산림 연구개발사업의 관리 등에 관한 규정

Transcription

1 보안과제( ), 일반과제( o ) S120910L 임업기술연구개발, 보조포함 고부가가치갈매보리수나무육종및기능성소재개발연구 Study on improvement and development of valuable materials with Vitamin trees(hippophae rhamnoides) 동국대학교산학협력단 산림청

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35 Table 1. Morphological characteristics of 5 seed sources of Hippophae rhamnolides Seed sources M1 M2 R1 R2 C Width (mm) 2.3± ± ± ± ±0.43 Length (mm) 5.3± ± ± ± ±0.45 No. of Seed (10g) Seed coat color brown, luster light brown, luster brown dark brown dark brown Seed shape medium small round long oval long oval long & narrow

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37 Fig 1-2. Effects of GA 3 and Filterpaper for Germination on White media

38 Table 1-2. Effects of treatments of Filterpaper and Carbon sources for germination on White media in H. rhamnoides. Carbon sources Filterpaper Germination(%) Contamination(%) stem Length(cm) root 3% sucrose 3% sugar O ± ±1.25 X ± ±1.24 O ± ±1.24 X ± ±1.39 Mean±standard deviation

39 A B C D Fig 1-3. Seed germination of H. rhamnoides in in vitro. A. excluding filterpaper, B. including filterpaper, C. 3% sucrose, D. 3% sugar

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41 Table 1-3. Effect of carbon source for germination from seed of different clone on WPM media in H. rhamnoides. Clone Carbon source Germination(%) Contamination(%) Mongol-1 Mongol-2 China Russia-1 Russia-2 Sucrose 82.4± ±0.8 Sugar 95.0± ±0.5 Sucrose 52.7± ±9.6 Sugar 67.2± ±1.1 Sucrose 61.8± ±3.0 Sugar 73.3± ±3.4 Sucrose 71.1± ±7.4 Sugar 78.2± ±7.02 Sucrose 26.7± Sugar 50.0± ±

42 Table 1-4. Effects of PGRs for the formation of somatic embryo and germination in SH media in H. rhamnoides. PGRs (mg/l) Kin IAA 0.5 Kin IAA 1.0 BA IAA 0.5 BA IAA 1.0 Somatic embryo formation(%) Somatic embryo germination(%)

43 Table 1-5. Effects of media and concentrations of GA3 on somatic embryo germination in H. rhamnoides. Media SH WPM Concentrations of GA 3 (mg/l) Germination of Somatic embryo(%)

44 A B C D Fig 1-4. Somatic embryo formation and germination in H. rhamnoides. (A) explant transfer to the media for somatic embryo formation (B) somatic embryo formation from explant after 3 weeks (C) initial stage of somatic embryo germination (D) elongation from germinated somatic embryo

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46 Table 1-6. Effects of PGRs for organogenesis from cotyledon(from in vitro) and leaf(from ex vitro) on WPM media in H. rhamnoides. Clone PGRs(mg/L) Induction of organogenesis(%) Germination rates BA Kin IAA cotyledon leaf cotyledon leaf ± ± ±7.0 Mongol ± ± ± ± ± ±9.0 Mongol-2 China ± ± ± ± ± ± ± ± ± ± ± ± ± Russia ± ± ± ± ± Russia ± ±

47 A B C D E F Fig 1-5. Organogenesis from leaf(from ex vitro) supplemented with 1.0 mg/l BA, 1.0 mg/l Kin and 5.0 mg/l IAA on WPM medium (A) and (B) Proliferation of adventitious bud with 1.0 mg/l BA, Kin and 5.0 mg/l IAA, (C) and (D) Early proliferation for shoot, (E) Root developed, and (F) Shoot development

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49 Table 1-7. Germination rates on storage duration in UR media. Storage period 0 6 month ago 1 year ago Weeks Germination(%) 66.9 ± ± ± ± ± ± 4.5 Mean ± standard deviation

50 A B C Fig 1-6. Effects of storage period for seed germination of H. rhamnoides in ex vitro. (A) 1 year, (B) 6 month, and (C) directly after harvest

51 A B C D Fig 1-7. Seed germination in ex vitro and acclimation. (A) germination on sand plastic pot, (B) germination on UR media, (C) and (D) transplant in field

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53 Fig 1-8. Effect of pot germination rates(%) on clone (M1) of H. rhamnoides

54 Fig 1-9. Effect of pot for length of shoot on clone (M1) of H. rhamnoides

55 Table 1-8. Different of weeks number of seedling from different 6 clones on H. rhamnoides. weeks M1 M2 M3 R1 R2 C

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57 Fig Seedling growth of H. rhamnoides in first year and second year in open ground

58 Fig Seedling growth reproduced by cutting with 1 year branch from 5 years stock trees

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60 Fig Different clones of chlorophyll contents on H. rhamnoides from leaf of grown the ex vitro condition

61 Fig Photosynthetic of 6 different clones of H. rhamonides

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63 Fig Antioxidative enzyme activities of H. rhamnoides on different clones

64 A B C D Fig Histological examination of the adventitious bud differentiation from leaf segment. (A) early stage cell division on the portion of adventitious bud formation, (B) meristem for adventitious bud in central-leaf, (C) cross-section from induction shoot, and (D) expansion of meristem

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68 Dry powder Hexane extraction Hexane extracts cake Ethanol extraction Ethanol extracts cake Hot water extraction Hot water extracts Methanol extraction cake waste Methanol extracts Water solubles Fig 2-1. Fractionation procedure of Sea buckthorn components

