. 2015 11 17 : : : : : : : : Li Meishan : : : : : : ( ) : : : - 2 -
Ⅰ. Ⅱ.. 4, 1. 2. 3, 2, 2 SCI 5 SCI. Ⅲ. - (in vitro in vivo) - - - - Ⅳ. 1. : in vitro activity-guided purification (C5) ferulic acid. in vivo, IRS-1, PI3K Akt GLUT4.,,, ph. 2. : ( ),,,,. in vitro.. - 3 -
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SUMMARY Ⅰ. Title Industrialization and Development of Foods for Prevention of Diabetes using Germinated Rough Rice Ⅱ. The purpose of the study and performance against targets The purpose of the this study was to investigated the antidiabetic effects of germinated rough rice through the isolation and identification of the antidiabetic substance and to developed a prototypes to serve as a functional food materials. Based on the results, we developed a formulation of the 4 cases of antidiabetic substances containing prototypes (powder, Sunsik, beverage and capsule) and 1 cases was commercialized. Optimized the germination and extraction conditions for high containing the antidiabetic substances and activities. The 2 cases was technology transferred to company. Also, three patents was pending, two case of media relations, two of SCI grade and five of KSCI grade of papers was published. Ⅲ. The contents and the scopes of the study - Investigation of the antidiabetic effects of germinated rough rice extracts - Isolation and identification of the antidiabetic substance from germinated rough rice - Investigation of the antidiabetic mechanism and standardization of active compound - Optimization of the germination and extraction conditions - Mass production methods study - Development of the prototype and optimum formulation setting Ⅳ. Results of the study Part 1 : Chungbuk National University Investigation of the in vitro and in vivo antidiabetic effects of germinated rough rice extracts through the antidiabetic related enzyme inhibition activity and glucose uptake on the 3T3-L1 adipocytes. We obtained active fraction from germinated rough rice and identified the ferulic acid as remarkable substance through the several purification steps using activity-guided purification method. We evaluated the in vivo antidiabetic effect using diabetic mouse model and revealed the antidiabetic mechanism in the cells and mouse model. The germinated rough rice extract activated the IRS-1, PI3K, Akt and GLUT4 gene expression. we investigated the content variation of the remarkable substance according to the manufacturing process and presented the standard and references. We evaluated the storage stability of remarkable - 5 -
substance and prototypes related the temperatures, light expose and ph. Part 2 : Fungi and Plants We optimized the germination conditions (temperature and period) and extraction methods (temperature and solvent). We developed a formulation of the 4 cases of antidiabetic substances containing prototypes (powder, Sunsik, beverage and capsule) and 1 cases was commercialized. The in vitro antidiabetic effect and remarkable substance content of the germinated rough rice prototypes was evaluated. Based on the in vivo study, the human equivalent dose of rough rice was expected and the Sunsik prototype was reformulated. Development of the prototype and optimum formulation setting and mass production methods study. The storage stability of prototypes were evaluated. Ⅴ. The outcomes of the study and further plans - Securing technique for separating the useful components contained in the germination rice - Data utilized for superior selection rice cultivars - Securing the patent related to extraction of the antidiabetic substance form germinated rice - Publishing the papers of antidiabetic effect of germinated rice extract - Health promotion in accordance with the functional food development - 6 -
CONTENTS Chapter 1. Introduction 9 Chapter 2. Overview of current research status of in this field 14 Chapter 3. Contents and results of research and development 16 Chapter 4. Achievement of goals and implications for related research 158 Chapter 5. Research and development outcomes and their application plans 160 Chapter 6. Scientific and technical information from abroad 163 Chapter 7. References 168-7 -
1 9 2 14 3 16 4 158 5 160 6 163 7 168 < >, 172-8 -
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GABA, γ-oryzanol,,.,..,,. /. 1.,. - (in vitro in vivo) - - - - 4, 1, 2. 3, 2, 3 SCI 4 SCI. - 10 -
2. ( ) O in vitro - - α-amylase - α-glucosidase - DPP-4 - β-cell line(hit-t15) insulin - 3T3-L1 O - (,,,, ) - (TLC, MPLC, Prep. HPLC, Open column chromatography ) - (LC/MS, NMR ) O in vitro - 3T3-L1 - FABP4, SCD1, PPARγ, C/EBPα, GLUT4, Akt O in vivo - db/db mouse -,, - in vitro in vitro in vivo 1 1 2 2 O in vivo -,, - FABP4, SCD1, PPARγ, C/EBPα, GLUT4, Akt O -, Open column chromatography, MPLC, prep. HPLC - HPLC O -,, ph - in vivo 3 3-11 -
O - (,,, ) - (,,, ) - 1 O -, : - - - - : - - - / - : - - - - - : - - - - - :,, -, - - O in vitro - α-amylase - α-glucosidase - DPP-4 in vitro O - -, - 1 2 2 O (Pilot ) - / - O - - 3 3-12 -
1. 1 2 3 O in vitro 100 O 100 O 100 O 100 O in vitro 100 O in vivo 100 O in vitro 100 O 100 O in vivo 100 O 100 O 100 O 100 O 100 2. 3 2 1 3 4 6 2 SCI SCI ( ) 3 1 1 2 5 8 2 (%) 100 50 100 66 125 133 100-13 -
,.,,,,.,,,,,,,,.,, 70%,,. 1.,. GABA. GABA,,,. - 14 -
,.,,,.,. (sulfonylurea drug) (biguanide). 2.,,,,,.,,,. - 15 -
1.. 2011 (Oryza sativa L.; Ilpum). Lee (2008)., 1 2 3. (37 ) (85%) (WGC-450, Dahan Inc, Seoul, Korea) 6, 1 2. ( ), 50 (WFO-459PD, EYELA, Tokyo, Japan) 2.. (FC2K, Kett Inc., Tokyo, Japan) (hull & sprout; HS) (Brown rice; BR), (Micro hammer cutter mill type-3, Culatti AG, Zurich, Switzerland) 80 mesh -20.. 10 80% (v/v) 80 3,, -20.. in vitro (1) α-glucosidase α-glucosidase Tibbot Skadsen (1996)., α-glucosidase ρ-nitrophenyl-α-d-glucopyranoside (ρnpg) 0.1 M sodium phosphate buffer (ph 7.0), 50 μl 0.35 unit/ml α-glucosidase 100 μl 37 10 1.5 mm ρnpg 50 μl 3 7 20. 1 M Na 2 CO 3 1 ml ELISA 405 nm (%), positive control acarbose (Sigma-Aldrich, St. Louis, MO, USA). - 16 -
(2) α-amylase α-amylase Lim (2005)., 125 μl 12 unit/ml pancreatin α-amylase 62.5 μl, 200 mm potassium phosphate buffer (ph 6.8) 62.5 μl 37 10 1% starch 125 μl 37 5. 48 mm DNS (3,5-dinitrosalicylic acid and 30% sodium potassium tartrate in 0.5 M NaOH) 125 μl 100 15, 3 ELISA 540 nm (%). (3) Dipeptidyl peptidase-4 Dipeptidyl peptidase-4 (DPP-4) DPP-4 Inhibitor Screening Kit (Cat. K780-100, Biovision Co., Milpitas, CA, USA)., DPP-4 enzyme 1 μl enzyme buffer 49 μl enzyme buffer DPP-4 Substrate 2 μl enzyme buffer 23 μl substrate. 96-Well plate 25 μl DPP-4 Enzyme buffer 50 μl 37 10 Substrate 25 μl 0 30 37 460 nm (%). kinetic 360 nm (4) 3T3-L1 ( ) 3T3-L1 3T3-L1 ATCC (American Type Culture Collection, Manassas, VA, USA), DMEM (Dulbecco's modified Eagle's medium) 10% BCS (bovine calf serum) 1% penicillin/streptomycin, 37, 5% CO 2 2 3. 3T3-L1 80 90% confluence 0.25% trypsin-edta suspension 6-well plate confluent 48. post-confluent IBMX (0.5 mm) dexamethasone (0.25 μm), insulin (5 μg/ml), 10% FBS differentiation medium. 2 5 μg/ml insulin 10% FBS DMEM. 4, 6 10% FBS DMEM. - 17 -
( ) 3T3-L1 2 3 2 10% FBS DMEM 7 10. PBS Serum-free DMEM 37 6 Krebs Ringer Phospate Hepes buffer (KRPH buffer; 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 1.3 mm CaCl 2, 118 mm NaCl, 5 mm KCl, 30 mm Hepes, ph 7.5) 2% BSA KRPH buffer 3T3-L1 2-Deoxyglucose Uptake Measurement kit (COSMO BIO CO., LTD, Tokyo, Japan)., 2% BSA KRPH buffer 1 um insulin, 1 mm 2-Deoxyglucose 37 20 200 um phloretin cooled PBS., 10 mm Tris-HCl buffer (ph 8.0) well 3 ml. Cell lyssate tube 80 15 4 15,000 g 20 tube 1 sample diluent buffer (1:4) kit solution 2-Deoxyglucose Uptake Measurement kit 420 nm, 25 30 30 5 kinetic (Zou, Wang & Shen, 2005).. (1) Angiotensin converting enzyme I Angiotensin converting enzyme I (ACE) Kwon (2006). ACE 5 mm HHL (Hippuryl-His-Leu) 0.3 M NaCl 0.1 M potassium phosphate buffer (ph 8.3), 0.2 mu ACE 0.1 M potassium phosphate buffer (ph 7.0). 100 μl 0.2 mu ACE 80 μl 5 mm HHL 100 μl 37 60. 1 M HCl 250 μl 0.45 μm syringe filters, HPLC (ACME 9000 system, Younglin, Anyang, Korea) ACE (%). C-18 column (Mightysil RP-18 GP column, 4.6 250 mm, Kanto Chemical, Tokyo, Japan), mobile phase A 10 mm phosphoric acid (ph 2.5), B methanol A:B 100:0 12 40:60, 19 0:100, 25 100:0 gradient system. Flow rate 1.0 ml/min, injection volume 20 μl, detector UV-detector (228 nm). - 18 -
(2) Xanthine oxidase Xanthine oxidase Stirpe Corte (1969)., 0.1 ml 0.1 M potassium phosphate buffer (ph 7.5) 0.6 ml, 2 mm xanthine 0.2 ml, 0.2 unit/ml xanthine oxidase 0.1 ml 37 5. 1 N HCl 1 ml uric acid ELISA 292 nm (%). (3) Pancreatic lipase Lipase Kim (2007)., porcine pancreatic lipase 0.3 mg 10 mm MOPS/1 mm EDTA (ph 6.8) 60 μl 100 mm Trice-HCl/ 5 mm CaCl 2 (ph 6.8) 800 μl enzyme buffer. Enzyme buffer 100 μl 37 15 10 mm ρ-npb 40 μl 37 15. ρ-npb ρ-nitrophenol ELISA 400 nm (%).. SPSS (Statistical Package for the Social Science, Ver. 12.0, SPSS Inc., Chicago, IL, USA) one-way ANOVA (analysis of variance) P<0.05 Duncan's multiple range test. 2.. (1) (Hull & sprout : HS) (Brown rice : BR) Table 1-1. HS 19.61%, 1 20.37% (p>0.05), 2 6 32.60% (p<0.05). HS BR 1 0.244 0.256 (p>0.05), 2 HS (p<0.05). - 19 -
Table 1-1. Changes in the contents of brown rice and hull & sprout with different germination time, and the ratio of hull & sprout and brown rice. Germination time (day) Brown rice (BR, %) Hull & sprout (HS, %) HS/BR 0 80.39±1.08 a1) 19.61±1.08 f 0.244±0.017 f 1 79.63±0.69 a 20.37±0.69 f 0.256±0.011 f 2 77.67±1.05 b 22.33±1.05 e 0.288±0.017 e 3 74.46±1.10 c 25.54±1.10 d 0.343±0.020 d 4 72.35±0.59 d 27.65±0.59 c 0.382±0.011 c 5 69.47±0.82 e 30.53±0.82 b 0.440±0.017 b 6 67.40±0.71 f 32.60±0.71 a 0.484±0.016 a 1) Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (2), (RR), (BR), (HS) Table 1-2, 1-3. BR(7.20 21.37%), RR(7.07 20.03%), HS(6.55 14.56%), (p<0.05). BR(6.56 17.36%), RR(6.31 16.21%), HS(5.31 11.59%), (p<0.05).. Table 1-2. Changes in the water extraction yields of rough rice, brown rice and hull & sprout with different germination time. Germination time (day) Rough rice (RR, %) Brown rice (BR, %) Hull & sprout (HS, %) 0 7.07±0.15 f1) 7.20±0.21 f 6.55±0.17 f 1 7.41±0.28 f 7.47±0.34 f 7.14±0.07 e 2 9.35±0.31 e 9.83±0.36 e 7.44±0.13 e 3 12.02±0.21 d 12.91±0.31 d 8.47±0.21 d 4 14.74±0.12 c 15.51±0.20 c 11.68±0.27 c 5 17.69±0.71 b 18.74±0.88 b 13.50±0.27 b 6 20.03±0.74 a 21.37±0.85 a 14.66±0.53 a 1) Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 20 -
Table 1-3. Changes in the ethanol extraction yields of rough rice, brown rice and hull & sprout with different germination time. Germination time(day) Rough rice (RR, %) Brown rice (BR, %) Hull & sprout (HS, %) 0 6.31±0.11 g 6.56±0.16 g 5.31±0.13 g 1 7.73±0.19 f 8.12±0.23 f 6.18±0.05 f 2 9.86±0.21 e 10.45±0.27 e 7.48±0.07 e 3 11.57±0.23 d 12.42±0.32 d 8.19±0.16 d 4 14.32±0.26 c 15.54±0.36 c 9.46±0.11 c 5 15.43±0.16 b 16.73±0.17 b 10.26±0.15 b 6 16.21±0.56 a 17.36±0.65 a 11.59±0.55 a 1) Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test.. in vitro (1) α-glucosidase α-glucosidase Fig. 1-1. 20 mg/ml 10 mg/ml 4 α-glucosidase (p<0.05), HS a-glucosidase (p<0.05). (Fig. 1-1. (A)) 20 mg/ml 4 HS 38.35% α-glucosidase, (Fig. 1-1. (B)) 10 mg/ml 5 HS 39.41%. - 21 -
