발간등록번호 전복소화분해시스템을활용한천연항알러지제품개발
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1 발간등록번호 전복소화분해시스템을활용한천연항알러지제품개발
2 : : : : : : 17 1
3 Ⅰ. 제목 : 전복소화분해시스템을활용한천연항알러지제품개발 (Development of a natural anti-allergic product using abalone-gastrointestinal digestion system) Ⅱ. 연구개발의목적및필요성 2
4 3
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6 Ⅲ. 연구개발내용및범위 5
7 Ⅳ. 연구개발결과 6
8 7
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10 Ⅴ. 연구성과및성과활용계획 9
11 10
12 11
13 Development of a natural anti-allergic product using abalone-gastrointestinal digestion system Summary u Abalone is a marine gastropod and an important fishery and food industrial resource that is massively maricultured in Asia, Africa, Australia, and America. Pacific abalone, H.discus hannai has been massively maricultured from the 1990s in the southwestern coast of South Korea. To meet the increasing demand of the Asian market, abalone mariculture has been expanding in land- and sea-based systems in S. Korea and the total yield was estimated at 7,580 tons of abalone in 2009 (The Korea Marine Institute). In addition, manufacturing products of abalone (dried, steamed, spiced abalone, and so on) have also been significantly increased. It is currently agreed that that marine organisms possess various bioactive natural components with many nutraceutical and pharmaceutical activities-related physiological functions such as antioxidant, anti-inflammatory, anti-allergic, antitumor, antimicrobial, antihypertension, anticoagulation, anti-cardiovascular disease, and etc. However, the health beneficial effects of abalone have rarely been reported, although a number of studies have been performed on the biological and physiological properties of abalones, considering pathology, genetics, and aquaculture technology. u Abalone intestine gastro-intestinal digests (AIGIDs) containing AIGID I ( kda), AIGID II (10-5 kda) and AIGID III (5-1 kda) were isolated from abalone gastro-intestinal digestion by an ultrafiltration membrane system. In vitro gastro-intestinal digestion which is consisted of two phase: gastric digestion (phase I) corresponding to a pepsin-hydrolysis, and intestinal digestion (phase II) corresponding to proteolysis by two enzymes (trypsin and alpha-chymotrypsin), was used for breakdown proteins into amino acids or oligopeptides with functional and nutritional properties. After treatment with proteases, hydrolysates were composed of individual amino acids and low molecular mass peptides. Advantageously, these products can withstand the conditions of physiological digestion and arrive at the blood stream to employ their bioactive effects after oral intake. 12
14 u First, we examined that antioxidant and anti-inflammatory effects of abalone Haliotis discus hannai in macrophage cells. The results exhibited that abalone intestine digest (AID) has higher antioxidant activities against lipid peroxidation, ROS stress and DNA damage in H 2 O 2 -treated RAW264.7 macrophages. In the lipopolysaccharide (LPS)-induced macrophages, AID suppresses LPS-induced production of nitric oxide (NO) via inducible nitric oxide synthase (inos) expression in a dose-dependent manner. It also significantly reduced the generation of proinflammatory cytokines, such as interleukin (IL-1β), tumor necrosis factor (TNF)-α, and IL-6. Furthermore, AID significantly suppresses phosphorylation of mitogen-activated protein kinases (MAPKs) such as ERK, JNK, and p38. These results indicated that AID inhibits oxidative damage by ROS and LPS-induced inflammatory response via blocking of MAPK signaling pathway in murine macrophages. Therefore, potent antioxidant and anti-inflammatory effects of abalone intestine as byproducts from fishery manufacturing might suggest possibility for high valuable utilization and application as nutraceutical and therapeutic substances. u To evaluate beneficial effects of H. discus hannai, an anti-inflammatory peptide (AAIP, abalone anti-inflammatory peptide) was purified from abalone intestines using consecutive HPLC purification system. In tandem MS analysis, the fragmentation results illustrate that the AAIP responsible for the nitric oxide (NO) inhibitory activity (IC 50 =55.8uM) has amino acid sequence as Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser (1175.2Da). To investigate anti-inflammatory