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73 Table 2-1. HPLC analysis conditions for the separation of SE components Standard compounds Sample preparation method Conditions of analysis EGCG & Epigallocatechin & Catechin & Gallocatechin Gallate & Epicatechin & Epicatechin Gallate - sample in 10 ml Volumetric flask - 1) Sonication after added 10ml MeOH 2) Sonication after added 10ml MeOH and 0.1% HCl - Centrifuge (3500 rpm, 10 min) & Syringe filter(0.45 μm) - Analysis 1. Equipment Model Agilent 1200 HPLC 2. Column ZORBAX Eclipse XDB-C x4.0mmI.D.,5μm 3. Guard column ZORBAX Eclipse XDB-C x4.6mmI.D.,5μm 4. Mobile phase 5. Detector UV 280 nm 6. Column Temp Flow rate 8. Injection volume 5 ~ 10 μl Conditions 1 : 0.1% TFA in water : Acetonitrile = 85 : 15 Conditions 2 : 0.1% TFA in water : Acetonitrile = 95 : 05 Conditions 1 : 0.35 ml/min Conditions 2 : 0.2 ml/min

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79 Fig 2-2. Fractionation procedure of Sea buckthorn fruits

80 Fig 2-3. Oil fractionation by column chromatography

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83 Fig 2-4. Solvent fractionation of Sea buckthorn materials

84 Fig 2-5. Bleaching of hexane solution of Sea buckthorn oil by adsorbents

85 Fig 2-6. Bleaching of hexane solution of Sea buckthorn oil by active carbon

86 Fig 2-7. Absorption spectra of the hexane solution of Sea buckthorn oil bleached by active carbon (190nm 800nm)

87 Fig 2-8. Effect of active carbon on the bleaching of Sea buckthorn oil

88 Fig 2-9. Absorption spectra of pigments removed by active carbon from the Sea buckthorn oil

89 Fig Bleaching of hexane solution of Sea buckthorn oil by acid clay

90 Fig Absorption spectra of the hexane solution of Sea buckthorn oil bleached by acid clay (190nm 800nm)

91 Fig Effect of acid clay on the bleaching of Sea buckthorn oil

92 Fig Absorption spectra of pigments removed by acid clay from the Sea buckthorn oil

93 Fig Absorption spectra of pigments recovered from the spent earth by ethanol

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95 Fig Fatty acid compositions of oil fractions recovered from the Sea buckthorn fruits (2008)

96 Table 2-2. Fatty acid compositions of various oil fractions recovered from Seabuckthorn fruits (2009) ID JH JHT JHP DH DHT DHP SH SHT SHP C14: C15: C16: C16: C16: C16: C17: C18: C18: C18: C18: C18: C20: C20: sample ID: JH: hexane extract of juice, JHT: neutral oil of JH, JHP: coloring compounds of JH DH: hexane extract of debris, DHT: neutral oil of DH, DHP: coloring compounds of DH SH: hexane extract of seed, SHT: neutral oil of SH, SHP: coloring compounds of SH

97 Fig Radical scavenging activity of Sea buckthorn components

98 Fig Protection effects of the extracts on cell damage induced by UVB (+UV, HaCaT, A; L-ascorbic acid 25µM)

99 Fig Protection effects of the extracts on cell damage induced by UVB (+UV, Fibroblast, A; L-ascorbic acid 25µM)

100 Fig Anti-inflammatory activity of extracts on UVB-induced TNF-α D; Dexamethasone 1µM

101 Fig Effect of extracts on the production of MMP-1. AS : Ascorbic acid 25μM

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103 Fig Protection effects of the SE extracts on cell damage induced by (+UV, Fibroblast, A; L-ascorbic acid 25µM)

104 Fig Collagen synthesis effect by SE extracts after UVB irradiation (AS : Ascorbic acid 25μM)

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106 Table 2-3. DPPH free radical scavenging activity of Sea buckthorn components Concentration (mg/ml) JH JE JW DH DE DW SH SE SW LH LE LW

107 Fig Composition of Sea buckthorn fractions

108 Fig Heat Stability of SE, LW fractions

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111 Fig TLC Separation of phenolic compounds

112 Fig TLC Separation of phenolic compounds

113 Table 2-4. The color developments of phenolic compounds by different visualization techniques Visualization No Vanillin AlCl 3 DPPH Light source white white white white t-cinnamic bk - - bk pgy - bk - - bk - - quercetin sbk br br bl sskybl br bk swt br bk br pk salicylic pbl skybl - bk pgy - bl skybl - pbl skybl - p-coumaric bk bl - bk gy ppp bk - - bk bl ppk ferulic bk skybl - pbl skybl ppp bk pbl pbr bk skybl pk gallic bk - bk bk bk pbr bk pbl gy bk gy pk catethin pbk - pbk sbk dpp pp bk pbl pbr pbk gy pk bk:black, bl:blue, gy:gray, pp:purple, wt:white, pk:pink, s:strong, p:pale, d:dark, sky:sky

114 Fig TLC Separation of SE fraction

115 Fig TLC Separation of ethanol fractions from Sea buckthorn

116 Fig TLC Separation of ethanol fractions from Sea buckthorn

117 Fig TLC Separation of fractionated SE

118 Fig TLC Separation of fractionated SE

119 Fig HPLC separation of SE fraction

120 Fig HPLC separation of SE fraction

121 a. 6.4 minute peak of condition 1 b minute peak of condition 2 c minute peak of condition 2 Fig UV absorption spectra of major peaks

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125 Fig ESI-MS of SE fraction

126 Fig Polyphenol contents of different ethanol extracts of Sea buckthorn seeds

127 Fig DPPH free radical scavenging activity of BHA and ascorbic acid

128 Fig DPPH free radical scavenging activity of the different ethanol extracts of Sea buckthorn seeds

129 Fig Effect of SHE and SEO on the cell viability in human fibroblasts by the MTT assay

130 Fig Cell counting using the BrdU assay

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133 Fig Effect of SHE and SEO on the collagen synthesis in human fibroblasts by the collagen assay

134 Fig Effect of SHE and SEO on the glycosaminoglycanollagen synthesis in human fibroblasts by the GAG assay

135 Table 2-5. DPPH free radical scavenging activity and total phenolic content (TPC) of the solvent-dependent extracts of Sea buckthorn seeds Compound Solvent Antioxidant activity TPC IC 50 /DPPH (μg/ml) a) (μg GAE/mg) b) SBSH Hexane ± a 6.70 ± 2.13c SBSE Ethanol 8.33 ± 0.39d ± 6.76a SBSW Water ± 3.93b ± 0.96b Ascorbic acid ± 0.29c - a) The antioxidant activity was evaluated as the content of the test sample required to decrease the b) absorbance at 517 nm by 50% in comparison to the control; Total reducing capacity of Sea buckthorn seed as determined by Folin-Cioculteu assay. TPC values are expressed as gallic acid equivalent (GAE)/mg in dry weight. Values are expressed as mean of triplicate determinations ± standard deviation; Different letters in the same column show significant differences from each other at P<0.05 level