Fig. 1-1. Changes in α-glucosidase inhibition activities of germinated rough rice extracts with different germination times and parts. (A) water extracts, (B) ethanol extracts. Different capital and small letters in the same row indicate a significant difference (p<0.05) among different germination times and parts, respectively. (2) α-amylase α-amylase Fig. 1-2. 2 mg/ml 0.5 mg/ml (Day 0 2) (Day 3 6) α-amylase, α-amylase BR HS. (Fig. 1-2. (A)) (Day 3 6) HS 72.96 76.75% α-amylase, (Fig. 1-2. (B)) HS 72.37 75.32% α-amylase. (3) Dipeptidyl peptidase-4 Dipeptidyl peptidase-4 (DPP-4) Fig. 1-3 4, 5, HS BR, DPP-4. (Fig. 1-3. (A)) HS 4 40.97% 34.09%, BR 6.87% 19.32% BR 2.93%. (Fig. 1-3. (B)) 0 HS DPP-4 34.09%, 4, 5 45.88 47.77%, BR 4, 5 4.01% 8.96%. - 22 -
Fig. 1-2. Changes in α-amylase inhibition activities of germinated rough rice extracts with different germination times and parts. (A) water extracts, (B) ethanol extracts. Different capital and small letters in the same row indicate a significant difference (p<0.05) among different germination times and parts, respectively. Fig. 1-3. Changes in dipeptidyl peptidase-4 (DPP-4) inhibition activities of germinated rough rice extracts with different germination times and parts. (A) water extracts, (B) ethanol extracts. Different capital and small letters in the same row indicate a significant difference (p<0.05) among different germination times and parts, respectively. (4) 3T3-L1-23 -
Table 1-4. Extraction yields of rough rice with different germination time, parts and extraction solvents. Treatments Parts Extraction Solvents Condition Sample code Yields (%) Brown rice 80% EtOH IP-BR_E 6.56±0.16 e1) Nongermination (BR) Water IP-BR_W 7.20±0.21 e Hull & sprout 80% EtOH IP-HS_E 5.31±0.13 f (HS) Water 80, 3 hr, IP-HS_W 6.55±0.17 e Brown rice 80% EtOH 1 times GIP-BR_E 17.36±0.65 b Germination (BR) Water GIP-BR_W 21.37±0.85 a (6 day) Hull & sprout 80% EtOH GIP-HS_E 11.59±0.55 d (HS) Water GIP-HS_W 14.66±0.53 c 1) Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. μ μ μ, (Fig. 1-5). - 24 -
Fig. 1-4. Effect of rough rice extracts with different germination time, parts and extraction solvents on the cell viability and glucose uptake by 3T3-L1 adipocyte. Different small letters in the same row indicate a significant difference (p<0.05). Fig. 1-5. Effect of brown rice (IP-BR), germinated brown rice (IP-GBR) and germinated rough rice extracts (GIP-BR and GIP-HS) on the cell viability and glucose uptake by 3T3-L1 adipocyte. Different small letters in the same row indicate a significant difference (p<0.05).. (1) Angiotensin converting enzyme I angiotensin converting enzyme 1 (ACE) Fig. 1-6 5, ACE HS BR (p<0.05). (Fig. 1-6. (A)) HS 5 40.23% 29.38%, BR 5 4.63% 0.56%. (Fig. 1-6. (B)), HS 43.16 58.60% - 25 -
ACE. Fig. 1-6. Changes in angiotensin converting enzyme 1 (ACE) inhibition activities of germinated rough rice extracts with different germination times and parts. (A) water extracts, (B) ethanol extracts. Different capital and small letters in the same row indicate a significant difference (p<0.05) among different germination times and parts, respectively. (2) Xanthine oxidase xanthine oxidase (XO) Fig. 1-7. Xanthine oxidase XO, (p<0.05). BR HS (p<0.05). (Fig. 1-7. (A)) HS 45.17% XO, HS 36.54 42.58% XO., (Fig. 1-7. (B)) 1 HS 64.49% 62.02%, 6 50.83%. (3) Pancreatic lipase lipase Fig. 1-8.,, lipase (p<0.05). HS BR (p<0.05). (Fig. 1-8. (A)) BR 4.83 7.36% lipase, HS 4 36.78% 31.72% (p<0.05)., (Fig. 1-8. (B)) BR - 26 -
(day 4 6) 12.43 13.70%, 5 HS 55.17% lipase. Fig. 1-7. Changes in xanthine oxidase (XO) inhibition activities of germinated rough rice extracts with different germination times and parts. (A) water extracts, (B) ethanol extracts. Different capital and small letters in the same row indicate a significant difference (p<0.05) among different germination times and parts, respectively. Fig. 1-8. Changes in pancreatic lipase inhibition activities of germinated rough rice extracts with different germination times and parts. (A) water extracts, (B) ethanol extracts. Different capital and small letters in the same row indicate a significant difference (p<0.05) among different germination times and parts, respectively. - 27 -
1.. 3T3-L1 3T3-L1 ATCC (American Type Culture Collection, Manassas, VA, USA), DMEM (Dulbecco's modified Eagle's medium) 10% BCS (bovine calf serum) 1% penicillin/streptomycin, 37, 5% CO 2 2 3. 3T3-L1 80 90% confluence 0.25% trypsin-edta suspension 6-well plate confluent 48. post-confluent IBMX (0.5 mm) dexamethasone (0.25 μm), insulin (5 μg/ml), 10% FBS differentiation medium. 2 5 μg/ml insulin 10% FBS DMEM. 4, 6 10% FBS DMEM. 2 3 2 10% FBS DMEM 7 10. PBS Serum-free DMEM 37 6 Krebs Ringer Phospate Hepes buffer (KRPH buffer; 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 1.3 mm CaCl 2, 118 mm NaCl, 5 mm KCl, 30 mm Hepes, ph 7.5) 2% BSA KRPH buffer 3T3-L1 2-Deoxyglucose Uptake Measurement kit (COSMO BIO CO., LTD, Tokyo, Japan)., 2% BSA KRPH buffer 1 um insulin, 1 mm 2-Deoxyglucose 37 20 200 um phloretin cooled PBS., 10 mm Tris-HCl buffer (ph 8.0) well 3 ml. Cell lyssate tube 80 15 4 15,000 g 20 tube 1 sample diluent buffer (1:4) kit solution 2-Deoxyglucose Uptake Measurement kit 420 nm, 25 30 30 5 kinetic (Zou, Wang & Shen, 2005). - 28 -
. 2011 (Oryza sativa L.; Ilpum). Lee (2008)., 1 2 3. (37 ) (85%) (WGC-450, Dahan Inc, Seoul, Korea) 6, 1 2. 50 (WFO-459PD, EYELA, Tokyo, Japan) 2.., (FC2K, Kett Inc., Tokyo, Japan) (hull & sprout; HS) (Brown rice; BR), (Micro hammer cutter mill type-3, Culatti AG, Zurich, Switzerland) 80 mesh -20. 1 kg 20 80% (v/v) impeller type (PL-SS20, Punglim Co. Ltd., Seoul, Korea) 400 rpm 12. 6 (Adventec No. 2) 45. 10%. n-hexane, chloroform, ethyl acetate n-butanol 3.. (1) 1 Methanol 36 260 mm ethyl acetate. 200 mg/ml MPLC system ethyl acetate methanol 10 100:0, 40 50:50, 50 0:100, 60 0:100 50 ml/min, 100 ml. ethyl acetate methanol 50:50 TLC /. (2) 2 10 ml ethyl acetate, n-butanol water 30 ml 3. - 29 -
(3) 2 Methanol 36 260 mm ethyl acetate. 120 mg/ml ethyl acetate methanol 10 100:0, 40 50:50, 50 0:100, 60 0:100 50 ml/min, 100 ml. ethyl acetate methanol 75:25 TLC /. (4) 1 25 mg/ml. Ethyl acetate methanol 75:25 / 5. 40 ml 30 3. (5) 2 10 mg/ml. Ethyl acetate hexane 50:50. / UV 254 365 nm 6. 40 ml 30 3. (6) HPLC HPLC system(acme 9100 series)., ODS column(5 μm, 46 250 mm, Agilent Eclipse XDB-C18, Agilent Technologies, USA), 0.1% acetic acid acetonitrile (A), 0.1% acetic acid water (B) A B 8 : 92, 2 10 : 90, 27 30 : 70, 50 90 : 10, 51 100 : 0, 60 100 : 0, 70 8 : 92. 10 μl 1.0 ml/min, 280 nm.. 1 (GIP-HS) 3T3-L1-30 -
., silica gel TLC (GIP-HS_E_B_S3_E_S5_T5_T1) 10 μg/ml 146.98%. trans-cinnamic acid (1.14 mg/g fraction) in vitro in vivo 3T3-L1. trans-cinnamic acid phenolic acid caffeic acid, p-coumaric acid ferulic acid. 2, 3 2. (GIP-HS),.. LC-ESI-MS. LC Agilent 1100 series (Agilent Technologies, USA), column ZORBAX 300SB_C18 (1 150 mm, 3.5 μm, Agilent, USA). Mobile phase 0.2% TFA water A, 0.2% TFA acetonitrile B A B 95 : 5, 5 95 : 5, 7 85 : 15, 25 50 : 50, 27 5 : 95, 34 5 : 95, 35 95 : 5, 45 95 : 5 35 μl/min flow rate. MS Hybrid Quadrupole TOF LC/MS/MS Mass Spectrometer (AB Sciex Instrument, CA, USA) ESI mode. Scan range 200~2,000 MHz, scan rate 1 sec/scan, source temperature 120. D 2 O 1 H nuclear magnetic resonance (NMR) 13 C NMR (500, 900 MHz), 2D-HMBC (two Dimension Heteronuclear Multiple Bond Correlation) spectra Advance 500 spectrometer (Bruker Analytic GmbH, Karlsruhe, Germany).. SPSS (Statistical Package for the Social Science, Ver. 12.0, SPSS Inc., Chicago, IL, USA) one-way ANOVA (analysis of variance) P<0.05 Duncan's multiple range test. - 31 -
2.. 3T3-L1 (GIP-HS_E) Table 2-1. 11.59%, 6.50%, 0.76% 1.12%. 97.51%. Table 2-1. Extraction yields of 1 st solvent fractionation fractions of germinated rough rice with various fractionation solvents. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 11.59±0.55 Hull & sprout fraction of germinated Ilpum (GIP-HS) Hexane GIP-HS_E_H 1.62±0.12 b1) Chloroform GIP-HS_E_C 1.14±0.08 bc Ethyl acetate GIP-HS_E_E 0.76±0.02 c Butanol GIP-HS_E_B 1.12±0.07 bc Water GIP-HS_E_W 6.50±0.42 a 1) Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (GIP-HS_E) 1 3T3-L1 100 μg/ml Fig. 2-1. 500 μg/ml 116.54%, 100 μg/ml (80.43%),, 113.09, 109.32 116.61% (p<0.05). (GIP-HS_E_B). - 32 -
Fig. 2-1. Effects of 1 st solvent fractionation fractions of germinated rough rice with various fractionation solvents on glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-1. Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test.. (1) 1 silica gel 3T3-L1 (GIP-HS_E_B) Table 2-2. S2 0.375% 40%. 1 69.74%. 1 3T3-L1 50 μg/ml Fig. 2-2. 100 μg/ml 121.29%, 1 50 μg/ml S6 (100.72%) (p<0.05). S2 S3 125.08% 132.24% (p<0.05). 1 3 (GIP-HS_E_B_S3). - 33 -
Table 2-2. Extraction yields of 1 st silica gel column chromatography fractions of germinated rough rice with different ethyl acetate concentrations. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 11.59±0.55 Butanol GIP-HS_E_B 1.12±0.07 GIP-HS_E_B_S1 0.087 Hull & sprout fraction GIP-HS_E_B_S2 0.375 of germinated Ilpum GIP-HS_E_B_S3 0.114 Ethyl acetate : (GIP-HS) GIP-HS_E_B_S4 0.108 Methanol GIP-HS_E_B_S5 0.054 GIP-HS_E_B_S6 0.028 GIP-HS_E_B_S7 0.016 Fig. 2-2. Effects of 1 st silica gel column chromatography fractions of germinated rough rice with different ethyl acetate concentrations on glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-2. Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (2) 2 3T3-L1 1 (GIP-HS_E_B_S3), 2 Table 2-3. 0.016%, 0.053% 0.050%. 2 95.46%. - 34 -
3 2 3T3-L1 50 μg/ml Fig. 2-3. 1 3 50 μg/ml 137.56%, 2 50 μg/ml 137.40% 1 3 (p>0.05), 178.74% 146.19% (p<0.05). 2 3 (GIP-HS_E_B_S3_E). Table 2-3. Extraction yields of 2 nd solvent fractionation fractions of germinated rough rice with various fractionation solvents. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 11.59±0.55 Butanol GIP-HS_E_B 1.12±0.07 Hull & sprout fraction EtOAc : MeOH GIP-HS_E_B_S3 0.114 of germinated Ilpum Ethyl acetate GIP-HS_E_B_S3_E 0.016 (GIP-HS) Butanol GIP-HS_E_B_S3_B 0.053 Water GIP-HS_E_B_S3_W 0.050 Fig. 2-3. Effects of 2 nd solvent fractionation fractions of germinated rough rice with various fractionation solvents on glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-3. Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 35 -