effect of AAIP on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and elucidated the molecular mechanism. The results show that the AAIP peptide suppresses LPS-induced production of nitricoxide (NO) via inducible nitricoxide synthase (inos) expression in a dose-dependent manner. It also significantly reduced the gene transcription of proinflammatory cytokines, such as interleukin(il-1β), tumor necrosis factor (TNF-α), and IL-6. Furthermore, AAIP significantly suppresses phosphorylation of mitogen-activated proteinkinases (MAPKs) such as p-p38 and p-jnk. These results indicated that abalone intestine-derived anti-inflammatory peptide,a AIP inhibits LPS-induced inflammatory response via blocking of MAPK pathway in murine macrophages. u Allergic diseases, such as asthma, atopic dermatitis, allergic rhinitis and food allergies, are typified by an undesirable reaction to harmless environmental allergens. Allergic diseases are characterized by infiltration and accumulation of lymphocytes, basophils, eosinophils, and mast cells to the inflamed site of lesions. Mast cells are one of the critical effecter cells in the pathogenesis of hypersensitivity and allergic responses, but 13
15 also play important roles in host defense against parasites. Mast cells are commonly found at sites exposed to the external environment, namely the skin, the airways and the gastrointestinal tract. Mast cells are recognized as granular cells of the connective tissue and key cells in autoimmune disorders, and key players in the establishment of innate immunity as well as modulators of adoptive immune responses. The aim of the present study was to examine whether abalone modulates inflammatory response, as well as to elucidate its mechanism of action in stimulated human mast cell line (HMC-1). Cells were stimulated with phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) in the presence or absence of AIGIDs. Abalone attenuated the PMACI-stimulated gene expression and production of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, and histamine releases in HMC-1 cells. In activated HMC-1 cells, phosphorylation of c-jun N-terminal kinase (JNK) decreased after pretreatment with AIGIDs. In addition, AIGIDs suppressed PMACI-induced IkB-a phosphorylation and degradation, leading to inhibition of nuclear factor (NF)-κB activation. AIGIDs suppressed the production of TNF-α, IL-6, histamine and through a decrease in the intracellular levels of JNK, as well as activation of NF-κ B. These results indicated that AIGIDs has a modulatory effect on mast cell-mediated inflammatory allergic diseases. u In addition, this study was to determine whether treatment with AIGID III (including AAIP, abalone anti-inflammatory peptide) results in significant effects in atopy dermatitis NC/Nga mouse model. In this study, we investigated the anti-atopy activity of AIGID III on the mice. This finding suggests that AIGID III might be useful candidate in treating atopy dermatitis. 14
16 Contents Chapter 1. Abstract of research and development subject Chapter 2. condition Domestic and foreign technical development present Chapter 3. Research and development contents and results 15
17 16
18 Chapter 4. Research and development results 17
19 18
20 β α κ 19
21 20
22 Chapter 5. Tagret achievement contribution about related field Chapter 6. Research and development in result and practical use plan Chapter 7. Foreign Scientific & Technologic Information collected in research and development process 21
23 Reference 22
24 제 1 장연구개발과제의개요 3 0 제 2 장국내외기술개발현황 3 9 제 3 장연구개발수행내용및결과
25 24
26 제 4 장연구개발결과
27 26
28 β α κ 27
29 제 5 장목표달성도및관련분야에의기여
30 제 6 장연구개발성과및성과활용계획 제 7 장연구개발과정에서수집한해외과학기술정보 제 8 장참고문헌
31 제 1 장 연구개발과제의개요 30
32 31
33 32
34 33
35 34
36 35
37 Phase II: Intestinal digestion Phase II: Intestinal digestion Trypsin, Trypsin, α-chymotrypsin, α-chymotrypsin, lipase, lipase, bile bile salt salt mixture, mixture, ph ph 朶 狡 ( 悛 峯 ) Marine organism 逅 Gastric? 몸 digests? 뜯 ph/thermometer 쨉 Stirrer? 넷? 償 Pump Pump 償 Concentration /? dialysis Phase Phase I: I: Gastric Gastric digestion digestion 悛삣왑穡 Pepsin, Pepsin, ph ph Enzyme 왑鳧 reactor? system Reservoir 衒嘉 Tank 욉퓌 Lyophilization Supplier Tank 疱嘉 償 Pump ph/temp 쨉랖櫻 controller 楕?? Gastro-Intestinal digests 36
38 37
39 38
40 제 2 장 국내외기술개발현황 39
41 40
42 41
43 42