136 Fig Effect of SBSE concentrations on the cytotoxicity in human fibroblasts by the MTT assay. Data are expressed as percentage of live cells compared to untreated controls. *P < 0.01 vs exposed controls and **P < vs exposed controls

137 Fig Effect of SBSE on the cell viability of skin fibroblasts exposed to UVB radiation at 30 mj/cm2,determined using the MTT assay. Data are expressed as percentage of live cells compared to unexposed controls. *P < 0.01 vs exposed controls and **P < vs exposed controls

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139 Fig SBSE blocked the UVB-induced increase of IL-1β expression in cultured fibroblasts

140 Fig SBSE inhibited UVB-induced increase in IL-6 and COX-2 expression in human dermal fibroblasts

141 Fig SBSE inhibited UVB-induced increase in TNF-α expression in human Keratinocytes

142 Fig SBSE inhibited UVB-induced expression of MMP-1 in human dermal fibroblasts

143 Fig SBSE stimulated the synthesis of type I procollagen in human dermal fibroblasts

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145 Crushing to mesh for sea buckthorn seed ò Mixing of ethanol and crushed sea buckthorn seed to a concentration of 5%(w/v) in reactor ò Extraction for 18 hr at ò Separation of supernatant ò Vacuum filtering after celite addition ò Vacuum evaporation to a dry weight of % at 60 ò Storage at for hr after addition of celite in evaporated extract ò Vacuum filtering ò Packaging Fig The extraction process of Sea buckthorn seeds

146 Table 2-6. Comparison of the extraction yield, antioxidant activity (IC 50 ),and stability at the different steps of the separation process used for ethanol extraction of Sea buckthorn seeds CR CE CE-CS Extraction yield(%) IC 50 ( μg /ml) Phase separation in stability test during 70 days under room temperature CR : the crude ethanol extract without separation CE : the extract separated by the use of celite CE-CS : the extract filtering CR, which contains celite, after the storage of 16 h in refrigerator of 4 IC 50 : 50% inhibition concentration

147 Antioxidant activity (%) CR CE CE-CS Concentration of SBS extract (ug/ml) Fig Antioxidant activity at different steps of ethanol extraction from Sea buckthorn seeds (CR : the crude ethanol extract without separation, CE : the extract separated by the use of celite, CE-CS : the extract filtering CR, which contains celite, after the storage of 16 h in refrigerator of 4 )

148 Antioxidant activity (%) Antioxidant activity (%) SBS extract BHA 0 day (SBS Vitamic extract) C 70 days (SBS extract) 0 day (BHA) 70 days (BHA) 0 day (Vitamic C) 70 days (Vitamin C) Concentration (ug/ml) Concentration (ug/ml) Fig Effect of storage time on the antioxidant activity Fig Comparison of the SBS extract with other antioxidant agents

149 100 SBS extract 90 Antioxidant activity (%) Control 1 h treatment at 100 O C 1 h treatment at 150 O C 1 h treatment at 200 O C Concentrations of SBS extract (ug/ml) BHA Antioxidant activity (%) Control 1 h treatment at 100 O C 1 h treatment at 150 O C 1 h treatment at 200 O C Concentrations of BHA (ug/ml) Vitamin C Antioxidant activity (%) Control 1 h treatment at 100 O C 1 h treatment at 150 O C 1 h treatment at 200 O C Concentrations of vitamin C (ug/ml) Fig Effect of temperature on the antioxidant activity

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152 Bactericidal ratio (%) Control SBS extract Vitamin C BHA Time (hr) Fig Comparisons of the antibacterial activities of the SBS extract, vitamin C and BHA

153 Bactericidal ratio (%) O C 25 O C Time (hr) Fig Effect of culture temperature on the antibacterial activities (E.coli 200 rpm)

154 Bactericidal ratio (%) ug/ml 500 ug/ml 1000 ug/ml 2000 ug/ml Time (hr) Fig Effect of SES extract concentration on the antibacterial activities (E.coli 200 rpm)

155 Bactericidal ratio (%) E. coli P. aerusinosa S. aureus B. subtilis Time (hr) Fig Antibacterial activity against other bacteria

156 Fig Prototype status of cosmetics adding ethanol extract from Sea buckthorn seeds

157 Fig Comparison of heat stability in the each LW fraction

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159 시약과

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163 Fig 3-1. Physical composition of Sea buckthorn fruit

164 Fig 3-2. Condition of GC-MS analysis

165 Fig 3-3. SAFE (Solvent Assisted Flavour Evaporation)

166 Fig 3-4. SPME (Solid Phase Micro Extraction)

167 Table 3-1. Method of making Home-brewed beer by adding Sea buckthorn fruit 맥주제조기계 맥주제조방법 맥주제조방법 단계 발효및숙성 생수를발효조에 을채운후 를모두혼합함 생수 를넣고효모를넣음 상온에서일주간숙성시킴 단계 차숙성 단계가끝나면 에서 일간숙성시킴 단계 차숙성 맥주 에설탕 넣고흔든후상온에서 주일간숙성시킴 갈매보리수나무열매착즙액첨가 단계 발효및숙성 발효조에생수 를채움 뜨거운물에맥주원액캔을담아둠 생수 에부스터를넣어녹인후끓임 끓인부스터에맥주원액캔을부어혼합후발효조에넣음 발효조에 리터의생수를넣은후 가되면효모를넣고상온에서 주간발효시킴 단계 차숙성 맥주 에설탕 넣고 주간숙성시킴 갈매보리수나무열매착즙액첨가 단계 차숙성 에서 주간숙성시킴