(3) 2 silica gel 3T3-L1 2 (GIP-HS_E_ B_S3_E) 2 Table 2-4. S2 S3 0.0037% 0.0046%, S1, S4 S5 0.0019%, 0.0022% 0.0021%. 2 91.27%. 3 2 3T3-L1 25 μg/ml Fig. 2-4. 2 50 μg/ml 139.84%, 2 25 μg/ml S1~S3 105.14%, 98.20% 90.96% (p>0.05), S4 S5 163.62% 172.57% (p<0.05). 2 3 5 (GIP-HS_E_B_S3_E_S5). Table 2-4. Extraction yields of 2 nd silica gel column chromatography fractions of germinated rough rice with different ethyl acetate concentrations. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 11.59±0.55 Butanol GIP-HS_E_B 1.12±0.07 EtOAc : MeOH GIP-HS_E_B_S3 0.114 Hull & sprout fraction of germinated Ilpum (GIP-HS) Ethyl acetate GIP-HS_E_B_S3_E 0.016 GIP-HS_E_B_S3_E_S1 0.0019 GIP-HS_E_B_S3_E_S2 0.0037 EtOAc : MeOH GIP-HS_E_B_S3_E_S3 0.0046 GIP-HS_E_B_S3_E_S4 0.0022 GIP-HS_E_B_S3_E_S5 0.0021-36 -
Fig. 2-4. Effects of 2 nd silica gel column chromatography fractions of germinated rough rice with different ethyl acetate concentrations on glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-4. Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (4) 1 TLC 3T3-L1 2 (GIP-HS_E_B_S3_E_S5) 1 Table 2-5. T1 0.00077%, 0.00020~0.00039%. 1 96.00%. Table 2-5. Extraction yields of 1 st thin layer chromatography fractions of germinated rough rice with different elution distances. Sample Solvent Sample code Yields (%) 80% Ethanol GIP-HS_E 11.59±0.55 Butanol GIP-HS_E_B 1.12±0.07 EtOAc : MeOH GIP-HS_E_B_S3 0.114 Ethyl acetate GIP-HS_E_B_S3_E 0.016 Hull & sprout fraction EtOAc : MeOH GIP-HS_E_B_S3_E_S5 0.0021 of germinated Ilpum GIP-HS_E_B_S3_E_S5_T1 0.00077 (GIP-HS) GIP-HS_E_B_S3_E_S5_T2 0.00036 EtOAc : MeOH GIP-HS_E_B_S3_E_S5_T3 0.00020 GIP-HS_E_B_S3_E_S5_T4 0.00039 GIP-HS_E_B_S3_E_S5_T5 0.00029-37 -
3 5 1 3T3-L1 10 μg/ml Fig. 2-5. 2 5 25 μg/ml 141.51%, 1 10 μg/ml T1 111.14% (p>0.05), T2~T4 51.39%, 51.12% 47.89%. T5 162.31% (p<0.05). 1 3 5 5 (GIP-HS_E_B_S3_E_S5_T5). Fig. 2-5. Effects of 1 st thin layer chromatography fractions of germinated rough rice with different elution distances on glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-5. Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (5) 2 TLC 3T3-L1 1 (GIP-HS_E_B_S3_E_S5_T5) 2 Table 2-6. T6 0.000061%, 0.000020~0.000055%. 1 90.59%. - 38 -
Table 2-6. Extraction yields of 2 nd thin layer chromatography fractions of germinated rough rice with different elution distances. Sample Solvent Sample code Yields (%) 80% Ethanol GIP-HS_E 11.59±0.55 Butanol GIP-HS_E_B 1.12±0.07 EtOAc : MeOH GIP-HS_E_B_S3 0.114 EtOAc GIP-HS_E_B_S3_E 0.016 Hull & sprout fraction of germinated Ilpum (GIP-HS) EtOAc : MeOH GIP-HS_E_B_S3_E_S5 0.0021 EtOAc : MeOH GIP-HS_E_B_S3_E_S5_T5 0.00029 GIP-HS_E_B_S3_E_S5_T5_T1 0.000044 GIP-HS_E_B_S3_E_S5_T5_T2 0.000031 EtOAc : Hexane GIP-HS_E_B_S3_E_S5_T5_T3 0.000051 GIP-HS_E_B_S3_E_S5_T5_T4 0.000020 GIP-HS_E_B_S3_E_S5_T5_T5 0.000055 GIP-HS_E_B_S3_E_S5_T5_T6 0.000061 3 5 5 2 3T3-L1 10 μg/ml Fig. 2-6. 1 5 10 μg/ml 150.82%, 2 10 μg/ml T3 102.59% (p>0.05), T3 71.19%. T2, T5 T6 112.59%, 132.84% 119.46% (p<0.05)., T1 146.98% 1 5 (p>0.05), pioglitazone (1 μg/ml) 119.90%. 2 3 5 5 2 1 (GIP-HS_E_B_S3_E_S5_T5_T1) HPLC. - 39 -
Fig. 2-6. Effects of 2 nd thin layer chromatography fractions of germinated rough rice with different elution distances on glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-6. Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (6) 2 TLC HPLC 1 T5 2 HPLC chromatogram Fig. 2-7. 17.32, (Fig. 2-6). 2 T1 12 (32.72 min) UV spectrum (212 and 280 nm) trans-cinnamic acid (Fig. 2-8), T1 1.14 μg/mg. trans-cinnamic acid, in vitro in vivo. 3T3-L1 GIP-HS_E_B_S3_E_S5_T5_T1, transcinnamic acid phenolic compound., HPLC chromatogram UV UV. - 40 -
[A] [D] [B] [E] [C] [F] Fig. 2-7. HPLC chromatograms of 2 nd thin layer chromatography fractions of germinated rough rice. [A]: T1, [B]: T2, [C]: T3, [D]: T4, [E]: T5, and [F]: T6 fraction. Sample code is shown in Table 2-6. - 41 -
[A] [B] [C] [D] Fig. 2-8. Comparison with HPLC chromatograms of 2 nd thin layer chromatography #1 fractions and phenolic compounds found in standard mixture and UV spectrums of cinnamic acid in standard mixture and germinated rough rice. [A]: phenolic compound standard mixture, [B]: GIP-HS_E_B_S3_E_S5_T5_T1, [C]: UV spectrum of trans-cinnamic acid (12) in standard mixture at 32.72 min, [D]: UV spectrum of trans-cinnamic acid in GIP-HS_E_B_S3_E_S5_T5_T1 at 32.72 min. - 42 -
. (1) 1 silica gel (GIP-HS_E_H) 200 mg 2 ml hexane methanol (MeOH) dichloromethane (DCM) (36 260 mm). MeOH, DCM, hexane 0~8 2:98:0, 24 20:80:0, 32 100:0:0, 40 100:0:0 50 ml/min, 20 100% MeOH 100% hexane 200 ml 12. DCM MeOH (20:1, v/v) silica gel TLC. TLC /. 1 (GIP-HS_E_H_S1~S5) Table 2-7. GIP-HS_E_H S1 S3 36.53% 21.75% (p<0.05), S2, S4 S5 8.86%, 11.13% 9.71%., hexane 10%. Table 2-7. The extraction yields of 1 st silica gel column chromatography fractions of germinated rough rice active fraction (GIP-HS_E_H) for the glucose uptake by 3T3-L1 adipocytes. Sample Fraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hexane GIP-HS_E_H 13.52±0.58 Hull & sprout fraction s1 GIP-HS_E_H_S1 36.53±0.84 a1) of germinated Ilpum s2 GIP-HS_E_H_S2 8.86±0.42 e (GIP-HS) s3~s6 GIP-HS_E_H_S3 21.75±0.35 b s7~s9 GIP-HS_E_H_S4 11.13±0.27 c s10~s12 GIP-HS_E_H_S5 9.71±0.31 d 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. 1 (GIP-HS_E_H_S1~S5) 3T3-L1 Fig. 2-9. GIP-HS_E_H 10 μg/ml 168.47%, S5. S1 10 25 μg/ml - 43 -
, 50 μg/ml 132.34% GIP-HS_E_H. S2 S3 GIP-HS_E_H. S4 GIP-HS_E_H. S5 10 μg/ml 209.94% GIP-HS_E_H. 1 μg/ml 131.11% (data not shown). S5 hexane (GIP-HS_E_H) C18 resin. Fig. 2-9. Effects of germinated rough rice active fraction (GIP-HS_E_H) and their 1st silica gel column chromatography fractions (GIP-HS_E_H_S1~S5) with different concentrations on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-7. (2) C18 (GIP-HS_E_H) 500 mg 2 ml hexane methanol (MeOH) C18 (36 260 mm). MeOH, water, hexane 0:100:0 30 50:50:0, 40 100:0:0, 50 100:0:0, 50 100:0:0, 60 0:0:100 50 ml/min. DCM MeOH (20:1, v/v) silica gel TLC. - 44 -
C18 (GIP-HS_E_H_C1~C5) Table 2-8. GIP-HS_E_H C4 28.71% (p<0.05), C2 C3 22.58% 23.43% (p>0.05). C1 5.07%, hexane C5 12%. Table 2-8. The extraction yields of C18 column chromatography fractions of germinated rough rice active fraction (GIP-HS_E_H) for the glucose uptake by 3T3-L1 adipocytes. Sample Fraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hexane GIP-HS_E_H 13.52±0.58 Hull & sprout c1 GIP-HS_E_H_C1 5.07±0.02 d1) fraction of germinated Ilpum c2 GIP-HS_E_H_C2 22.58±0.20 b (GIP-HS) c3 GIP-HS_E_H_C3 23.43±1.10 b c4 GIP-HS_E_H_C4 28.71±0.87 a c5 GIP-HS_E_H_C5 12.10±0.06 c 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. C18 (GIP-HS_E_H_C1~C5) 3T3-L1 Fig. 2-10. GIP-HS_E_H 10 μg/ml 158.79%, C18 10 μg/ml C1 C4 94.53% 103.29% (p>0.05). C2 C3 135.53% 140.41%, GIP-HS_E_H. C5 194.69% GIP-HS_E_H (p<0.05). GIP-HS_E_H_C5 3T3-L1 Fig. 2-11, 5 μg/ml 147.27% 10 μg/ml 145.54% GIP-HS_E_H (p>0.05). C5 C18 methanol hexane 3T3-L1. GIP-HS_E_H_C5, GIP-HS_E_H_C5 10 μg/ml. - 45 -
Fig. 2-10. Effects of germinated rough rice active fraction (GIP-HS_E_H) and their C18 column chromatography fractions (GIP-HS_E_H_C1~C5) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-8. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. Fig. 2-11. Effects of germinated rough rice active fraction (GIP-HS_E_H) and their C18 column chromatography fraction (GIP-HS_E_H_C5) with different concentrations on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-8. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. - 46 -
(3) 1 TLC (GIP-HS_E_H_C5) 100 mg 1 ml hexane silica gel TLC plate (Silica gel 60 F 254 Glass plate, Merck KGaA, Darmstadt, Germany) 1 μl. DCM MeOH (5:95, v/v) TLC plate chamber TLC plate 16 cm TLC plate / UV 254 365 nm silica gel MeOH 40 ml 30 3. 1 (GIP-HS_E_H_C5_T1~T3) Table 2-9. GIP-HS_E_H_C5 T3 45.3%, T1 T2 36.0% 13.3%. Table 2-9. The extraction yields of 1 st thin layer chromatography fractions of germinated rough rice active fraction (GIP-HS_E_H_C5) for the glucose uptake by 3T3-L1 adipocytes. Sample Fraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hull & sprout fraction of germinated Ilpum (GIP-HS) Hexane GIP-HS_E_H 13.52±0.58 C5 GIP-HS_E_H_C5 12.10±0.06 t1 GIP-HS_E_H_C5_T1 36.0 t2 GIP-HS_E_H_C5_T2 13.3 t3 GIP-HS_E_H_C5_T3 45.3 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. 1 (GIP-HS_E_H_C5_T1~T3) 3T3-L1 Fig. 8. GIP-HS_E_H GIP-HS_E_H_C5 10 μg/ml 115.55% 135.64%, TLC 5 μg/ml T1 T3 106.72% 99.82% (p>0.05). T2 126.94%, 10 μg/ml GIP-HS_E_H_C5 (135.64%) (p>0.05). GIP-HS_E_H_C5_T2. - 47 -
Fig. 2-12. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5) and their 1 st thin layer chromatography fractions (GIP-HS_E_H_C5_T1~T3) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-9. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. (4) Preparative HPLC (GIP-HS_E_H_C5_T2) 100 mg/ml 0.45 μm syringe filter Preparative HPLC (SP930D, Younglin, Anyang, Korea). HECTOR-M (250 21.2 mm, RStech Co., Daejeon, Korea), acetonitrile, 3.0 ml/min, UV-detector (Younglin instrument, 203 nm), 100 μl 8 (Fig. 2-13). main minor peak 4,. (GIP-HS_E_H_C5_T2_P1~P4) Table 2-10. GIP-HS_E_H_C5_T2 22~26%. (GIP-HS_E_H_C5_T2_P1~P4) 3T3-L1 Fig. 2-14. GIP-HS_E_H_C5 GIP-HS_E_H_C5_T2 10 5 μg/ml 148.59% 131.96%, Prep. HPLC 5 μg/ml P1 P2 113.87% 117.01%. P3 P4 135.79% 165.53% (p<0.05), (p>0.05). 4-48 -
GIP-HS_E_H_C5_T2. Fig. 2-13. Preparative high performance liquid chromatography (Prep. HPLC) chromatogram of germinated rough rice active fraction (GIP-HS_E_H_C5_T2) for the glucose uptake by 3T3-L1 adipocytes. Table 2-10. The extraction yields of preparative high performance liquid chromatography (Prep. HPLC) fractions of germinated rough rice active fraction (GIP-HS_E_H_C5_T2) for the glucose uptake by 3T3-L1 adipocytes. Sample Fraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hexane GIP-HS_E_H 13.52±0.58 Hull & sprout C5 GIP-HS_E_H_C5 12.10±0.06 fraction of t1 GIP-HS_E_H_C5_T2 13.3 germinated Ilpum p1 GIP-HS_E_H_C5_T2_P1 26.48 (GIP-HS) p3, p4 GIP-HS_E_H_C5_T2_P2 22.33 p6, p7 GIP-HS_E_H_C5_T2_P3 24.61 p2, p5, p8 GIP-HS_E_H_C5_T2_P4 26.58 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. - 49 -
Fig. 2-14. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5_T2) and their preparative high performance liquid chromatography fractions (GIP-HS_E_H_C5_T2 _P1~P4) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-10. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. (GIP-HS_E_H_C5_T2_P1~P4) 3T3-L1 Fig. 2-15. GIP-HS_E_H_C5_T2 Prep. HPLC 153.41% 148.20% (Fig. 2-15[B]). 2 5 μg/ml (p<0.05), 6 (P2+P3) 211.75% (Fig. 2-15[A]). 3 5 μg/ml (p<0.05), 2 (P1+P2+P4) 183.41% (Fig. 2-15[B]). (GIP-HS_E_H_C5_T2)., silica gel C18 GIP-HS_E_H_C5. - 50 -
[A] [B] Fig. 2-15. Effects of combination fractions with preparative high performance liquid chromatography fractions (GIP-HS_E_H_C5_T2 _P1~T4) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-10. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. - 51 -