44 제 3 장 연구개발수행내용및결과 43
45 44
46 μ μ μ 45
47 λ 46
48 Phase Phase II: II: Intestinal Intestinal digestion digestion Trypsin, Trypsin, α-chymotrypsin, α-chymotrypsin, lipase, bile salt mixture, ph 7.8 lipase, bile salt mixture, ph 7.8 朶 狡 ( 悛 峯 ) Marine organism 逅 Gastric? 몸 digests? 뜯 ph/thermometer 쨉 Stirrer? 넷? 償 Pump Pump 償 Concentration /? dialysis Phase Phase I: I: Gastric Gastric digestion digestion 悛삣왑穡 Pepsin, ph 2.2 Pepsin, ph 2.2 Enzyme 왑鳧 reactor? system Reservoir 衒嘉 Tank 욉퓌 Lyophilization Supplier Tank 疱嘉 償 Pump ph/temp 쨉랖櫻 controller 楕?? Gastro-Intestinal digests 47
49 α μ μ μ 48
50 49
51 μ α 50
52 μ μ μ μ μ μ 51
53 μ μ μ α μ 52
54 μ μ λ λ μ μ μ μ μ μ μ μ μ μ μ 53
55 γ 54
56 μ μ 55
57 β α β α α α 56
58 α β α α β 57
59 58
60 59
61 60
62 61
63 62
64 μ μ μ μ μ μ μ μ μ μ μ μ 63
65 64
66 (2) Bacillus subtilis,,. 65
67 .,,,, 5. 66
68 67
69 68
70 γ 69
71 70
72 71
73 α β γ γ 72
74 73
75 74
76 75
77 76
78 77
79 78
80 79
81 50 40 Polyphenols (mg/g) Muscle Intestine 80
82 30 Contents of ondroitin sulfate (%) Abalone Mussel Oyster Skate fish Sea cucumber 81
83 82
84 83
85 α 84
86 85
87 86
88 70 60 Degree of hydrolysis (%) Substrate concentration (%) Degree of hydrolysis (%) Substrate concentration (%) 87
89 80 Degree of hydrolysis (%) Substrat/Enzyme (W/W) 100 Degree of hydrolysis (%) Substrat/Enzyme (W/W) 88
90 80 70 Degree of hydrolysis (%) Time (hr) 100 Degree of hydrolysis (%) Time (hr) 89
91 90
92 β α β α 91
93 60 50 Degree of hydrolysis (DH %) A B C D E Commercial Enzymes 92
94 93
95 60 50 Degree of Hydrolysis (%) ph 70 Degree of hydrolysis (%) Temperature 94
96 70 60 Degree of hydrolysis (%) Substrate concentration (%) 80 Degree of hydrolysis (%) Substrat/Enzyme (W/W) 95
97 80 70 Degree of hydrolysis (%) Time (hr) 96
98 97
99 98
100 99
101 100
102 101
103 102
104 α 103
105 α α 104
106 105
107 μ μ μ μ μ 106
108 μ μ λ λ μ 107
109 μ μ μ μ μ μ μ μ μ μ μ 108
110 DPPH Hydroxyl Superoxide Peroxyl Scavenging activity (%) Concentration (ug/ml) μ RAW264.7 BAB Cell viability (%) Concentration (ug/ml) μ 109
111 12000 Control Blank 100 ug/ml 500 ug/ml 1000 ug/ml RAW264.7 cell 9000 DCF fluoresce intensity Incubation time (mim) μ μ μ μ μ μ μ μ μ μ 110
112 γ μ μ 111
113 μ 112
114 μ 113
115 114
116 β α β α α α α β α α β 115
117 β α 116
118 β α 117
119 118
120 κ κ κ κ κ κ κ α κ κ κ κ κ α γ κ κ 119
121 κ κ κ 120
122 μ μ μ μ μ 121
123 122
124 123
125 124
126 μ 125
127 126
128 β 127
129 128
130 μ 129
131 130
132 β 131
133 132
134 133
135 μ 134
136 135
137 μ μ μ 136
138 137
139 138
140 139
141 140
142 γ α β α β α β 141
143 142
144 α α α 143
145 144
146 7. 145
147 . 1, 2 (esp. ). 10 kda 1 kda,. 10 kg (wet weight), (AIGID III) 1.4±0.4 kg, HPLC AIGID III 8.80±0.11 mg/g AIGID III..(table. 25) * Data are expressed as mean ± SD of duplicate determinations. 146
148 . (3 ) 147
149 . (Offer Spec.) 148
150 149
151 150
152 . ( 3 ton/ ) 151
153 8..., ( ),,,. 152
154 (1) AIGID III,,,.,,,,,,,,,,...,,,, 153
155 ,.,. 154
156 μ 100 COX-2 inhibition activity(%) Control Atopy lotion(mg) Nirite(mM) LPS+ LPS- Control Atopy lotion(mg) 155
157 μ μ μ μ μ μ μ 120 Cell viability(% of control) LPS+ LPS- Control Atopy lotion(mg) 100 COX-2 inhibition activity(%) Atopy lotion A B C D E 156
158 μ Moisturizing Effect(%) hr Bacillus subtilis., 157
159 ,.. 158
160 159
161 160
162 제 5 장 목표달성도및관련분야에의기여도 161
163 162
164 제 6 장 연구개발성과및성과활용계획 163
165 164
166 165
167 제 7 장 연구개발과정에서수집한해외과학기술정보 166
168 167
169 168
170 169
171 170
172 제 8 장 참고문헌 171
173 172
174 173
175 174
176 175
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The e-business Studies Volume 17, Number 6, December, 30, 2016:275~289 Received: 2016/12/02, Accepted: 2016/12/22 Revised: 2016/12/20, Published: 2016/12/30 [ABSTRACT] SNS is used in various fields. Although
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DBPIA-NURIMEDIA
The e-business Studies Volume 17, Number 4, August, 30, 2016:319~332 Received: 2016/07/28, Accepted: 2016/08/28 Revised: 2016/08/27, Published: 2016/08/30 [ABSTRACT] This paper examined what determina
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醫 史 學 제18권 제2호(통권 제35호) 2009년 12월 Korean J Med Hist 18ː57 72 18ː173-188 Dec. Dec. 2009 2009 C 大 韓 醫 史 學 會 ISSN ISSN 1225 505X 1225-505X 지방병 연구와 식민지배 : 1927년 영흥 및 해남지역 에메틴 중독사건을 중심으로 신 규 환* 1. 머리말 2. 일제의
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