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170 Fig 3-5. H. rhamnoides berry juice

171 Table 3-2. The questionnaire of functional jellies contained H. rhamnoides juice sensory test. Sensory test methods Sensory characteristics Appearance Interest : The higher samples get grades, the better assessors feel characteristics. Suitability : 4 points mean the most suitable intensity of samples' characteristics, while higher points than 4 mean intensity is strong and lower points than 4 mean intensity is weak. Grades Little Not good So Too bad Bad Little good Good bad not bad good Taste Too bad Bad Little bad Not good not bad Little good Good So good Flavor Too bad Bad Little bad Not good not bad Little good Good So good Overall Color Little Not good So Too bad Bad Little good Good bad not bad good Too Little Little Too Weak Suitable Strong weak weak strong strong Sweet-ness Too weak Weak Little weak Suitable Little strong Strong Too strong

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173 Table 3-3. The questionnaire of functional beverages containing H. rhamnoides juice sensory test. Sensory test methods Sensory characteristics Appearance Taste Flavor Overall Color Sweet-ness Sourness Interest : The higher samples get grades, the better assessors feel characteristics. Suitability : 4 points mean the most suitable intensity of samples' characteristics, while higher points than 4 mean intensity is strong and lower points than 4 mean intensity is weak. Grades Little Not good So Too bad Bad Little good Good bad not bad good Little Not good So Too bad Bad Little good Good bad not bad good Little Not good So Too bad Bad Little good Good bad not bad good Little Not good So Too bad Bad Little good Good bad not bad good Too Little Little Too Weak Suitable Strong weak weak strong strong Too Little Little Too Weak Suitable Strong weak weak strong strong Too Little Little Too Weak Suitable Strong weak weak strong strong

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179 Fig 3-6. Physical composition of Sea buckthorn fruit

180 Fig Total flavonoid content of the each samples

181 Table 3-4. Comparision of total flavonoid content of the each samples Type Catechin equivelnet (mg/l) Standard deviation Sea buckthorn Fermented Enzyme (Enzyme+Lactic acid bacteria) Fermentation Natural Fermentation Lactic acid bacteria fermentation Laurel fruit extract

182 Table 3-5. Chemical composition of Sea buckthorn fruit Chemical composition Average(%) Standard deviation Moisture Crude Fat Crude Protein - - Reducing Sugar Crude Fiber

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184 Fig 3-8. GC chromatogram for volatile compounds of Sea buckthorn fruit with SAFE

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186 Table 3-6. volatile compounds of Sea buckthorn fruit by analyzing SAFE peak Possible compound no. 1) I Retention % of total Time (min) 1 ethyl butylrate ethyl 2-methylbutyrate ethyl 3-methylbutyrate pentanol ethyl hexanoate trans-beta-ocimene methybutyl 3-methylbutyrate propylhexnoate ethyl heptanoate methylpenta 3-ol, methyl octanoate ethyl octanoate butyric acid methylbutyl hexanoate hepten-1-ol,2 methyl methyl 3-hydroxybutyrate methyl benzoate methylbutyric acid, benzyl alcohol ethyl hexanoate methylbutyl benzoate phenyl ethanol pentacosane heptasosane * ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

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188 (a) polydimetysiloxane (PDMS) SPME fiber chromatogram Fig 3-9. Amount of adsorption according to fiber type (b) carbowax/divinylbenzene (DVB/CAR/PDMS) SPME fiber chromatogram Fig 3-9. Amount of adsorption according to fiber type

189 Fig GC chromatogram for volatile compounds of Sea buckthorn fruit with SPME

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191 Table 3-7. Volatile compounds of Sea buckthorn fruit with SPME peak No. 1) 2) Possible compound 1) I Retention Time (min) % of total 1 ethyl2-methylbuturate ethyl hexanoate isoamylbutyrate 2) * methylbutyl 2-methylbutyrate methylbutyl 3-methylbutylate 3, propylhexanoate methyl 5-hepten 2-one * ethyl hex-2-enoate isobutyl hexanoate * hexanol dodecamethylcyclohexasiloxane methyl octanoate ethyl octanoate butanoic acid * methylbutyl octanoate methly but-2-enoate * ethyl oct-4-enoate ethyl sorbate ethyl oct-3-enoate propyl octanoate ethyl 4,7-octadienoate (Z) ethyl nonanoate ethyl oct-2-enoate (E) methylbenzoate ethyl decanoic acid butanoic acid ethyl E-4-decenoate ethyl benzoate ethyl dec-4-noate methyl diethyl borinic acid * methyl decadienoate * ethylphenyl aceate phenylethyl acetate ethyl dodecanoate hexanoicacid decahydro naphthalene * methyl 1-butanol, benzoate * I mean Kovats retention index on DB-WAX * mean no identified in Kovats index

192 총산 초산으로서 N 소비량 N 의 검체량 ml

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195 Table 3-8. Using Sea buckthorn fruit for 15 home-brewed beers and control GA GA5g GAS5% GAS3% GAS1% GA5% GA3% GA1% AA AAS5% AAS3% GL GLS3% PA PAS3% Con Control(Con), Golden Ale(GA), Golden Ale + sugar5g(ga5g), Golden Ale (GAS) Amber Ale(AA), Amber Ale (AAS), Golden Larger(GL), Golden Larger (GLS), Pale Ale(PA), Pale Ale (PAS)

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197 Table 3-9. Total acid content of 16 kinds of beers Beer Total acid Beer Total acid GA AA GA5g AAS5% GA5% 0.27 AAS3% GA3% GL GA1% 0.18 GLA3% 0.18 GAS5% PA 0.14 GAS3% PAS3% GAS1% Con

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199 Table Total sugar content of 16 kinds of beers Beer Total sugar(%) Beer Total sugar(%) GA 2.99±0.32 1) AA 1.60±0.02 GA5g 2.82±0.37 AAS5% 1.78±0.07 GA5% 2.81±0.18 AAS3% 1.47±0.06 GA3% 3.15±0.19 GL 2.67±0.12 GA1% 2.82±0.43 GLS3% 2.67±0.01 GAS5% 1.97±0.73 PA 2.92±0.06 GAS3% 2.83±0.57 PAS3% 2.35±0.12 GAS1% 3.02±0.49 Con 2.55±

