibited sleep-inducing effect of ME extracts. Therefore, the result demonstrated that ME extracts and induced sleep via the GABAergic system in mice. In conclusion, ME extract and its component might induce sleep in mice through GABAergic pathway.
- Issued Date
- 2014
- Awarded Date
- 2014. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권, 김학준
- Table Of Contents
- Abstract i
List of Tables vi
List of Figures vii
List of Abbreviations ix
Chapter 1 1
General Information 1
1. Introduction for Insomnia 2
2. Sleep structure and function 3
3. Role of GABA in sleep induction and maintenance 7
4. Current treatment methods and their limitation 9
5. Scope of the study 11
Chapter 2 13
Effects of blue mussel (Mytilus edulis) water extracts on pentobarbital-induced sleep and changes in sleep architecture. 13
1. INTRODUCTION 14
2. MATERIALS AND METHODS 22
2.1. Drugs 22
2.2. Animals 22
2.3. Pentobarbital-induced sleep test 22
2.4. Analysis of sleep architecture and profile 24
2.5. Statistical analysis 27
3. RESULTS 28
3.1. Screening for hypnotic activity 28
3.2. Effects of ME extract on pentobarbital-induced sleep in mice 28
3.3. Effect of ME extract onsub-hypnotic dose of pentobarbital-induced sleep in mice 30
3.4. Effects of ME extract on sleep latency and the amounts of NREMS and REMS 33
3.5. Effects of ME extract on the time spent in each sleep stage 33
3.6. Effects of ME extract on the mean duration of each sleep stage and power density in NREMS 37
4. DISCUSSION 40
Chapter 3 42
Isolation of bioactive compounds from blue mussel Mytilus edulis hot water extract and mechanism of compound on sleep induction. 42
1. INTRODUCTION 43
2. MATERIALS AND METHODS 43
2.1. Intruments, Chemicals and reagents 43
2.2. Drugs 44
2.3. Preparation of blue mussel hot water extract 44
2.4. Animals 44
2.5. Pentobarbital-induced sleep test 45
2.6. Isolation, and structure elucidation of bioactive compuonds from Mytilus edulis. 45
2.7. Binding affinity of BHT 46
2.8. Cell culture and cytotoxicity determination 46
2.9. Western blot analysis 47
2.10. Determination of BHT in ME extract 48
2.11. Binding assay of BHT on GABA receptor 49
2.12. Statistical analysis 49
3. RESULTS 50
3.1. Extraction of bioactive compounds from Mytilus edulis. 50
3.2. Bioactive compound from Mytilus edulis. 53
3.3. Spectroscopic properties of compounds isolated from Mytilus edulis. 53
3.4. Spectroscopic properties of compounds isolated from Blue mussel Mytidus edulis. 56
3.5. Effect of 4-Methyl-2,6-di-tert-butylphenol on pentobarbital-induced sleep test. 62
3.6. Verification of the in vivo GABAergic mechanism of ME extract 64
3.7. Verification of the in vivo GABAergic mechanism of hexane fraction 66
3.8. Verification of the in vivo GABAergic mechanism of BHT 67
3.10. Effect of of GABA and BHT on cytokine in Normal Human Astrocytes (NHA) 76
3.11. Binding of BHT on GABA receptor in NHA 76
3.12. Concentration of BHT in ME extract 77
4. DISCUSSIONS AND CONCLUSION 81
SUMMARY 87
References 89
Acknowledgments 103
- Degree
- Doctor
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