(5) (C5) (GIP-HS_E_H) C18 (GIP-HS_E_H_C5) GC 14 8 (Table 2-10), C5 Fig. 2-16. GIP-HS_E_H oleic acid 27.60%, plamitic acid palmitoleic acid 23.75%, myristic acid, stearic acid linolenic acid. GIP-HS_E_H_C1~C5 C5 palmitic acid 36.46%, linoleic acid 32.17%. c9,11t-conjugated linoleic acid (CLA), 0.26%. Fig. 2-16. Gas chromatogram of germinated rough rice active fractions (GIP-HS_E_H_C5) on the glucose uptake by 3T3-L1 adipocytes. Peak 1 : myristic acid, Peak 2 : palmitic acid, Peak 3 : stearic acid, Peak 4 : oleic acid, Peak 5 : linoleic acid, Peak 6 : linolenic acid, Peak 7 : c9, t11-conjugated linoleic acid. CLA PPARγ GLUL4, c9,t11-cla PI3-kinase AS160 GLUT4, t10,c12-cla AMPK GLUT4. C5 linoleic acid C5 c9,t11-cla 3T3-L1 Fig. 2-17. Linoleic acid 5 μg/ml 150.09% C5, CLA 1 μg/ml 180.09% C5. CLA. - 52 -
Table 2-10. The fatty acid compositions of germinated rough rice active fraction (GIP-HS_E_H) and their C18 column chromatography fractions (GIP-HS_E_H_C1~C5) for the glucose uptake by 3T3-L1 adipocytes. Fatty acid GIP-HS_E_H Fatty acid composition (%) C1~4 GIP-HS_E_H_ Myristic acid (14:0) 0.50 ± 0.01 e1) 0.40 ± 0.01 e 2.67 ± 0.01 e Palmitic acid (16:0) 23.75 ± 0.04 b 19.18 ± 0.03 c 36.46 ± 0.04 a Palmitoleic acid (16:1) 23.75 ± 0.03 b 19.18 ± 0.01 c nd Stearic acid (18:0) 2.07 ± 0.01 d 1.68 ± 0.02 d 4.34 ± 0.03 d Oleic acid (18:1) 27.60 ± 0.02 a 28.26 ± 0.04 b 19.67 ± 0.05 c Linoleic acid (18:2) 20.27 ± 0.04 c 29.66 ± 0.05 a 32.17 ± 0.04 b Linolenic acid (18:3) 2.07 ± 0.01 d 1.65 ± 0.02 d 2.64 ± 0.03 f c9,t11-cla (18:2) nd nd 0.26 ± 0.01 g t10,c12-cla (18:2) nd nd nd Total 100.00 100.00 100.00 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. nd : not detected. C5 Fig. 2-17. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5), linoleic acid and conjugated linoleic acid with different concentrations on the glucose uptake by 3T3-L1 adipocytes. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. - 53 -
(6) (C5) (GIP-HS_E_H_C5), KOH 3T3-L1 Fig. 2-18. 10 M KOH 1 μg/ml 136.59%, 5 μg/ml C5 (p>0.05).. Fig. 2-18. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5) and their unsaponifiable matters (USM) with different KOH concentrations on the glucose uptake by 3T3-L1 adipocytes. The small letters on the error bars means significant difference at p<0.05 by Duncan's multiple test. (7) γ-oryzanol phytosterol (GIP-HS_E_H) C18 (GIP-HS_E_H_C1~C5) 10 M KOH (USM) γ-oryzanol Table 2-11. γ-oryzanol cycloartenyl ferulate, 2,4-methylene cycloartenyl ferulate, campesteryl ferulate, sitosteryl ferulate campestanyl ferulate γ-oryzanol. GIP-HS_E_H 470.97 mg/100 g, C18 (GIP-HS_E_H_C1~C4) 806.07 mg/100 g, (GIP-HS_E_H_C5) 107.61 mg/100 g γ-oryzanol. 139.53 mg/100 g, 255.76 mg/100 g 14.94 mg/100 g γ-oryzanol - 54 -
, ferulate phytosterol. C5 γ-oryzanol γ-oryzanol, phytosterol. Table 2-11. The γ-oryzanol contents of germinated rough rice active fraction (GIP-HS_E_H) and their C18 column chromatography fractions (GIP-HS_E_H_C1~C5) for the glucose uptake by 3T3-L1 adipocytes. γ-oryzanol content (μg/100 g each fraction) Sample code 2,4-methylene Cycloartenyl Campesteryl Sitosteryl Campestanyl Total -cycloartenyl ferulate ferulate ferulate ferulate ferulate GIP-HS_E_H 68.30 c1) 56.00 c 94.00 b 89.03 b 163.63 b 470.97 b GIP-HS_E_H_C1~4 115.37 a 96.74 c 125.91 a 156.62 a 311.43 a 806.07 a GIP-HS_E_H_C5 17.14 e 11.90 e 56.45 c 16.10 e 6.01 e 107.61 e GIP-HS_E_H_USM 42.50 d 42.93 d 3.17 e 20.90 d 30.03 d 139.53 d GIP-HS_E_H_C1~4_USM 74.63 b 76.31 b 6.14 d 40.55 c 58.14 c 255.76 c GIP-HS_E_H_C5_USM 7.82 f 6.98 f nd nd 0.13 f 14.94 f 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. nd : not detected. total free phytosterol Table 2-12. total free phytosterol 41.23 6.31 mg/100 g, free phytosterol 15.31%. total free phytosterol 55.02 9.13 mg/100 g, free phytosterol 16.59%. BR 76.10 mg/100 g HS 14.49 mg/100 g phytosterol BR. 24.25 mg/100 g total phytosterol, (GIP-HS_E_H) 154.50 mg/100 g total phytosterol, free phytosterol 20.36%. GIP-HS_E_H GIP-HS_E_H_C5 total phytosterol free phytosterol 1004.41 mg/100 g 23.04%. - 55 -
Table 2-12. Changes in the total and free phytosterol contents of rough rice with different isolation procedure for the glucose uptake by 3T3-L1 adipocytes. Phytosterol content Isolation procedure Sample code (mg/100 g each fraction) Free phytosterol Total Free (% of total) Germination IP 41.23 6.31 15.31 GIP 55.02 9.13 16.59 Partition GIP-BR 76.10 12.06 15.85 GIP-HS 14.90 2.46 16.48 Extraction GIP-HS_E 24.25 4.10 16.89 GIP-HS_E_H 154.50 31.45 20.36 GIP-HS_E_C 17.46 3.13 17.92 Solvent fractionation GIP-HS_E_E 11.11 1.75 15.79 GIP-HS_E_B nd nd nd GIP-HS_E_W nd nd nd GIP-HS_E_H_C1 18.83 3.01 16.00 GIP-HS_E_H_C2 49.70 8.11 16.33 C18 column chromatography GIP-HS_E_H_C3 70.03 11.73 16.74 GIP-HS_E_H_C4 15.16 2.39 15.79 GIP-HS_E_H_C5 1004.41 231.40 23.04 1) Any means in the same column followed by the different superscripts are significantly different (p<0.05) by Duncan's multiple test. nd : not detected. phytosterol, total free phytosterol 3T3-L1 (Table 2-13), total free phytosterol 0.785 (p<0.01) 0.851 (p<0.01), free phytosterol (0.924, p<0.01). total phytosterol. Table 2-13. Correlation coefficients among total and free phytosterol content, ratio of free phytosterol and glucose uptake by 3T3-L1 adipocytes. Free phytosterol content Free phytosterol ratio Glucose uptake Total phytosterol content 0.999 *** 0.628 * 0.785 ** Free phytosterol content 1.000 0.756 * 0.851 ** Free phytosterol ratio 1.000 0.924 ** Glucose uptake 1.000 *, **, *** and ns are indicates significant differences among factors by t-test and there are means p<0.05, p<0.01, p<0.001 and not significant, respectively. - 56 -
(8) (C5) Diaion HP-20 (GIP-HS_E_H) 200 mg 2 ml methanol (MeOH) Diaion HP-20 (36 260 mm). 0% MeOH, 50% MeOH, 100 MeOH 100% CHCl 3 1 L.. Diaion Hp-20 (GIP-HS_E_H_C5_D1~D4) Table 2-14. GIP-HS_E_H_C5 D4 50.57% (p<0.05), D1 22.13%, D2 D3 17.15% 9.15%. Table 2-14. Extraction yields of Diaion HP-20 column chromatography fractions of GIP-HS_E_H_C5. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hexane GIP-HS_E_H 13.52±0.58 Hull & sprout fraction C18 GIP-HS_E_H_C5 12.10±0.06 of germinated Ilpum 0% methanol GIP-HS_E_H_C5_D1 22.13±0.85b (GIP-HS) 50% methanol GIP-HS_E_H_C5_D2 17.15±0.12c 100% methanol GIP-HS_E_H_C5_D3 9.15±0.08d 100% chloroform GIP-HS_E_H_C5_D4 50.57±1.04a 1)Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. Diaion Hp-20 (GIP-HS_E_H_C5_D1~D4) 3T3-L1 Fig. 2-19. GIP-HS_E_H_C5 5 μg/ml 144.74%, Diaion Hp-20 5 μg/ml D3 204.39% (p<0.05). D1 D4 165.79% 159.65% (p>0.05), D2 140.35%. D3 GIP-HS_E_H_C5_D3. - 57 -
Fig. 2-19. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5) and their Diaion HP-20 column chromatography fractions (GIP-HS_E_H_C5_D1~4) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-14. The small letters on the error bars means significant difference at P<0.05 by Duncan's multiple test. (9) Prep. HPLC (GIP-HS_E_H_C5_D3) 100 mg/ml 0.45 μm syringe filter Preparative HPLC (SP930D, Younglin, Anyang, Korea). HECTOR-M (250 21.2 mm, RStech Co., Daejeon, Korea), acetonitrile A, water B 0-10 9:95, 30 30:70, 50 100:0, 90 100:0, 100 5:95, 3.0 ml/min, UV-detector (Younglin instrument, 203 nm), 100 μl 4. (GIP-HS_E_H_C5_D3_P1~P4) Table 2-15. GIP-HS_E_H_C5_D3 P4 54.08% (p<0.05), P3 26.21%, P2 P1 15.14% 4.57%. (GIP-HS_E_H_C5_D3_P1~P4) 3T3-L1 Fig. 2-20. GIP-HS_E_H_C5 GIP-HS_E_C5_D3 5 μg/ml 136.21% 163.55%. Prep. HPLC 5 μg/ml P3 1194.85% (p<0.05). P2 P4 141.30% - 58 -
156.52% (p>0.05), P1 122.83%. D3 GIP-HS_E_H_C5_D3_P3. Table 2-15. Extraction yields of preparative HPLC fractions of GIP-HS_E_H_C5_D3. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hexane GIP-HS_E_H 13.52±0.58 C18 GIP-HS_E_H_C5 12.10±0.06 Hull & sprout fraction Diaion HP-20 GIP-HS_E_H_C5_D3 9.15±0.08 of germinated Ilpum p1 GIP-HS_E_H_C5_D3_P1 4.57±0.12d (GIP-HS) p2 GIP-HS_E_H_C5_D3_P2 15.14±0.09c p3 GIP-HS_E_H_C5_D3_P3 26.21±0.17b p4 GIP-HS_E_H_C5_D3_P4 54.08±1.11a 1)Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. Fig. 2-20. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5_D3) and their preparative HPLC fractions (GIP-HS_E_H_C5_D3_P1~4) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-15. The small letters on the error bars means significant difference at P<0.05 by Duncan's multiple test. - 59 -
(10) Recycling prep. HPLC (GIP-HS_E_H_C5_D3_P3) 100 mg/ml 0.45 μm syringe filter Recycling preparative HPLC (JAI, Tokyo, Japan). HECTOR-M (250 21.2 mm, RStech Co., Daejeon, Korea), methanol, 3.0 ml/min, UV-detector (Younglin instrument, 203 nm), 100 μl 2. Re. HPLC (GIP-HS_E_H_C5_D3_P3_R1~R2) Table 2-16. GIP-HS_E_H_C5_D3_P3 R1 23.42%, R2 76.58%. Re. HPLC (GIP-HS_E_H_C5_D3_P3_R1, R2) 3T3-L1 Fig. 2-20. GIP-HS_E_H_C5 D3 P3 5 μg/ml 139.19%, 156.89% 174.89%. Re. HPLC 5 μg/ml R1 169.64% P3 (p>0.05), R2 123.20%. R2. Table 2-16. Extraction yields of recycling HPLC fractions of GIP-HS_E_H_C5_D3_P3. Sample Extraction Sample code Yields (%) 80% Ethanol GIP-HS_E 28.88±1.00 Hexane GIP-HS_E_H 13.52±0.58 Hull & sprout fraction C18 GIP-HS_E_H_C5 12.10±0.06 of germinated Ilpum Diaion HP-20 GIP-HS_E_H_C5_D3 9.15±0.08 (GIP-HS) Prep. HPLC GIP-HS_E_H_C5_D3_P3 26.21±0.17 r1 GIP-HS_E_H_C5_D3_P3_R1 23.42±0.42b r2 GIP-HS_E_H_C5_D3_P3_R2 76.58±1.48a 1)Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 60 -
Fig. 2-21. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5_D3_P3) and their recycling HPLC fractions (GIP-HS_E_H_C5_D3_P3_R1~2) on the glucose uptake by 3T3-L1 adipocytes. Sample code is shown in Table 2-15. The small letters on the error bars means significant difference at P<0.05 by Duncan's multiple test.. (GIP-HS_E_H_C5_D3_P3_R1) HPLC chromatogram (Fig. 2-22 [A]). LC-ESI-MS 194 (Fig. 2-22 [B]), 1 H-NMR (Fig. 2-22 [C]) 13 C-NMR (Fig. 2-22 [D]) ferulic acid (Fig. 2-22 [E]).. phenolic acid ferulic acid (C5) phenolic acid Fig. 2-23 [A]. C5 caffeic acid, trans-cinnamic acid, p-coumaric acid, ferulic acid, gallic acid sinapic acid, gallic acid 10 μg/ml 186.8% ferulic acid 103.5%. ferulic acid gallic acid 1, 5 10 μg/ml ferulic acid 5 μg/ml 174.7%, gallic acid 10 μg/ml 169.8%. C5 ferulic acid C5, ferulic acid C5. - 61 -
Fig. 2-22. Analytical HPLC chromatogram [A], ESI-LC-MS chromatogram [B], 13C-NMR [C] and 1H-NMR [D] spectrum and structure [E] of reference substance for anti-diabetic effect from germinated rough rice extract. [A] [B] Fig. 2-23. Effects of active fraction (GIP-HS_E_H_C5) and major phenolic acids on the glucose uptake by 3T3-L1 adipocytes with different concentrations. The small letters on the error bars means significant difference at p<0.05 by Duncan s multiple range test. - 62 -