200 Table Total reducing sugar content of 16 kinds of beers Beer Reducing sugar(%) Beer Reducing sugar(%) GA 1.28±0.34 1) AA 1.22±0.06 GA5g 1.39±0.28 AAS5% 1.05±0.01 GA5% 1.13±0.36 AAS3% 1.14±0.07 GA3% 1.31±0.34 GL 0.89±0.02 GA1% 1.28±0.31 GLS3% 0.91±0.02 GAS5% 0.93±0.35 PA 1.00±0.02 GAS3% 1.26±0.30 PAS3% 0.88±0.03 GAS1% 1.20±0.34 Con 0.74±

201 Table Ethanol content of 16 kinds of beers

202 Fig a) Sensory evaluation b) Sensory evaluation paper

203 Table Sensory evaluation of golden larger and pale ale(40 panels) 1) Type Beer sample GL GLS3% PA PAS3% Con Total preference 3.38 b 2.78 b 3.58 b 3.15 b 6.03 a Color 4.79 bc 3.98 c 5.88 a 4.18 bc 4.98 ba Flavor 3.62 b 3.90 b 4.38 b 3.65 b 6.20 a Taste 3.13 b 2.90 b 3.23 b 3.30 b 5.88 a Sourness 3.38 cb 3.10 c 4.05 b 3.38 cb 5.73 a Sweetness 3.56 b 3.53 b 3.48 b 3.60 b 5.49 a Bitterness 3.77 b 3.90 b 3.85 b 4.09 b 5.65 a Sparkling 3.00 cb 2.73 c 3.15 cb 3.75 b 5.46 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test)

204 Table Sensory evaluation of golden larger and pale ale(16men panels) 1) Type Beer sample GL GLS3% PA PAS3% Con Total preference 3.75 b 2.69 b 3.50 b 2.88 b 6.06 a Color 4.69 a 4.00 a 5.56 a 3.81 a 4.88 ba Flavor 4.00 a 4.13 b 4.63 b 3.31 b 6.38 a Taste 3.38 b 2.69 b 2.94 b 2.75 b 5.94 a Sourness 3.38 b 3.31 b 4.06 b 3.00 b 6.00 a Sweetness 3.31 b 3.38 b 3.00 b 3.13 b 5.81 a Bitterness 3.44 b 3.44 b 3.63 b 3.13 b 5.81 a Sparkling 2.81 b 2.50 b 3.44 b 2.28 b 5.69 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test)

205 Table Sensory evaluation of golden larger and pale ale(24 women panels) 1) Type Beer sample GL GLS3% PA PAS3% Con Total preference 3.13 b 2.83 b 3.63 b 3.33 b 6.00 a Color 4.87 b 3.96 b 6.08 a 4.42 b 5.04 a Flavor 3.35 b 3.75 b 4.21 b 3.88 b 6.08 a Taste 2.96 b 3.04 b 3.34 b 3.67 b 5.83 a Sourness 3.39 b 2.96 b 4.04 b 3.63 b 5.54 a Sweetness 3.74 b 3.63 b 3.79 b 3.92 b 5.27 a Bitterness 4.00 b 4.21 b 4.00 b 4.73 ba 5.54 a Sparkling 3.13 b 2.88 b 2.96 b 4.00 b 5.31 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test)

206 Table Sensory evaluation of Amber ale(41 panels) 1) Type Beer sample AA AAS5% AAS3% Con Total preference 2.39 b 2.12 b 2.27 b 5.71 a Color 4.20 b 3.20 c 3.04 c 6.71 a Flavor 2.76 b 3.37 b 3.17 b 6.00 a Taste 2.37 b 2.05 b 2.34 b 5.46 a Sourness 3.98a 4.24 a 4.37 a 4.87 a Sweetness 3.44 b 3.26 b 3.44 b 5.07 a Bitterness 4.37 a 4.29 a 3.89 a 4.63 a Sparkling 3.02 b 2.78 b 2.24 b 4.98 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test)

207 Table Sensory evaluation of Amber ale(15 men panels) 1) Type Beer sample AA AAS5% AAS3% Con Total preference 2.27 b 2.33 b 2.27 b 5.33 a Color 3.53 b 3.27 b 3.13 b 6.27 a Flavor 2.38 b 3.47 b 3.00 b 6.12 a Taste 2.33 b 2.40 b 2.33 b 5.33 a Sourness 4.53 a 4.73 a 4.73 a 4.67 a Sweetness 2.80 b 2.93 ba 3.53 ba 4.53 a Bitterness 4.67 a 4.20 a 3.80 a 4.07 a Sparkling 3.67 ba 2.73 b 2.33 b 5.13 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test)

208 Table Sensory evaluation of Amber ale(26 women panels) 1) Type Beer sample AA AAS5% AAS3% Con Total preference 2.46 b 2.00 b 2.27 b 5.92 a Color 4.58 b 3.15 c 3.00 a 6.96 a Flavor 2.38 b 3.31 b 3.00 b 6.12 a Taste 2.38 b 1.85 b 2.35 b 5.54 a Sourness 3.65 a 3.96 a 4.15 a 5.00 a Sweetness 3.81 b 3.46 b 3.38 b 5.38 a Bitterness 4.19a 4.35a 3.92a 4.96 a Sparkling 2.65 b 3.81 b 2.19 b 4.88 a 1) Values in the sharing a common letter are not significantly different (p<0.05, Duncan s multiple range test)

209

210 Fig GC Chromatogram for volatile compounds of Control beer by SAFE

211

212 Table volatile compounds of Control beer with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol isoamyl acetate isoamyl alcohol ethyl hexanoate methylbut-2-en-1-ol ethyl heptanoate acetic acid furfural heptanol ethyl octanoate linalool octanol propanoic acid ,5-dimethyl 2(5H)furanone butyrolactone ethanol * butyric acid ethyl decanoate furanmethanol butanoic acid alpha-terpineol methlythiopropanal delta-cadinene citronellol phenyl ethylacetate hexyl but-2-enoate hexanoic acid benzyl alcohol phenyl ethylalcohol acetylpyrrole octanoic acid pyrrole-2-carboxaldehyde cis-ethyl cinnamate phenoxyethanol decanoic acid phenol dodecanoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

