검색 상세

Characterization and Purification of β−secretase Inhibitory Peptide from Sea Cucumber ( Holothuria spinifera ) Enzymatic Hydrolysate

해삼 가수분해물 유래의 β−secretase 저해활성 펩타이드 정제 및 특성

초록/요약 도움말

Alzheimer’s disease (AD) is widely known as a progressive neurodegenerative disorder, mainly found in the elderly population. Progressive neurodegeneration in AD leads to cognitive functional and behavioural alterations while affecting public health. In this study, we focused on the Amyloid cascade hypothesis, where enzymatic cleavage of amyloid precursor protein (APP) by β-secretase and the formation of amyloid-beta (Aβ) peptides lead to neuron cell degradation. Sea cucumber and their dried products were delicacies mainly in China and most Asian countries over many centuries. After harvesting, holothurians are converted into dried form termed as “bêche-de-mer” to prevent the autolysis. Our study we investigated the bioactivity of dried Holothuria spinifera enzymatic hydrolysates prepared by alcalase, α-chymotrypsin, neutrase, papain and trypsin at their optimum conditions. Dried H. spinifera body wall is composed of 79.77% protein, 14.80% carbohydrates,2.96% ash, 1.69% lipid and 0.78% moisture. Glycine was the most abundant amino acid present followed by glutamic acid, alanine and aspartic acid at rates of 178.89, 140.89, 94.07 and 93.14 g/kg. Trypsin hydrolysate of dried H. spinifera exhibited significantly high β-secretase inhibitory activity (IC50 81.94 µg/mL) after 6 h of hydrolysis. By precipitating polysaccharides, trypsin hydrolysate (IC50 703.53 µg/mL) was further purified on solid-phase by ion-exchange chromatography (IEC) and reverse-phase HPLC. The SPE-F2 obtained from solid-phase extraction (SPE) exhibited the highest β-secretase inhibitory activity with an IC50 value of 302.10 µg/mL. Further purification on RP-HPLC produced active fraction (HPLC-F3) with IC50, 116.61 µg/mL. Subsequently, four peptides were identified from the HPLC-F3 as YPIEHGIVTNWDDM*EK, IEELEEEIEAER, EYVEETTGDEYVSLK, YPIEHGIVTNWDDMEK. Predominant peptide presented in HPLC-F3 was IEELEEEIEAER. Further, the effect of HPLC-F3 (2.5, 5, 12.5, 25, 37.5, 50 µg/ mL) was tested in a cellular level in SH-SY5Y neuroblastoma cells. HPLC-F3 with bioactive peptides reduced cellular level β-secretase enzyme, Aβ and soluble amyloid precursor protein beta (sAPPβ) protein levels. Besides, bioactive fraction protected SH-SY5Y cells from the Aβ induced oxidative stress and neuron cell apoptosis by decreasing the phosphorylation level of c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (p-38 MAPK). Thus, the study suggests H. spinifera as a potential source in functional foods and biomedicine industries.

more

목차 도움말

Table of Contents
List of Tables iii
List of Figures iv
Abstract 1
1. Introduction 3
2. Materials and methods 7
2.1 Materials 7
2.2 Sample preparation 7
2.3 Proximate analysis of dried H. spinifera body wall 8
2.4 Amino acid analysis 8
2.5 Enzymatic hydrolysis of H. spinifera body wall……………9
2.6 Measurement of β-secretase inhibitory activity 12
2.7 Purification on an ion-exchange column 13
2.8 Purification on reversed-phase HPLC 13
2.9 Identification of β-secretase inhibitory peptide sequence (LC-MS/MS) 14
2.10 Cell culture and differentiation 14
2.11 MTT assay 15
2.12 Western blot analysis 15
2.13 Statistical analysis 16
3. Results and discussion 17
3.1 Proximate composition of dried sea cucumber 17
3.2 Amino acid composition 19
3.3 Enzymatic hydrolysis of H. spinifera body wall 21
3.4 β-secretase inhibitory activity of hydrolysates 23
3.5 Purification on an ion-exchange column 27
3.6 Purification on reversed-phase HPLC 27
3.7 Identification of β-secretase inhibitory peptide sequence 30
3.8 Cell culture and differentiation 38
3.9 MTT assay 41
3.10 Western blot analysis 41
4. Conclusion 45
5. Abbreviations 46
6. References 48
7. Acknowledgement 55

more
반출 Meta View 목록