1.. (GIP-HS_E_H_C5; C5).,. Pioglitazone hydrochloride (pioglitazone, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan).. 3T3-L1 ATCC (American Type Culture Collection, Manassas, VA, USA), DMEM (Dulbecco's modified Eagle's medium) 10% BCS (bovine calf serum) 1% penicillin/streptomycin, 37, 5% CO 2 2 3. 3T3-L1 80 90% confluence 0.25% trypsin-edta suspension 6-well plate confluent 48. post-confluent IBMX (0.5 mm) dexamethasone (0.25 μm), insulin (5 μg/ml), 10% FBS differentiation medium. 2 5 μg/ml insulin 10% FBS DMEM. 4, 6 10% FBS DMEM. 2 3 2 10% FBS DMEM 7 10. PBS Serum-free DMEM 37 6 Krebs Ringer Phospate Hepes buffer (KRPH buffer; 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 1.3 mm CaCl 2, 118 mm NaCl, 5 mm KCl, 30 mm Hepes, ph 7.5) 2% BSA KRPH buffer 3T3-L1 2-Deoxyglucose Uptake Measurement kit (COSMO BIO CO., LTD, Tokyo, Japan)., 2% BSA KRPH buffer 1 um insulin, 1 mm 2-Deoxyglucose 37 20 200 um phloretin cooled PBS., 10 mm Tris-HCl buffer (ph 8.0) well 3 ml. Cell lyssate tube 80 15-63 -
4 15,000 g 20 tube 1 sample diluent buffer (1:4) kit solution 2-Deoxyglucose Uptake Measurement kit 420 nm, 25 30 30 5 kinetic 2005). (Zou, Wang & Shen,. (Immuno-fluorescence method) 3T3-L1 8 well chamber slide 2 7. 2 10% FBS high glucose DMEM. serum free DMEM 3% bovine serum albumin (BSA) 12, 5. 5 1 50 μg/ml 10. phosphate buffer saline (PBS) 4% formaldehyde in PBS 10. 10% normal goat serum in 0.1% PBS-tween 20, 1% BSA, 0.3 M glycine 1 blocking, 1 GLUT-4 (1:100, abcam Plc. Boston, MA.) pirs-1 (1:100, Santa cruz biotechnology, Inc. Dallas, Texas) 4 overnight, 2 Alexa Fluor 488 goat-anti-rabbit IgG (1:1000, green, abcam Plc. Boston, MA) 1. DAPI stain, abcam Alexa Fluor 594WGA (red).. Western blot assay 3T3-L1 75 cm 2 flask, 2 7. 2 10% FBS high glucose DMEM. serum free DMEM 3% bovine serum albumin (BSA) 12, 5. 5 1 50 μg/ml 10. phosphate buffer saline (PBS) lysis buffer (50 mm Tris (ph 8.0), 150 mm NaCl, 0.02% sodium azide, 100 μg/ml PMSF, 1 μ g/ml Aprotinin, 1% Triton X-100) 12,000 rpm. Bradford, 30 μg 10% SDS-PAGE gel, protein PVDF membrane 80 V 1 transfer. 5% skim milk in TBS-Tween 20 1 blocking. IRS-1, pirs-1 (Tyr632) (Santa cruz biotechnology, Inc. Dallas, Texas), PI 3 kinase, AKT, pakt (Ser473), PPAR-γ (Cell signalling technology. Beverly, MA), GLUT4 (Santa cruz - 64 -
biotechnology, Inc. Dallas, Texas) 1 antibody 1 30, HRP-conjugated anti-mouse, anti-rabbit IgG (1:2000), anti-goat (1:3000) 2 antibody 1., ECL Kit (WEST-one TM, intronbiotechnology). 2.. (GIP-HS_E_H_C5, C5) (Pioglitazone, PG) 3T3-L1 Fig. 3-1. 1 μm 1 mm 2-deoxy-glucose-6-phosphate (control) C5 1 μg/ml, 5 10 μg/ml 147.27% 180.12%. PG 0.5, 1 0.2 μg/ml 106.20%, 149.88% 220.32%. C5 PG 3T3-L1 (p<0.05). Fig. 3-1. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5) and pioglitazone with different concentrations on the glucose uptake by 3T3-L1 adipocytes. Different capital and small letters in the same row indicate a significant difference (p<0.05) among concentrations by Duncan's multiple range test, respectively. *, **, *** and ns are indicates significant differences among factors by t-test and there are means p<0.05, p<0.01, p<0.001 and not significant, respectively. - 65 -
. Western blot assay 3T3-L1 3T3-L1 in vivo GLUT4 3T3-L1 IRS-1 PI3 kinase AKT. GLUT-4 translocation. (GIP-HS_E_H_C5, C5) (pioglitazone, PG) 3T3-L1 western blot. GLUT-4 C5 (Fig. 3-2), pirs-1 C5 (Fig. 3-3). western blot 1 ng/ml C5 pirs, PI3 kinase, pakt, GLUT-4 (Fig. 3-4). C5. pioglitazone PPAR-γ agonist. C5 PPAR-γ PPAR-γ. Fig. 3-2. Immunofluorescence study of the glucose transporter 4 (GLUT-4). A : control, B : pioglitazone 1 μg/ml, C : insulin 50 ng/ml, D, E and F : germinated rough rice extract (GIP-HS_E_H_C5) at concentrations of 1, 5 and 10 μg/ml, respectively. - 66 -
Fig. 3-3. Immunofluorescence study of the activated insulin receptor substrate 1 (IRS-1). A : control, B : pioglitazone 1 μg/ml, C : insulin 50 ng/ml, D, E and F : germinated rough rice extract (GIP-HS_E_H_C5) at concentrations of 1, 5 and 10 μg/ml, respectively. Fig. 3-4. Regulation of insulin receptor substrate 1 (IRS1), activated IRS1 (pirs1 (Tyr632) )), phosphoinositide 3-kinase (PI 3 Kinase), protein kinase B (AKT) and activated AKT (pakt (Ser473) ), glucose transporter 4 (GLUT4) and peroxisome proliferator-activated receptorγ (PPAR-γ) expression of germinated rough rice active fraction (GIP-HS_E_H_C5) and pioglitazone. - 67 -
1.. (GIP-HS_E_H_C5; C5).,. Pioglitazone hydrochloride (pioglitazone, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan).. 6 C57BLKS/J-db/db C57BLKS/J-db/m+ (Shizuoka Laboratory Animal Center; SLC, Shizuoka, Japan) ( ) 1, 3 1 / C57BLKS/J-db/db 150 mg/dl, 10. (220 200 145 mm 3 ) 2 3, 22±3 C, 50±20%, 10 15 /, 12 / (07:00 19:00) 150 300 Lux. (Teklad certified irradiated global 18% protein rodent diet, Harlan Laboratories, Inc., U.S.A.),. ( : CBNUA-623-13-01).. 2 C57BLKS/J-db/db 150 mg/dl 5. 1), 2), 3) C5 50 mg/kg, 4) C5 100 mg/kg, 5) (pioglitazone 5 mg/kg ). C57BLKS/J-db/m+ 6, C57BLKS/J-db/db C5 10, 7 (Table 4-1). C5 5% cremophor EL, pioglitazone 100 mg/ml dimethylsulfoxide (Sigma-Aldrich Inc., USA.) 1 ml 5 μl. 5% cremophor EL, 10 ml/kg - 68 -
. 1 / 9. Table 4-1. Experimental treatment groups for identification of anti-diabetic effect of germinated rough rice extract C5-fraction (GIP-HS_E_H_C5 ; C5) in C57BLKS/J-db/m+ mice and C57BLKS/J-db/db mice No. Treatment No. of animals G1 Normal control 6 G2 Positive control 10 G3 C5 50 mg/kg 10 G4 C5 100 mg/kg 10 G5 Pioglitazone 5mg/kg 7 G1 : C57BLKS/J-db/m+ mice, G2~G : C57BLKS/J-db/db mice. 1 /,,. (0 ) 1 / 9 10.. 1 /, 2., 48 1. Feed / Water intake = (A gram of the supplying 0 h) (A gram of the remaining 48 h) 2 Days The number of animal in a cage. 0 ( ), 1, 3, 5, 7 9 6. 16,, 1 (GlucoDr PlusTM AGM-3000, Allmedicus Co., Ltd., Anyang, Korea). 8 16., 1 (1 g/kg, Sigma-Aldrich Inc., St. Louis, MO. USA.). 0( ), 30, 60, 90 120. - 69 -
. / diethyl ether.,,,,.,,, 10% (neutral buffered formalin).,,,. serum separating tube (3,000 rpm, 15, 4 ). aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T Chol.), triglyceride, low density lipoproteins (LDL), high density lipoproteins (HDL)... (Insulin, ng/ml) Mouse Insulin ELISA kit (Shibayagi Co., Ltd., Gunma, Japan) kit manual..,, 3~4 μm, Hematoxylin & Eosin (HE). HE.., ±,,,, ±., GRE-C5 5 mg/kg Pioglitazone (one-way analysis of variance, ANOVA) (P<0.05)., Levene test Duncan test ( : 0.05 0.01), Games-Howell test ( : 0.05 0.01). - 70 -
. in vivo (1) 9 C57BLKS/J-db/m+ C57BLKS/J-db/db (gastrocnemius muscle),. (2) Western blot analysis 50 mg tube, protease inhibitor cocktail, phosphatase RIPA buffer (Thermo Fisher Scientific Inc., USA) homogenizer, (13,000 rpm, 10, 4 ). Detergent compatible bradford assay kit (Thermo Fisher Scientific Inc., USA), 6% 10% polyacrylamide gel. nitrocellulose membrane (GE Healthcare Whatman GmbH, Germany) transfer, 5% skim milk (Becton Dickinson and Company, U.S.A.) blocking. Blocking rabbit anti-insulin receptor substrate 1 (IRS1) IgG (Abcam, UK) 1:800, rabbit anti-phospho-insulin receptor substrate 1 Tyr608 (pirs1) IgG (EMD Millipore, USA) 1:800, rabbit anti-phosphatidylinositol 3 kinases (PI 3 K) IgG (Cell Signaling Technology, Inc., USA) 1:800, rabbit anti-akt IgG (Cell Signaling Technology, Inc., USA) 1:1,000, rabbit anti-phospho-akt Ser473 (pakt) IgG (Cell Signaling Technology, Inc., U.S.A.) 1:800, rabbit anti-glucose transporter type 4 (GLUT4) IgG (Abcam, UK) 1:2,500 rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) IgG (Cell Signaling Technology, Inc., USA) 1:2,500 1 4 16 (overnight). 1 goat anti-rabbit IgG HRP conjugate (Cell Signaling Technology, Inc., USA) 1:3,000 2 1, Luminata Crescendo Western HRP substrate (EMD Millipore, U.S.A.), Chemidoc (Lugen Sci. Inc., Korea). Blocking transfer membrane 5 3 4 washing tris buffered saline-0.1% tween 20, ph 7.6 (TBS-T), 1 2 data sheet 5% skim milk 5% BSA TBS-T. ImageJ (http://http://imagej.nih.gov/ij/) (intensity), C57BLKS/J-db/m+ 100. - 71 -
(3) (Immuno-fluorescence) (Leica Mikrosysteme Vertrieb GmbH, Germany) 8 μm, acetone:ethanol = 3:1 20 10. phosphate buffered saline (PBS, GE Healthcare HyClone Laboratories, USA) 3 washing, GLUT4 5% triton X-100 (Sigma Aldrich Co., LLC., USA) PBS 30. 5% bovine serum albumin (BSA; Sigma Aldrich Co., LLC., USA) PBS 1 blocking. rabbit anti-akt IgG 1:50, rabbit anti-pakt IgG 1:50 rabbit anti-glut4 IgG 1:200 1 Akt pakt 4 16 (overnight), GLUT4 4. 2 Akt GLUT4 goat anti-rabbit IgG fluorescein conjugate (EMD Millipore, USA) 1:1,000, pakt goat anti-rabbit IgG rhodamine conjugate (EMD Millipore, USA) 1:1,000. 2 1 μg/ml 4',6-diamidino-2 -phenylindole (DAPI; Sigma Aldrich Co., LLC., USA) 5, fluorescence mounting medium (Agilent Technologies DAKO, Denmark). Blocking TBS-T 5 3 4 washing, 1 2 5% normal goat serum (Vector Laboratories, Inc., USA) PBS. confocal microscopy (Carl Zeiss Microscopy GmbH, Germany),. 2..,. 28.9~32.9 g, 43.5~50.2 g (Fig. 4-1, p<0.01). 50 100 mg/ml C5 42.0~47.2 g 43.6~47.5 g,. 5 mg/kg pioglitazone 43.8~63.3 g, 5 9 (p<0.01). - 72 -
Fig. 4-1. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on changes of body weights in type II diabetic C57BLKS/J-db/db mice for 9 weeks. : Normal control group (C57BLKS/J-db/m+ mice), : Positive control group, : C5 50 mg/kg group, : C5 100 mg/kg group, : Pioglitazone 5mg/kg group. Data are mean ± standard deviation. ** Significantly different from positive control, P<0.01.. 1 3.1~4.3 g, 1 6.6~8.6 g (Table 4-2, p<0.01). 50 mg/ml C5 1 7.2~8.6 g, 3, 5 8 (p<0.05), 100 mg/ml C5 1 5.9~8.5 g, 9 (p<0.05). 5 mg/kg pioglitazone 1 4.1~8.4 g, 3, 6, 7 9 (p<0.05: 7, p<0.01: 3, 6 9 ). - 73 -
Table 4-2. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on changes of feed consumption in type II diabetic C57BLKS/J-db/db mice for 9 weeks. Feed consumption (g) Group Week 0 1 2 3 4 5 6 7 8 9 Normal control 4.3 ± 0.3** 4.1 ± 0.7** 3.9 ± 0.4** 3.9 ± 0.7** 3.5 ± 0.4** 3.3 ± 0.4** 3.7 ± 0.6** 3.8 ± 0.3** 3.1 ± 0.2** 3.3 ± 0.3** Positive control 8.6 ± 1.8 7.2 ± 0.9 6.6 ± 0.7 6.9 ± 0.6 6.6 ± 0.8 6.8 ± 1.3 7.6 ± 1.1 7.3 ± 1.1 6.6 ± 0.6 6.7 ± 0.9 C5 50 mg/kg 8.1 ± 1.0 7.7 ± 0.9 7.6 ± 0.8 7.7 ± 1.3* 7.4 ± 1.0 8.5 ± 1.4* 8.6 ± 1.1 8.2 ± 0.9 7.7 ± 0.9* 7.2 ± 0.8 C5 100 mg/kg 8.5 ± 0.5 8.1 ± 0.8 7.0 ± 0.8 7.0 ± 1.1 7.5 ± 0.7 7.3 ± 0.6 7.1 ± 1.4 6.6 ± 0.7 6.5 ± 0.7 5.9 ± 0.9** Pioglitazone 5 mg/kg 8.4 ± 0.4 7.9 ± 0.3 6.6 ± 0.3 5.9 ± 0.2** 6.5 ± 0.5 6.3 ± 0.4 5.8 ± 0.4** 5.7 ± 0.5* 5.8 ± 1.4 4.1 ± 1.2** Data are mean ± standard deviation. * Significantly different from positive control, P<0.05. ** Significantly different from positive control, P<0.01. 1 1.8~6.2 ml, 1 12.1~25.3 ml (Table 4-3, p<0.05: 5, p<0.01: ). 50 mg/ml C5 1 15.9~29.1 ml, 1 (p<0.05), 100 mg/ml C5 1 11.6~16.3 ml, 9 (p<0.05). 5 mg/kg pioglitazone 1 7.3~13.9 ml, 2, 7, 8 9 (p<0.05: 7 8, p<0.01: 3 9 ). - 74 -