213 Fig GC Chromatogram for volatile compounds of Pale ale(3%) by SAFE

214 Table Volatile compounds of Pale ale(3%) with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol isoamyl acetate isoamyl alcohol ethyl hexanoate methylpentanol methylbut-2-en-1-ol hexanol ethyl octanoate furfural heptanol ethyl octanoate Ethanol linalool octanol propanoic acid trans-caryophyllene ,5-dimethyl 2(5H)furanone butyrolactone butyric acid ethyl decanoate alpha-humulene methylbutyl octanoate ethyl benzoate furanmethanol ethyl succinate alpha-terpineol methlythiopropanol delta-cadinene citronellol phenyl ethylacetate hexanoic acid phenyl ethylalcohol malto acetylpyrrole octanoic acid pyrrole-2-carboxaldehyde cis-ethyl cinnamate benzoic acid methoxy-4-vinylphenol beta-eudesmol phenol decanoic acid dodecanoic acid benzoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

215

216 Fig GC Chromatogram for volatile compounds of Pale ale by SAFE

217 Table Volatile compounds of Pale ale with SAFE 1) peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol isoamyl acetate isoamyl alcohol ethyl hexanoate methylpentanol methylbut-2-en-1-ol hexanol ethyl octanoate furfural heptanol Ethanol linalool octanol propanoic acid trans-caryophyllene ,5-dimethyl 2(5H)furanone butyrolactone butyric acid ethyl decanoate alpha-humulene methylbutyl octanoate ethyl benzoate furanmethanol ethyl succinate alpha-terpineol methlythiopropanol delta-cadinene phenyl ethylacetate hexanoic acid phenyl ethylalcohol malto acetylpyrrole octanoic acid pyrrole-2-carboxaldehyde cis-ethyl cinnamate benzoic acid methoxy-4-vinylphenol beta-eudesmol phenol decanoic acid dodecanoic acid benzoic acid I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

218 Fig GC Chromatogram for volatile compounds of Golden ale(3%) by SAFE

219 Table Volatile compounds of Golden ale(3%) with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol isoamyl acetate isoamyl alcohol ethyl hexanoate methylpentanol methylbut-2-en-1-ol ethyl heptanoate ethyl octanoate furfural heptanol ethyl octanoate Ethanol linalool octanol propanoic acid trans-caryophyllene ,5-dimethyl 2(5H)furanone butyrolactone butyric acid ethyl decanoate alpha-humulene furanmethanol ethyl benzoate ethyl succinate alpha-terpineol methlythiopropanol delta-cadinene citronellol phenyl ethylacetate hexanoic acid phenyl ethylalcohol malto acetylpyrrole octanoic acid pyrrole-2-carboxaldehyde cis-ethyl cinnamate benzoic acid methoxy-4-vinylphenol beta-eudesmol phenol decanoic acid dodecanoic acid benzoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

220 Fig GC Chromatogram for volatile compounds of Golden ale by SAFE

221 Table Volatile compounds of Golden ale with SAFE peak no. Possible compound 1) I Retention Time (min) % of total 1 isobutyl alcohol isoamyl acetate isoamyl alcohol ethyl hexanoate methylpentanol methylbut-2-en-1-ol ethyl heptanoate ethyl octanoate furfural heptanol ethyl octanoate Ethanol linalool octanol propanoic acid trans-caryophyllene ,5-dimethyl 2(5H)furanone butyrolactone butyric acid ethyl decanoate alpha-humulene furanmethanol ethyl benzoate ethyl succinate alpha-terpineol methlythiopropanol delta-cadinene citronellol phenyl ethylacetate hexanoic acid phenyl ethylalcohol malto acetylpyrrole octanoic acid pyrrole-2-carboxaldehyde cis-ethyl cinnamate benzoic acid methoxy-4-vinylphenol beta-eudesmol phenol decanoic acid dodecanoic acid benzoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

222

223 Fig GC Chromatogram for volatile compounds of control beer by SPME

224 Table Volatile compounds of Control beer with SPME peak no. Possible compound 1) I Retention Time (min) % of total 1 2-methylpropyl acetate ethyl butyrate isobutyl alchol isoamyl acetate D-limonene isoamyl alcohol ethyl hexanoate octan-3-one benzaldehyde ethyl undecanoate ethyl octanoate acetic acid ethyl octanoate heptanol alpha-cubebene ethanol ethyl nonanoate linalool octanol propanoic acid trans-caryophyllene benzaldehyde ,5-dimethyl 2(5H)furanone undecan-2-one pyrrole ethyl decanoate alpha-humulene methylbutyl octanoate alpha-terpineol delta-cadinene citronellol ethyl dodecanoate hexanoic acid benzyl alcohol phenylethyl alcohol acetylpyrrole octanoic acid methyldiphenyl ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

225 Fig GC Chromatogram for volatile compounds of Pale ale(3%) by SPME

226 Table Volatile compounds of Pale ale(3%) with SPME peak no. Possible compound 1) I Retention Time (min) % of total 1 ethyl 2-methylbutyrate ethyl butyrate isobutyl alchol isoamyl acetate isoamyl alcohol ethyl hexanoate ethyl 3-methylbutyrate methyl hex-2-enoate ethyl undecanoate ethyl octanoate heptanol (E)-ethyl oct-4-enoate ethyl nonanoate linalool trans-caryophyllene acetic acid ethyl decanoate alpha-humulene ethyl benzoate methylbutyl octanoate furanmethanol butanoic acid (Z)-ethyl dec-4-enoate alpha-terpineol heptadec-1-ene delta-cadinene decanol citronellol phenylethyl acetate hexanoic acid ethyl dodecanoate butyl benzoate phenylethyl alcohol acetylpyrrole octanoic acid cis-ethyl cinnamate decanoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

227 Fig GC Chromatogram for volatile compounds of Pale ale by SPME

228 Table Volatile compounds of Pale ale with SPME peak no. Possible compound 1) I Retention % of total Time (min) 1 2-methylpropyl acetate ethyl butyrate isobutyl alchol isoamyl acetate isoamyl alcohol ethyl hexanoate hexyl acetate methyl hex-2-enoate benzaldehyde ethyl undecanoate ethyl octanoate heptanol propanoic acid ethyl nonanoate linalool octanol trans-caryophyllene ethyl decanoate methylbutyl octanoate furanmethanol tetradecanol delta-cadinene decanol citronellol phenylethyl acetate hexanoic acid benzyl alcohol phenylethyl alcohol acetylpyrrole octanoic acid decanoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

229 Fig GC Chromatogram for volatile compounds of Golden ale(3%) by SPME

230 Table Volatile compounds of Golden ale(3%) with SPME peak no. Possible compound 1) I Retention Time (min) % of total 1 ethyl 2-methylbutyrate ethyl butyrate isobutyl alchol isoamyl acetate isoamyl alcohol ethyl hexanoate ethyl 3-methylbutyrate methyl hex-2-enoate ethyl undecanoate ethyl octanoate heptanol (E)-ethyl oct-4-enoate ethyl nonanoate linalool trans-caryophyllene acetic acid ethyl decanoate alpha-humulene ethyl benzoate methylbutyl octanoate furanmethanol butanoic acid (Z)-ethyl dec-4-enoate alpha-terpineol heptadec-1-ene delta-cadinene decanol citronellol phenylethyl acetate hexanoic acid ethyl dodecanoate butyl benzoate phenylethyl alcohol acetylpyrrole octanoic acid cis-ethyl cinnamate decanoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

231 Fig GC Chromatogram for volatile compounds of Golden aleby SPME

232 Table Volatile compounds of Golden ale with SPME peak no. Possible compound 1) I Retention % of total Time (min) 1 2-methylpropyl acetate ethyl butyrate isobutyl alcohol isoamyl acetate D-limonene isoamyl alcohol ethyl hexanoate octan-3-one benzaldehyde methylbutyl butyrate ethyl heptanoate ethyl undecanoate ethyl octanoate heptanol alpha-cubebene ethyl nonanoate linalool trans-caryophyllene ethyl decanoate alpha-humulene methylbutyl octanoate furanmethanol delta-cadinene citronellol phenylethyl acetate ethyl dodecanoate hexanoic acid benzyl alcohol phenylethyl alcohol acetylpyrrole octanoic acid decanoic acid benzoic acid ) I mean Kovats retention index on DB-WAX 2) * mean no identified in Kovats index

233 Fig Total flavonoid content of each beer types

234

235

236 Fig DPPH free radical scavenging activity of Sea buckthorn fruit

237 Fig ABTS radical cation activity of the home-brewed beers

238 Fig Functional jellies containing H. rhamnoides berry juices (sample A : containing 10% juices, sample B : containing 20% juices, sample C : containg 30% juices)

239 Table Interest sensory test of functional jellies containing H. rhamnoides juice. Sensory characteristics Sample A Sample B Sample C Appearance 4.58±0.75 1) 5.26± ±0.72 Taste 5.11±0.97 b 3.74±0.96 a 4.58±1.04 b Flavor 5.05±1.05 b 3.79±0.77 a 3.42±1.31 a Overall 5.26±0.71 b 3.84±0.93 a 4.11±1.12 a 1) All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test

240 Table Suitability sensory test of functional jellies contained H. rhamnoides juice Sensory characteristics Sample A Sample B Sample C Color 2.74±0.85a 1) 4.16±0.49b 4.47±0.68b 1) Sweetness 3.89± ± ±0.57 All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test

241 Fig Functional beverages containing H. rhamnoides berry juices (sample A : containing 0% juices, sample B : containing 5% juices, sample C : containg 10% juices, sample D : containing 15% juices)

242 Table Interest sensory test of functional beverages contained H. rhamnoides juice Sensory characteristics Sample A Sample B Sample C Sample D Appearance 4.45±1.32 1) 4.39± ± ±1.33 Taste 4.65±1.26 c 3.81±1.57 ab 4.13±1.36 bc 3.23±1.29 a Flavor 4.55±1.10 b 3.81±1.26 a 3.58±1.10 a 3.45±1.43 a 1) Overall 4.61±1.10 c 4.32±1.30 bc 3.87±1.18 ab 3.35±1.36 a All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test

243 Table Suitability sensory test of functional beverages contained H. rhamnoides juice Sensory characteristics Sample A Sample B Sample C Sample D Color 4.74±1.14 b 1) 4.32±1.25 ab 3.90±1.25 a 3.84±1.30 a Sweetness 4.48±1.34 c 4.06±1.13 bc 3.77±1.18 ab 3.26±1.41 a 1) Sourness 3.55±1.16 a 4.48±1.10 b 4.55±1.10 b 5.23±1.21 c All values are mean ± S.D. a-c Means in the same rows bearing different superscript letters are significantly different(p<0.05) by Duncan's multiple range test

244 μ μ

245 μ μ

246 Fig DPPH radical scavenging activity of H. rhamnoides L fruit extract

247 Fig Concentration-dependent anti-oxidative effect of H. rhamnoides L fruit extract in MEF cells

248 Fig Morphological changes in MEF cells exposed to H 2 O 2 and H. rhamnoides L fruit extract

249 Fig Effect of H. rhamnoides L fruit extract treatment on cell cycle progression in oxidative damaged cells

250 Fig Effect of H. rhamnoides fruit extract treatment on cell cycle progression in oxidative damaged cells

251

252 Fig Effects of H. rhamnoides fruit extract treatment on apoptotic cell death assessing by flow cytometry

253 Fig Effects of the H. rhamnoides L. fruit extract in dysregulation cell cycle of the oxidative damaged cell (1; control group, 2; H 2 O 2, 3; H. rhamnoides, 4; Co-Treat)