Table 4-3. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on changes of water consumption in type II diabetic C57BLKS/J-db/db mice for 9 weeks. Water consumption (g) Group Week 0 1 2 3 4 5 6 7 8 9 Normal control 6.2 ± 0.7** 5.4 ± 1.5** 5.4 ± 1.2** 4.0 ± 0.9** 2.9 ± 0.5** 1.8 ± 0.1* 3.4 ± 1.7** 2.6 ± 0.3** 2.3 ± 0.1** 2.0 ± 0.3** Positive control 12.4 ± 2.1 12.1 ± 1.9 13.3 ± 2.9 17.3 ± 2.7 18.5 ± 3.4 20.3 ± 4.7 22.9 ± 5.4 22.9 ± 4.2 23.4 ± 3.9 25.3 ± 3.8 C5 50 mg/kg 15.9 ± 2.8 17.6 ± 3.2* 18.4 ± 4.1 23.0 ± 7.9 24.7 ± 7.8 27.2 ± 8.9 28.8 ± 10.1 29.1 ± 10.7 27.9 ± 9.9 27.4 ± 9.1 C5 100 mg/kg 11.6 ± 3.2 13.8 ± 1.7 13.1 ± 3.5 16.0 ± 4.1 16.3 ± 3.6 15.4 ± 3.8 16.0 ± 4.5 15.7 ± 4.8 15.3 ± 4.9 14.6 ± 5.3* Pioglitazone 5 mg/kg 13.9± 2.6 10.1 ± 3.0 8.2 ± 3.6** 8.8 ± 4.0 7.3 ± 4.2 7.4 ± 4.8 7.4 ± 4.8 7.7 ± 4.7* 7.7 ± 4.8* 8.0 ± 5.8** Data are mean ± standard deviation. * Significantly different from positive control, P<0.05. ** Significantly different from positive control, P<0.01.. 70.2~85.0 mg/dl, 175.2~512.9 mg/dl (Fig. 4-2, p<0.01). 50 C5 174.0~499.3 mg/dl,, 100 mg/ml C5 176.5~315.7 mg/dl, (p<0.05: 7 9 ). 5 mg/kg pioglitazone 181.1~182.7 mg/dl, 1 9 (p<0.05: 1, p<0.01: 3-9 ). - 75 -
Fig. 4-2. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on changes of blood glucose in type II diabetic C57BLKS/J-db/db mice for 9 weeks. : Normal control group (C57BLKS/J-db/m+ mice), : Positive control group, : C5 50 mg/kg group, : C5 100 mg/kg group, : Pioglitazone 5mg/kg group. Data are mean ± standard deviation. * Significantly different from positive control, P<0.05. ** Significantly different from positive control, P<0.01.. 8 glucose 0 ( ), 30, 60, 90 120 70.3 mg/dl, 155.3 mg/dl, 126.5 mg/dl, 119.3 mg/dl 102.2 mg/dl, 547.2 mg/dl, 737.2 mg/dl, 712.5 mg/dl, 654.6 mg/dl 636.0 mg/dl (Fig. 4-3, p<0.01). 50 mg/ml C5 475.1 mg/dl, 757.1 mg/dl, 675.0 mg/dl, 635.7 mg/dl 615.6 mg/dl, 100 mg/ml C5 419.1 mg/dl, 628.3 mg/dl, 585.6 mg/dl, 560.8 mg/dl 527.4 mg/dl,. 5 mg/kg pioglitazone 256.1 mg/dl, 385.1 mg/dl, 368.1 mg/dl, 364.1 mg/dl 349.1 mg/dl, 0 120 (p<0.05). - 76 -
Fig. 4-3. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on glucose tolerance test in type II diabetic C57BLKS/J-db/db mice for 9 weeks. : Normal control group (C57BLKS/J-db/m+ mice), : Positive control group, : C5 50 mg/kg group, : C5 100 mg/kg group, : Pioglitazone 5mg/kg group. Data are mean ± standard deviation. * Significantly different from positive control, P<0.05. ** Significantly different from positive control, P<0.01.., ( ) 1.476 g, 0.081 g 0.196 g,, 2.444 g, 0.067 g 0.204 g, (Table 4-4). 50 mg/kg C5, 2.153 g, 0.061 g 0.226 g, 100 mg/kg C5, 2.547 g, 0.063 g 0.196 g,. 5 mg/kg pioglitazone, 4.234 g, 0.086 g 0.184 g,., 4.83%, 0.27% 0.64%,, 5.25%, 0.15% 0.44%,. 50 mg/kg C5, 5.32%, 0.17% 0.58%, 100 mg/kg C5, 5.58%, 0.15% 0.46%,. 5 mg/kg pioglitazone, 7.02%, 0.14% 0.31%,. - 77 -
Table 4-4. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on absolute and relative organ weight in type II diabetic C57BLKS/J-db/db mice for 9 weeks. Absolute organ weight (g) Relative organ weight (%) Group Kidney Kidney Liver Spleen Liver Spleen (mean) (mean) Normal control 1.476 ± 0.114** 0.081 ± 0.014 0.196 ± 0.029 4.83 ± 0.40 0.27 ± 0.05 0.64 ± 0.07* Positive control 2.444 ± 0.498 0.067 ± 0.042 0.204 ± 0.041 5.25 ± 0.96 0.15 ± 0.13 0.44 ± 0.08 C5 50 mg/kg 2.153 ± 0.380 0.061 ± 0.051 0.226 ± 0.035 5.32 ± 0.74 0.17 ± 0.20 0.58 ± 0.22 C5 100 mg/kg 2.547 ± 0.809 0.063 ± 0.027 0.196 ± 0.038 5.58 ± 0.94 0.15 ± 0.06 0.46 ± 0.19 Pioglitazone 5mg/kg 4.234 ± 0.484** 0.086 ± 0.031 0.184 ± 0.025 7.02 ± 0.76** 0.14 ± 0.05 0.31 ± 0.08 Data are mean ± standard deviation. * Significantly different from positive control, P<0.05. ** Significantly different from positive control, P<0.01.. 4.83 ng/ml, 2.26 ng/ml,. 50 100 mg/kg C5 1.54 ng/ml 3.48 ng/ml, 5 mg/kg pioglitazone 8.43 ng/ml, (Fig. 4-4). - 78 -
Fig. 4-4. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on serum insulin concentrations in type II diabetic C57BLKS/J-db/db mice for 9 weeks. : Normal control group (C57BLKS/J-db/m+ mice), : Normal control group (C57BLKS/J-db/m+ mice), : Positive control group, : GRE-C5 50 mg/kg group, : GRE-C5 100 mg/kg group, : Pioglitazone 5mg/kg group.. aspartate transaminase (AST) 43.5 U/L, alanine transaminase (ALT) 28.3 U/L, total cholosterol (T Chol.) 119.7 mg/kg, triglyceride 94.2 mg/kg, low density lippoproteins (LDL) 14.3 mg/kg high density lippoproteins (HDL) 106.2 mg/kg, AST 310.3 U/L, ALT 252.4 U/L, T Chol. 191.6 mg/kg, triglyceride 133.6 mg/kg, LDL 31.2 mg/kg HDL 120.0 mg/kg, AST, ALT, T Chol. HDL (Table 4-5, p<0.05: HDL, p<0.01: AST, ALT T Chol.). 50 mg/kg C5 AST 330.1 U/L, ALT 197.8 U/L, T Chol. 140.9 mg/kg, triglyceride 145.9 mg/kg, LDL 12.0 mg/kg HDL 114.1 mg/kg, 100 mg/kg C5 AST 277.2 U/L, ALT 195.6 U/L, T Chol. 174.3 mg/kg, triglyceride 105.1 mg/kg, LDL 31.1 mg/kg HDL 110.8 mg/kg, 50 mg/kg C5 T Chol.. 5 mg/kg pioglitazone AST 243.4 U/L, ALT 293.1 U/L, T Chol. 186.1 mg/kg, triglyceride 111.7 mg/kg, LDL 48.7 mg/kg HDL 111.0 mg/kg,. - 79 -
Table 4-5. Effects of germinated rough rice active fraction (GIP-HS_E_H_C5; C5) on serum biochemistry in type II diabetic C57BLKS/J-db/db mice for 9 weeks. Serum biochemistry Group AST ALT T Chol. Triglyceride LDL HDL (U/L) (U/L) (mg/dl) (mg/dl) (mg/dl) (mg/dl) Normal control 43.5 ± 2.4** 28.3 ± 1.3** 119.7 ± 4.2** 94.2 ± 8.1 14.3 ± 1.3 106.2 ± 3.4* Positive control 310.3 ± 45.0 252.4 ± 42.1 191.6 ± 11.9 133.6 ± 9.8 31.2 ± 6.8 120.0 ± 0.0 C5 50 mg/kg 330.1 ± 63.1 197.8 ± 28.7 140.9 ± 8.0* 145.9 ± 16.6 12.0 ± 2.8 114.1 ± 3.7 C5 100 mg/kg 277.2 ± 54.4 195.6 ± 16.9 174.3 ± 20.9 105.1 ± 13.3 31.1 ± 9.5 110.8 ± 5.8 Pioglitazone 5mg/kg 243.4 ± 57.1 293.1 ± 56.3 186.1 ± 25.7 111.7 ± 10.7 48.7 ± 11.9 111.0 ± 9.0 Data are mean ± standard deviation. * Significantly different from positive control, P<0.05. ** Significantly different from positive control, P<0.01..,, C5 (Fig. 4-5)., 50 mg/kg 100 mg/kg., β-cell (pancreatic islet) (pancreatic acinus), (renal corpuscle), (proximal or distal tubule) (cortex) (medullar). - 80 -
[ Liver ] [ Pancreas ] [ Kidney ] Fig. 4-5. Representative observations of histopathological lesions of liver, pancreas and kidney in C57BLKS/J-db/m+ mice (A) and C57BLKS/J-db/db mice (B-E). A: Normal control group, B: Positive control group, C: C5 50 mg/kg group, D: C5 100 mg/kg group, E: Pioglitazone 5 mg/kg group. - 81 -
. C57BLKS/J-db/m+ 2 C57BLKS/J-db/db 40%., 2 2, AST ALT. 5 mg/kg piolgitazone pioglitazone., pioglitazone. C5 50 mg/kg,.., 100 mg/kg 30%, 7 9, 14~23%. 5 mg/kg pioglitazone 3.7 pioglitazone, 50 100 mg/kg C5 C5. 50 100 mg/kg C5, 50 100 mg/kg C5., C5 100 mg/kg C57BLKS/J-db/db 2, 2. - 82 -
., (GIP-HS_E_H_C5) (Table 4-6), C5 (GIP) 0.16%, (GIP-HS) 0.47%. (GIP-HS_E) (GIP-HS_E_H) 1.64% 12.10%. C5 100 mg/kg C57BLKS/J-db/db 2. (100 mg/kg/day in mouse) FDA Draft Guideline., K m factor 3 K m factor 37 60 kg 1 C5 486.5 mg., C5 Table 16. Table 4-6. Changes in the extraction yields of rough rice and isolated active fractions wigh different isolation procedure for the glucose uptake by 3T3-L1. Purification steps Weight (g) Yield (% of each fraction) GIP HS E H C5 D3 P3 GIP (4 day) 1,000.000 After polishing 992.608 99.261 BR 657.008 65.701 HS 335.599 33.560 After grinding of HS 331.352 33.135 98.734 GIP-HS_E 96.922 9.692 28.880 GIP-HS_E_H 13.103 1.310 3.904 13.519 GIP-HS_E_H_C5 1.585 0.158 0.472 1.635 12.096 GIP-HS_E_H_C5_D3 0.145 0.015 0.043 0.150 1.107 9.149 GIP-HS_E_H_C5_D3_P3 0.038 0.004 0.011 0.039 0.290 2.398 26.207 GIP-HS_E_H_C5_D3_P3_R1 0.009 0.001 0.003 0.009 0.068 0.562 6.138 23.421 Table 4-7. Changes in the human equivalent dose of rough rice and isolated active fractions with different isolation procedure for improving the anti-diabetic. Active fraction Human equivalent dose (g/60 kg/day) Yield (%) GIP-HS_E_H_C5 0.4865 GIP-HS_E_H 4.0220 12.10 GIP-HS_E 29.7502 1.64 GIP-HS 103.0126 0.47 GIP 306.9511 0.16-83 -
. in vivo C57BLKS/J-db/m+ (hetero; ) C57BLKS/J-db/db 9 (GIP-HS_E_H_C5, C5) 50 mg/kg 100 mg/kg, pioglitazone 5 mg/kg,. IRS1 100% C57BLKS/J-db/db ( ) 31%, C5 100 mg/kg 81% (Fig. 4-6, 4-7). pirs (100%) C5 50 mg/kg 100 mg/kg 127% 142% (Fig. 4-6, 4-7). PI3K (100%) 165%, C5 50 mg/kg 100 mg/kg 190% 301% (Fig. 4-6, 4-8). PI3K Akt pakt GRE-C5, Akt (100%) 78% C5 50 mg/kg 100 mg/kg 97% 108%, pakt (100%) 50% C5 50 mg/kg 100 mg/kg 105% 180% (Fig. 4-6, 4-8). Akt, Akt C5 Akt (Fig. 4-9), pakt C5 pakt (Fig. 4-10). GLUT4 (100%), 65%, C5 50 mg/kg 100 mg/kg 85 94% (Fig. 4-6, 4-8). GLUT4, C5 GLUT4 (translocation) (Fig. 4-10). pioglitazine 5-84 -
mg/kg,, (Fig. 4-11). (C5) 2 C57BLKS/J-db/db IRS1/PI3K pathway, Akt GLUT4,. Fig. 4-6. Effects of active fraction from germinated rough rice on the expressions of the insulin receptor substrate 1 (IRS1), phospho-irs1 (pirs1) Tyr 608, phosphatidylinositol 3 kinases (PI3K), Akt, phospho-akt (pakt)ser473, glucose transporter type 4 (GLUT4) and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) in skeletal muscles of C57BLKS/J-db/db mice or C57BLKS/J-db/db mice. Normal control group: C57BLKS/J-db/m+ mice, Positive control group: C57BLKS/J-db/db mice, db/db: C57BLKS/J-db/db mice. - 85 -
Fig. 4-7. Effects of germinated rice extract C5 fraction (GRE C5) on the expression levels of the insulin receptor substrate 1 (IRS1) and phospho-irs1 Tyr608 (pirs1) in skeletal muscles of C57BLKS/J-db/db mice or C57BLKS/J-db/db mice. : Normal control group (C57BLKS/J-db/m+ mice), : Positive control group (C57BLKS/J-db/db mice), : C57BLKS/J-db/db mice + GRE-C5 50 mg/kg group, : C57BLKS/J-db/db mice + GRE-C5 100 mg/kg group, : C57BLKS/J-db/db mice + Pioglitazone 5mg/kg group. Fig. 4-8. Effects of germinated rice extract C5 fraction (GRE C5) on the expression levels of phosphatidylinositol 3 kinases (PI3K), Akt, phospho-akt Ser473 (pakt) and glucose transporter type 4 (GLUT4) in skeletal muscles of C57BLKS/J-db/db mice or C57BLKS/J-db/db mice. : Normal control group (C57BLKS/J-db/m+ mice), : Positive control group (C57BLKS/J-db/db mice), : C57BLKS/J-db/db mice + GRE-C5 50 mg/kg group, : C57BLKS/J-db/db mice + GRE-C5 100 mg/kg group, : C57BLKS/J-db/db mice + Pioglitazone 5mg/kg group. - 86 -
Fig. 4-9. Representative observations of skeletal muscles stained with Fluorescein sothiocyanate (FITC) for Akt in C57BLKS/J-db/db mice or C57BLKS/J-db/db mice. A: Normal control group (C57BLKS/J-db/m+ mice), B: Positive control group (C57BLKS/J-db/db mice), C: C57BLKS/J-db/db mice + GRE-C5 50 mg/kg group, D: C57BLKS/J-db/db mice + GRE-C5 100 mg/kg group, E: C57BLKS/J-db/db mice + Pioglitazone 5mg/kg group. Green (FITC): Akt, Blue (DAPI): nucleus. - 87 -
Fig. 4-10. Representative observations of skeletal muscles stained with rhodamine for phospho-akt ser473 (pakt) in C57BLKS/J-db/db mice or C57BLKS/J-db/db mice. A: Normal control group (C57BLKS/J-db/m+ mice), B: Positive control group (C57BLKS/J-db/db mice), C: C57BLKS/J-db/db mice + GRE-C5 50 mg/kg group, D: C57BLKS/J-db/db mice + GRE-C5 100 mg/kg group, E: C57BLKS/J-db/db mice + Pioglitazone 5mg/kg group. Green (FITC): Akt, Blue (DAPI): nucleus. - 88 -