254

255 Fig Protective effect of H. rhamnoides fruit extract on DNA damaged cells induced by DNA alkylating agent (MMS)

256 Fig Effect of H. rhamnoides fruit extract on cytotoxicity in three different human cancer cell lines (A; AGS, B; HepG2, C; HeLa)

257 Fig Apoptotic cell death increases according to H. rhamnoides fruit extract treatment times

258 Fig Expression profiles of characteristic intrinsic apoptotic proteins (Bax, Bcl-2) after exposure to H. rhamnoides fruit extract in AGS cell for various times (24, 48, 72h)

259

260 Fig H. rhamnoides fruit extract inhibits AGS cell proliferation through the cell cycle arrest

261 Fig Anti-proliferative effect through G2/M cell cycle arrest by 72 h administration times

262 Fig Effect of H. rhamnoides fruit extract on related to G2/M cell cycle checkpoint regulating proteins

263 Fig Expression of caspase-dependent apoptotic proteins exposure to H. rhamnoides extract in AGS cells after 72h administration times

264

265 Fig Bacteria for examination of antibacterial performance. (A ; E. coli, B ; B. cereus, C ; S. aureus)

266 Table Selection of bacteria and cultivation conditions. Bacteria Culture media time of culture temp. Gram negative bacteria Escherichia coli (ATCC 25922) 18 h Trypticase Gram positive bacteria Bacillus cereus (ATCC 14579) Soy 16 h Broth 37 Gram positive bacteria Staphylococcus aureus (ATCC 25923) 15 h

267 Fig Antibacterial effect of H. rhamnoides extract against E. coli

268 Control H. rahmnoides treatment Fig Clear zone of H. rhamnoides extract against E. coli

269 Table Examination of forming clear zone against E. coli. Clear zone (mm) Kanamycin H. rhamnoides treatment 17.3 ±

270 Fig Antibacterial effect of H. rhamnoides L. extract against B. cereus

271 Control H. rhamnoides treatment Fig Clear zone of H. rhamnoides extract against B. cereus

272 Table H. rhamnoides fruit extract form clear zone against B. cereus. Clear zone(mm) Kanamycin H. rhamnoides treatment 20.3 ± ±

273 μ

274 Fig Antibacterial effect of H. rhamnoides extract against S. aureus

275 Control H. rhamnoides treatment Fig Clear zone of H. rhamnoides extract against S. aureus

276 Table Examination of forming clear zone against S. aureus. Clear zone (mm) Kanamycin H. rhamnoides treatment 17.3 ±

277 Fig Set the effective minimal concentration of inhibition bacterial performance

278 Table Minimal concentration of antibacterial effectiveness. Minimum antibacterial concentration (μg/ml) H. rhamnoides treatment E. coli B. cereus S. aureus - >

279 Table Examination of antibacterial performance against gram positive and gram negative bacteria. Clear zone (mm) H. rhamnoides treatment E. coli B. cereus S. aureus : No clear zone, + : 7 9 mm Clear zone, ++ : 9 12 mm Clear zone, +++ : > 12 mm Clear zone

280 Fig Large gel 2-DE separation of total protein lysate of MEF cell

281 Table Comparative proteomic analysis of oxidative stress and H. rhamnoides extract using 2-DE. Spo t Accession number Protein name PI M. W (kda) Sequence covered Score ratio Reported function 1 gi peroxiredoxin % up Prx4 plays a role in inhibition of NF-kB (nuclear factor 'kb) function as a cytosolic factor Arp2/3 complex is 2 gi Actin-related protein % down major role in the regulation of the actin cytoskeleton Ratio: ratio of H. rhamnoides L. / H 2 O

282 Fig The peptide mass fingerprinting of peroxiredoxin

283

284 Fig Large gel 2-DE separation of total protein lysate of AGS cells

285

286

287

288

289 Fig 4-1. Climate information of investigate site

290 Table 4-1. Germination properties of H. rhamnoides seeds at different temperatures. Temperature ( ) GP (%) MGT (day) GR GPI

291 Table 4-2. Quadratic regression equations, r2 value and cardinal temperatures (Tb, Tm and To) for influence of temperature on H. rhamnoides seed germination. Characteristic Regression equations r 2 T b a T m T o a GP y = x x * Base(T b ), maximum(t m ), and optimum(t o ) temperatures ( ), respectively. * : p<

292 Table 4-3. Regression equations, r2 and cardinal temperatures (Tb, Tm and To) by linear sub- and supra-optimal models for germination rate in H. rhamnoides seed as a function of temperature response. a Characteristic Function type Model r 2 T b a GR Sub-optimal Supra-optimal y = 0.290x y = x * * - Base(T b ), maximum(t m ), and optimum(t o ) temperatures ( ), respectively. * : p<0.05. T m T o

293 Fig Regression analysis models of GP and GR in H. rhamnoides seed as a function of temperature response

294

295

296 Table 4-4. Growth characteristics of H. rhamnoides seedling according to light conditions. Light condition Stem length(cm) Root collar diameter(mm) Survival(%) full light % light % light % light

297 Fig 4-3. Growth characteristics of H. rhamnoides seedling according to light quality conditions

298 Fig 4-4. Electrical conductivity in leachate of H. rhamnoides seed by seed age

299 Fig 4-5. Inorganic compounds in leachate of H. rhamnoides seed by seed age

300 Table 4-5. Change of soil properties before and after H. rhamnoides seedling planting Classification Rep. ph Before After OM (%) T-N (%) EP (mg/kg) CEC (cmol + /kg) EC (Ms/m) Salinity (%) Mean Mean

301

302 Fig 4-6. Germination of H. rhamnoides seeds with different irrigation intervals

303 Fig 4-7. Height and survival of H. rhamnoides seedlings with different irrigation intervals

304 Fig 4-8. Growth of H. rhamnoides seedlings with different irrigation intervals

305 Fig 4-9. Height and root collar diameter of H. rhamnoides seedlings by various fertilization condition

306 Fig Chlorophyll contents of H. rhamnoides seedlings by various fertilization condition