Fig. 4-11. Representative observations of skeletal muscles stained with Fluorescein isothiocyanate (FITC) for glucose transporter type 4 (GLUT4) in C57BLKS/J-db/db mice or C57BLKS/J-db/db mice. A: Normal control group (C57BLKS/J-db/m+ mice), B: Positive control group (C57BLKS/J-db/db mice), C: C57BLKS/J-db/db mice + GRE-C5 50 mg/kg group, D: C57BLKS/J-db/db mice + GRE-C5 100 mg/kg group, E: C57BLKS/J-db/db mice + Pioglitazone 5mg/kg group. Green (FITC): Akt, Blue (DAPI): nucleus. - 89 -
1.. 2011. 1, 2,, 7.6%, 18.6%. 596 kg/10 a 27.7 g.,.,. 6.4%, 18.9%. 534 kg/10 a, 21.3 g. 500 g 20 3. 32, 35 37, 85%, 1 1, 1 3 10. 1 4,. 50 2.. (FC2K, Kett Inc., Tokyo, Japan) (hull & sprout; HS) (Brown rice; BR), (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland) 80 mesh -20..,., 80%, 60, 70 80, 3.. HS BR 20 g 400 ml n-hexane shaking water bath 30 12 (400 rpm). 400 ml 2 2 (total lipid). - 90 -
. Folin-Ciocalteu phenol reagent (Dewanto, Xianzhong & Liu, 2002). 100 μl 2% Na 2 CO 3 2 ml 3 50% Folin-Ciocalteu reagent 100 μl 30 750 nm. gallic acid (Sigma-Aldrich Co., St. Louis, MO, USA), g mg gallic acid.. 4 g 80% methanol 40 ml 1 3.. 10% methanol 5 ml 10 ml diethyl ether : ethyl acetate (1:1) 3. methanol 0.45 μm membrane filter. HPLC system(acme 9100 series)., ODS column (5 μm, 46 250 mm, Agilent Eclipse XDB-C18, Agilent Technologies, USA), 0.1% acetic acid acetonitrile (A), 0.1% acetic acid water (B) A B 8 : 92, 2 10 : 90, 27 30 : 70, 50 90 : 10, 51 100 : 0, 60 100 : 0, 70 8 : 92. 10 μl 1.0 ml/min, 280 nm.. 0.5 g (methanol:heptane:bezene:2,2-dimethoxypropane: H 2 SO 4 = 37:36:20:5:2 (v/v)) 2 ml 80 20 hexane. Gas chromatography (Agilent 6850 GC, Agilent Technologies, Palo Alto, CA, USA), Varian VF-WaxMS (30m 300 μm, 0.25 μm, Varian, Palo Alto, CA, USA), flame ionization detector. 250, 300, 120 5 10 230 20. Carrier gas N 2 (99.999%), 1.3 ml/min, 1 μl, split ratio 10:1. myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, conjugated linoleic acid (9c,11t 10t,12c) Sigma-Aldrich peak area. - 91 -
. 2 g 30 ml methanol 10 ml 10 M KOH 90 60. 50 ml. 50 ml diethyl ether 3 ether 50 ml. Ether.. (γ-oryzanol) 2 g 50 ml tube dichloromethane methanol (2:1, v/v) 40 ml 30. 10 (3,000 rpm) (Whatman No. 2). 30 ml 20 ml 2. isoprpanol 1 ml syringe filter (PVDF, 0.45 μm ) HPLC (Acme 9000 system, Younglin, Anyang, Korea). Mightysil RP-18 GP column (4.6 250 mm, 5 μm, Denwato, Kyoto, Japan), methanol : acetonitrile : dichloromethane:acetic acid 50 : 44 : 3 : 3 50, 1.4 ml/min, 20 μ L, UV detector 325 nm.. (phytosterol) total phytosterol free phytosterol., chloroform 0.1 ml 0.6 ml acetic acid 0.4 ml 2.5% ferric chloride 10. 0.2 ml 550 nm. β -sitosterol, total free phytosterol β-sitosterol.. SPSS (Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, USA) one-way ANOVA (Analysis of variation) Duncan s multiple range test (p=0.05). Pearson's correlation analysis. - 92 -
2.. (1) 20 3, 32, 35 37 4, Table 5-1~5-3. 32 1.8~2.2 cm, 90%, 1.6~2.1 cm, 85%. 35 1.7~2.5 cm, 93%, 1.6~2.3 cm, 90%. 37 1.7~2.8 cm, 95%, 1.7~2.5 cm, 92%,. 20 3, 32, 35 37 4,, 4 1.7~2.8cm, 90~95%, 1.6~2.5cm, 85~92%,. Table 5-1. Changes in the sprout length and germination raito of Shindongjin and Ilpum rough rice with different germination time at 32. Time 0 day 1 day 2 day 3 day 4 day Varieties Sindongjin Shape Sprout length 0.5~0.8cm 0.8~1.2cm 1.3~1.7cm 1.8~2.2cm Germination ratio 25% 60% 85% 90% Ilpum Shape Sprout length 0.4~0.7cm 0.8~1.1cm 1.1~1.5cm 1.6~2.1cm Germination ratio 15% 35% 70% 85% - 93 -
Table 5-2. Changes in the sprout length and germination raito of Shindongjin and Ilpum rough rice with different germination time at 35. Time 0 day 1 day 2 day 3 day 4 day Varieties Sindongjin Shape Sprout length 0.4~0.9cm 0.8~1.2cm 1.1~1.8cm 1.7~2.5cm Germination ratio 30% 65% 85% 93% Ilpum Shape Sprout length 0.4~0.8cm 0.7~1.2cm 1.1~1.7cm 1.6~2.3cm Germination ratio 25% 45% 75% 90% Table 5-3. Changes in the sprout length and germination raito of Shindongjin and Ilpum rough rice with different germination time at 37. Time 0 day 1 day 2 day 3 day 4 day Varieties Sindongjin Shape Sprout length 0.5~0.8cm 0.8~1.2cm 1.3~1.7cm 1.8~2.2cm Germination ratio 25% 60% 85% 90% Ilpum Shape Sprout length 0.4~0.7cm 0.8~1.1cm 1.1~1.5cm 1.6~2.1cm Germination ratio 15% 35% 70% 85% - 94 -
(2) (32, 35 37 ) (0~4 ) (hull & sprout; HS ) Table 5-4. (control) 3 (soaked) 5.12% 5.17%, 5.24% 5.34% (p>0.05).,., 37 /4 15.62% 16.69% (p<0.05). Table 5-4. Changes in the ethanol extraction yields of hull & sprout with different germination temperatures and times. Treatment Extraction yield (%) Temp. ( ) Time (day) Shindongjin Ilpum Control 5.12 ± 0.20 j 5.24 ± 0.10 j Soaked (0 day) 5.17 ± 0.10 j 5.34 ± 0.05 j 32 35 37 1 6.12 ± 0.12 i 6.67 ± 0.12 i 2 6.71 ± 0.18 h 7.22 ± 0.11 h 3 7.57 ± 0.36 g 8.20 ± 0.14 g 4 9.09 ± 0.17 e 11.13 ± 0.16 e 1 7.74 ± 0.23 fg 8.17 ± 0.16 g 2 8.24 ± 0.08 f 9.65 ± 0.10 f 3 11.83 ± 0.32 c 12.21 ± 0.28 d 4 13.49 ± 0.25 b 14.52 ± 0.28 b 1 10.72 ± 0.53 d 11.31 ± 0.34 e 2 10.93 ± 0.71 d 12.84 ± 0.35 c 3 13.20 ± 0.22 b 14.60 ± 0.25 b 4 15.62 ± 0.29 a 16.69 ± 0.32 a* * p<0.05 indicates significant differences between groups by Student's t-test. a-c Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 95 -
. (1) 3T3-L1. (32, 35 37 ) (0~4 ) (hull & sprout; HS ) Table 5-5. (control) 3 (soaked) 1.25 mg/g 1.40 mg/g, 1.73 mg/g 1.87 mg/g (p<0.05).,., 37 /4 4.45% 4.63%, 35 /4, 37 /3-4 (p<0.05). Table 5-5. Changes in the total polyphenol content of hull & sprout with different germination temperatures and times. Treatment Total polyphenol content (mg/g) Temp. ( ) Time (day) Shindongjin Ilpum Control 1.25 ± 0.01 i 1.73 ± 0.06 k Soaked (0 day) 1.40 ± 0.10 hi 1.87 ± 0.06 j 32 35 37 1 1.49 ± 0.06 h 2.37 ± 0.03 i 2 1.68 ± 0.07 g 2.59 ± 0.04 h 3 2.36 ± 0.08 e 2.92 ± 0.05 g 4 2.61 ± 0.12 d 3.18 ± 0.07 f 1 1.89 ± 0.10 f 2.70 ± 0.02 h 2 2.29 ± 0.05 e 3.47 ± 0.10 e 3 2.77 ± 0.11 d 3.71 ± 0.07 d 4 3.39 ± 0.14 c 4.19 ± 0.08 c** 1 2.43 ± 0.04 e 3.16 ± 0.07 f 2 3.22 ± 0.17 c 3.61 ± 0.17 d* 3 3.87 ± 0.06 b 4.34 ± 0.12 b** 4 4.45 ± 0.23 a 4.63 ± 0.06 ans * p<0.05, ** p<0.01, *** p<0.001 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test. a-k Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 96 -
(2) (32, 35 37 ) (0~4 ) (hull & sprout, HS ) HPLC. 14 (gallic acid, chlorogenic acid, catechin, caffeic acid, coumaric acid, rutin, ferulic acid, salicylic acid, naringin, hesperidin, myricetin, cinnamic acid, naringenin kaemperol), in vitro in vivo caffeic acid, coumaric acid, ferulic acid cinnamic acid. Table 5-6. Caffeic acid (control) 3 (soaked) 0.73 0.77 μg/g, 37 /4 3.48 μg/g. p-coumaric acid 3.44 4.15 μg/g,, 37 4, 2 3 10.97 20.73 μg/g. Ferulic acid 3.08 3.75 μg/g, 4, 37 /2 26.77 μg/g. cinnamic acid 11.74 13.92 μg/g, 37 4, 3 90.86 μg/g. Table 5-7. Caffeic acid 35 /3 5.96 μg/g, 37. p-coumaric acid, 32 37 4, 37 /3 22.16 μg/g. Ferulic acid coumaric acid, 37 /2 49.55 μg/g. Cinnamic acid, 35 37 4 37 /3 110.75 μg/g., 4,. 37, 3. - 97 -
Table 5-6. Changes in major phenolic compounds content of hull & sprout from Shindongjin with different germination temperatures and times. Treatment Temp. Time Caffeic p-coumaric acid acid ( ) (day) Ferulic acid trans-cinnamic acid Control 0.73 k 3.44 j 3.08 ai 11.74 j Soaked (0 day) 0.77 k 4.15 j 3.75 ai 13.92 j 1 0.92 j 5.60 i 5.46 h 19.18 i 32 2 1.10 i 6.80 h 7.05 g 26.40 h 3 1.68 g 9.57 g 15.44 d 42.62 f 4 2.03 e 12.16 e 12.51 f 45.10 ef 1 1.37 h 6.82 h 7.57 g 31.35 g 35 2 2.04 e 10.94 f 13.08 ef 45.08 ef 3 2.96 c 15.69 c 22.42 b 65.31 d 4 3.28 b 20.73 b 16.20 d 70.37 c 1 1.87 f 14.91 c 13.94 e 46.96 e 37 2 2.80 d 21.97 a 26.77 a 70.72 c 3 3.31 b 21.84 a 23.06 b 90.86 a 4 3.48 a 13.78 d 19.74 c 77.84 b Unit : μg/g hull & sprout, a-k Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. Table 5-7. Changes in major phenolic compounds content of hull & sprout from Ilpum with different germination temperatures and times. Treatment Temp. Time Caffeic p-coumaric acid acid ( ) (day) Ferulic acid trans-cinnamic acid Control 1.11 m 3.78 k 6.66 l 25.45 k Soaked (0 day) 1.20 l 4.40 j 7.37 l 31.25 j 1 1.56 k 7.25 i 10.26 k 44.49 i 32 2 1.98 j 8.14 h 19.49 h 50.69 h 3 2.25 h 9.64 f 23.46 e 66.26 f 4 2.60 g 9.19 g 16.20 j 67.58 f 1 2.12 i 8.98 g 17.72 i 54.87 g 35 2 3.09 f 13.28 e 20.58 g 78.75 e 3 5.96 a 16.35 c 32.02 b 99.23 bc 4 4.27 b 17.50 b 22.29 f 98.09 c 1 2.53 g 14.83 d 27.38 d 66.65 f 37 2 3.38 e 17.51 b 49.55 a 101.64 b 3 3.84 d 22.16 a 29.91 c 110.75 a 4 3.93 c 17.85 b 31.51 b 89.02 d Unit : μg/g hull & sprout, a-k Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 98 -
. 37 3., (Table 5-8)., 80%. Table 5-9. 80%, 80, cinnamic acid. 80% 80 80 80% 70 3. Table 5-8. Changes in the extraction yields and total polyphenol content of hull & sprout from Ilpum rough rice with extraction solvents and temperatures. Treatment Extraction yields Total polyphenol Solvent Temp. ( ) (%) content (mg/g) 60 12.41 ± 0.28 c 1.48 ± 0.04 e Water 70 14.25 ± 0.09 b 2.45 ± 0.10 d 80 16.75 ± 0.07 a 3.25 ± 0.08 c 60 11.50 ± 0.34 d 2.33 ± 0.12 d 80% Ethanol 70 12.60 ± 0.14 c 3.53 ± 0.07 ab 80 14.60 ± 0.25 b 4.34 ± 0.12 a a-k Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. Table 5-9. Changes in major phenolic compounds content of hull & sprout from Ilpum rough rice with different extraction solvents and temperatures. Treatment Caffeic p-coumaric Ferulic trans-cinnamic Solvent Temp. ( ) acid acid acid acid 60 0.893 f 5.795 e 8.978 e 34.355 d Water 70 1.788 d 10.823 c 21.200 c 65.709 b 80 1.640 e 9.537 d 17.673 d 63.314 b 60 1.896 c 11.130 c 20.548 c 50.026 c 80% 70 3.320 a 17.184 a 48.628 a 99.763 a Ethanol 80 2.914 b 15.937 b 30.887 b 99.285 a Unit : μg/g hull & sprout, a-k Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 99 -
., Table 5-10. 1.25% 1.34%, 3 1.27% 1.36%.. 32, 35 37 1.44~2.47%, 1.45~2.50% 1.67~2.56%, 1.55~2.65%, 1.56~2.68% 1.79~2.75%. Table 5-10. Changes in the total lipid contents of rough rice with different germination temperatures and times. Treatment Total lipid content (%, d.b.) Temp. ( C) Time (day) Shindongjin Ilpum Control 1.25 ± 0.02 Bg 1.34 ± 0.01 Ag Soaked (0 day) 1.27 ± 0.03 Bg 1.36 ± 0.02 Ag 1 1.44 ± 0.01 Bf 1.55 ± 0.02 Af 32 2 1.96 ± 0.04 Bd 2.10 ± 0.04 Ad 3 2.26 ± 0.03 Bc 2.43 ± 0.03 Ac 4 2.47 ± 0.07 Bab 2.65 ± 0.05 Ab 1 1.45 ± 0.02 Bf 1.56 ± 0.02 Af 35 2 1.99 ± 0.02 Bd 2.13 ± 0.05 Ad 3 2.44 ± 0.04 Bb 2.62 ± 0.04 Ab 4 2.50 ± 0.07 Ba 2.68 ± 0.06 Ab 1 1.67 ± 0.06 Be 1.79 ± 0.05 Ae 37 2 1.99 ± 0.04 Bd 2.14 ± 0.08 Ad 3 2.50 ± 0.05 Ba 2.68 ± 0.02 Ab 4 2.56 ± 0.09 Ba 2.75 ± 0.04 Aa Results are expressed as the mean ± SD of triplicate samples (n=3). Different capital and small letters in the same row indicate a significant difference (p<0.05) among cultivar and germination conditions by Duncan's multiple range test, respectively. - 100 -
. total free phytosterol phytosterol Table 5-11. 38.43% 38.45%,. 32, 35 37 total phytosterol 40.75~50.55%, 42.55~52.70% 46.45~52.70%, free phytosterol 6.23~8.16%, 6.70~8.54% 7.55~8.59%., free phytosterol phytosterol., 37 3 53.34% 8.90% phytosterol. Table 5-11. Changes in the total and free phytosterol contents and ratio of free phytosterol of Shindongjin with different germination temperatures and times. Treatment Phytosterol content (mg/100 g rough rice) Free phytosterol Temp. Time (% of total) Total Free ( ) (day) Control 38.43 ± 0.12 i 5.87 ± 0.02 i 15.27 h Soaked (0 day) 38.45 ± 0.14 i 5.86 ± 0.04 i 15.24 h 1 40.75 ± 0.15 h 6.23 ± 0.02 h 15.29 h 32 2 46.31 ± 0.11 g 7.14 ± 0.02 g 15.42 g 3 49.18 ± 0.21 e 7.91 ± 0.03 e 16.07 e 4 50.55 ± 0.09 de 8.16 ± 0.02 de 16.13 de 1 42.55 ± 0.12 fg 6.70 ± 0.01 fg 15.74 fg 35 2 49.10 ± 0.15 f 7.83 ± 0.04 f 15.95 f 3 51.70 ± 0.17 ef 8.30 ± 0.02 ef 16.05 ef 4 52.70 ± 0.12 c 8.54 ± 0.03 c 16.20 c 1 46.45 ± 0.11 bc 7.55 ± 0.04 bc 16.26 bc 37 2 51.83 ± 0.15 cd 8.39 ± 0.02 cd 16.19 cd 3 53.34 ± 0.14 a 8.90 ± 0.03 a 16.69 a 4 52.70 ± 0.15 b 8.59 ± 0.05 b 16.31 b Results are expressed as the mean ± SD of triplicate samples (n=3). Different small letters in the same row indicate a significant difference (p<0.05) among germination conditions by Duncan's multiple range test. - 101 -
phytosterol Table 5-12. 41.23% 41.25%,,. 32, 35 37 total phytosterol 43.72~54.24%, 45.65~56.55% 49.84~56.54%, free phytosterol 6.70~8.77%, 7.20~9.18% 8.12~9.24%., free phytosterol, 37 3 57.23% 9.57% phytosterol. Table 5-12. Changes in the total and free phytosterol contents and ratio of free phytosterol of Ilpum with different germination temperatures and times. Treatment Phytosterol content (mg/100 g rough rice) Free phytosterol Temp. Time (% of total) Total Free ( ) (day) Control 41.23 ± 0.12 k 6.31 ± 0.02 k 15.30 i Soaked (0 day) 41.25 ± 0.09 k 6.30 ± 0.02 k 15.27 i 1 43.72 ± 0.08 j 6.70 ± 0.04 j 15.32 i 32 2 49.69 ± 0.12 h 7.68 ± 0.03 h 15.46 h 3 52.77 ± 0.03 f 8.50 ± 0.06 f 16.11 de 4 54.24 ± 0.14 e 8.77 ± 0.07 e 16.17 d 1 45.65 ± 0.08 i 7.20 ± 0.05 i 15.77 g 35 2 52.68 ± 0.12 f 8.42 ± 0.06 f 15.98 f 3 55.47 ± 0.17 d 8.92 ± 0.04 d 16.08 e 4 56.55 ± 0.09 c 9.18 ± 0.05 c 16.23 cd 1 49.84 ± 0.11 g 8.12 ± 0.07 g 16.29 c 37 2 55.61 ± 0.14 cd 9.02 ± 0.05 cd 16.22 cd 3 57.23 ± 0.11 a 9.57 ± 0.04 a 16.72 a 4 56.54 ± 0.12 b 9.24 ± 0.06 b 16.34 b Results are expressed as the mean ± SD of triplicate samples (n=3). Different small letters in the same row indicate a significant difference (p<0.05) among germination conditions by Duncan's multiple range test. - 102 -
total phytosterol Table 5-13. F 14810.00(p<0.001), 5958.96(p<0.001), 291.53(p<0.001) total phytosterol., F 14884.30(p<0.001), 5985.58(p<0.001), 293.71(p<0.001) total phytosterol. 37 3. Table 5-13. Analysis of variance for total phytosterol yield of germinated rough rice. Factor df S.S 1) M.S 2) F-value X 1 2 115.27 57.64 5958.96 *** Shindongjin X 2 3 429.74 143.25 14810.00 *** X 3 6 16.92 2.82 291.53 *** X 1 2 132.68 66.34 5985.58 *** Ilpum X 2 3 494.90 164.97 14884.30 *** X 3 6 19.53 3.26 293.71 *** 1) Sum of square, 2) Mean square, *** : p<0.001 X 1 : Germination temperature ( C), X 2 : Germination time (day), X 3 : X 1 X 2. total phytosterol phytosterol 37 3 total phytosterol Table 5-14. Hexane 30 12 3 Control 53.34% 57.23%., 12 18., total phytosterol. total phytosterol Table 5-15. F 18160.50(p<0.001), 16669.30(p<0.001), 11376.90(p<0.001) total phytosterol. 30 12. - 103 -
Table 5-14. Changes in the total phytosterol content and yield of germinated rough rice with different cultivar and extraction conditions. Treatment Total phytosterol content Total phytosterol yield (mg/100 g rough rice) (%) Temp. Time ( ) (h) Shindongjin Ilpum Shindongjin Ilpum Control 53.34 B 57.23 A 6 19.08 Bi 20.44 Ai 35.77 i 35.71 NSi 30 12 25.22 Bc 27.08 Ac 47.28 c 47.32 NSc 18 26.55 Ba 28.51 Aa 49.78 a 49.81 NSa 6 19.87 Bg 21.31 Ag 37.25 g 37.24 NSg 40 12 24.99 Bd 26.80 Ad 46.85 d 46.83 NSd 18 25.65 Bb 27.53 Ab 48.08 b 48.11 NSb 6 21.41 Be 22.99 Ae 40.14 e 40.17 NSe 50 12 20.33 Bf 21.83 Af 38.11 f 38.15 NSf 18 18.91 Bh 20.30 Ah 35.45 h 35.47 NSh Results are expressed as the average of triplicate samples (n=3). Different capital and small letters in the same row indicate a significant difference (p<0.05) among cultivar and extraction conditions by Duncan's multiple range test, respectively. NS : not significant. Table 5-15. Analysis of variance for total phytosterol yield of germinated rough rice. Factor df S.S 1) M.S 2) F-value X 1 2 235.96 117.98 16669.30 *** X 2 2 257.07 128.54 18160.50 *** X 3 4 322.09 80.52 11376.90 *** 1) Sum of square, 2) Mean square, *** : p<0.001 X 1 : Extraction temperature ( C), X 2 : Extraction time (h), X 3 : X 1 X 2-104 -
1.. 37 3, 50 15, (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland), 10.5 cm x. 2.7 cm 5 g. 100 g 60, 100, 120 140 mesh 250>, 150, 125, <106 μm. (Colorimeter CR-300, Minolta, Tokyo, Japan) (L), (a) (b). 5g 30 ml 0.2 M phosphate buffer (ph 6.9), 320 unit α-amylase 25 14. 80. 2 g 20 ml 1 200 rpm 4,000 rpm 15. 2 g 30 ml 80 shaking water bath 200 rpm 30 (4,000 rpm, 15 min) 80.. 37 3, 50 15, (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland)., 10 12 cm, 8.5 10.5 cm Retort Pouch 35 g.,,,,,,, 14, 5 : 30, 10 : 25, 15 : 20 (w/w). - 105 -
. 100 g 60, 100, 120 140 mesh 250>, 150, 125, <106 μm. (Colorimeter CR-300, Minolta, Tokyo, Japan) (L), (a) (b). 5g 30 ml 0.2 M phosphate buffer (ph 6.9), 320 unit α-amylase 25 14. 80. 2 g 20 ml 1 200 rpm 4,000 rpm 15. 2 g 30 ml 80 shaking water bath 200 rpm 30 (4,000 rpm, 15 min) 80. 35 g 200 g (20~50, ) 10.. 37 3, 50 15. 200g 2 L 1. 30 1 L, 1. 100%, 80% 60% 0.2%.. ph ph meter(orion 4 STAR, Thermo Scientific, USA), (Colorimeter CR-300, Minolta, Tokyo, Japan) (L), (a) (b). (Viscometer, RVT DV-II, Brookfield, USA), spindle No 1 100 rpm., (20~50, ) 10. - 106 -
. 37 3, 50 15. (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland) 80 mesh.,,,,,,, 14 5 : 30, 10 : 25, 15 : 20 (w/w) SiO2(1%), (1.5%), 500 mg (52A/52A)... 20 g 100 ml 20 cm 20., Adobe photoshop CS2 H D.. 20 ml, (37 ). (ph 1.2) NaCl 5g HCl 24 ml 1 L, (ph 6.8) 0.2 M KH 2 PO 4 250 ml 0.2 N NaOH 118 ml 1 L.. SPSS (Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, USA) one-way ANOVA (Analysis of variation) Duncan s multiple range test (p=0.05). Pearson's correlation analysis. - 107 -
2.. 37 3, 50 15, (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland), 10.5 cm x 2.7 cm 5 g (Fig. 6-1, 6-2). Fig. 6-1. The powders of germinated rough rice Shindongjin and Ilpum. Shindongjin Ilpum Fig. 6-2. The prototype of germinated rough rice Shindongjin and Ilpum powders. - 108 -
(1) Fig. 6-3. 250 μm 7.31%, 150 μm 39.90%, 125 μm 41.64%, 106 μm 8.25% 2.91% 125~150 μm. 250 μm 9.77%, 150 μm 49.69%, 125 μm 33.41%, 106 μm 5.68% 1.46% 125~150 μm.,. Fig. 6-3. The size distributions of germinated rough rice Shindongjin and Ilpum powders. (2) Table 6-1. L 96.99, a -4.81, b 7.15, L 98.10, b -4.57, a 6.46.,,. - 109 -
Table 6-1. The color of germinated rough rice Shindongjin and Ilpum powders. Shindongjin Ilpum Lightness (L) 96.99 ± 0.15 98.10 ± 0.07 *** Redness (a) -4.81 ± 0.02-4.57 ± 0.02 *** Yellowness (b) 7.15 ± 0.09 *** 6.46 ± 0.01 *** p<0.001 indicates significant differences between groups by Student's t-test. (3),,,, Table 6-2. (enzymatic digestibility) 23.83%, 24.17%. (water binding capacity) 116.32%, 117.57%. (solubility) 6.73%, 5.96%, (swelling power) 3.12%, 3.00%.,,. Table 6-2. The enzymatic digestibility, water binding capacity, solubility and swelling power of germinated rough rice Shindongjin and Ilpum powders. Shindongjin Ilpum Enzymatic digestibility (%) 23.83 ± 1.37 ns 24.17 ± 0.45 Water binding capacity (%) 116.32 ± 3.38 ns 117.57 ± 1.83 Solubility (%) 6.73 ± 0.49 ns 5.96 ± 0.79 Swelling power (%) 3.12 ± 0.46 ns 3.00 ± 0.44 ns indicates no significant differences between groups by Student's t-test. (4) Table 6-3. 37 3 caffeic acid 7.45 8.76 μg/g, p-coumaric acid 14.21 15.02 μg/g, ferulic acid 10.44 12.71 μg/g, trans-cinnamic acid 53.76 64.91 μg/g. - 110 -
Table 6-3. The contents of major phenolic compounds of germinated rough rice Shindongjin and Ilpum powders. Shindongjin Ilpum Caffeic acid 7.45 8.76 * p-coumaric acid 14.21 15.02 * Ferulic acid 10.44 12.71 * trans-cinnamic acid 53.76 64.91 ** Unit : μg/g powder * p<0.05, ** p<0.01 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test.. 37 3, 50 15, (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland)., 10 12 cm, 8.5 10.5 cm Retort Pouch 35 g.,,,,,,, 14, 5 : 30 (w/w), 10 : 25, 15 : 20 (Fig. 6-4, 6-5). Fig. 6-4. The Sunsik powders of germinated rough rice Shindongjin and Ilpum.with different sub-material ratios. - 111 -
Ratio (germinated rough rice : sub-material) Shindongjin Ilpum 5 : 30 10 : 25 15 : 20 Fig. 6-5. The prototype of germinated rough rice Shindongjin and Ilpum Sunsik powders with sub-material different ratios. - 112 -
(1) Fig. 6-6. 5 g (5:30) 150 μm 125 μm 28.63% 44.72%, 10 g (10:25) 53.18% 33.98%, 15 g (15:20) 43.52% 40.22% 125~150 μm. 5 g (5:30) 150 μm 125 μm 33.56% 32.46%, 10 g (10:25) 51.20% 33.61%, 15 g (15 : 20) 34.95% 16.02% 125~150 μm, 106 μm 16.02%. Shindongjin Ilpum Fig. 6-6. The size distributions of germinated rough rice Shindongjin and Ilpum Sunsik powders with different sub-material ratios. (2) Table 6-4. 5 g (5:30) 98.18, -3.51, 13.96, 10 g (10:25) 95.89, -3.63, 12.62, 15 g (15:20) 100.33, -4.10, 12.16. 5 g (5:30) 96.09-3.35, 15.53, 10 g (10:25) 99.88, -3.81, 11.98, 15 g (15:20) 98.86, -3.68, 13.22,. - 113 -
Table 6-4. The color of germinated rough rice Shindongjin and Ilpum Sunsik powders with different sub-material ratios. Ratio (germinated rough rice : sub-material) Shindongjin Ilpum Lightness (L) Redness (a) Yellowness (b) 5 : 30 98.18 ± 2.30 b 96.09 ± 0.13 c*** 10 : 25 95.89 ± 0.15 c 99.88 ± 0.05 a*** 15 : 20 100.33 ± 0.25 a 98.86 ± 0.03 b*** 5 : 30-3.51 ± 0.06 a -3.35 ± 0.02 a*** 10 : 25-3.63 ± 0.02 b -3.81 ± 0.01 c*** 15 : 20-4.10 ± 0.04 c -3.68 ± 0.02 b*** 5 : 30 13.96 ± 0.11 a 15.53 ± 2.84 a*** 10 : 25 12.62 ± 0.02 b 11.98 ± 0.01 c*** 15 : 20 12.16 ± 0.05 c 13.22 ± 0.02 b*** *** p<0.001 indicates significant differences between groups by Student's t-test. a-c Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (3),,,, Table 6-5. (enzymatic digestibility) 29.11% 25.51%, 26.16~26.88%. (water binding capacity) 146.12% 134.80%, 161.01% 145.51%. (solubility) 5.27~3.13%, 4.24% 2.30%. (swelling power) 2.77% 1.98%, 2.73% 1.70%. (p>0.05), (10 : 25 15 : 20)... - 114 -
Table 6-5. The enzymatic digestibility, water binding capacity, solubility and swelling power of germinated rough rice Shindongjin and Ilpum Sunsik powders with different sub-material ratios. Items / Ratios Shindongjin Ilpum 5 : 30 29.11 ± 1.92 a 26.37 ± 2.93 abns Enzymatic digestibility (%) 10 : 25 27.73 ± 1.67 ab 26.16 ± 6.88 bns 15 : 20 25.51 ± 3.26 b 26.88 ± 1.32 ans 5 : 30 146.12 ± 3.75 a 161.01 ± 1.60 a** Water binding capacity (%) 10 : 25 140.73 ± 1.94 b 148.33 ± 4.98 bns 15 : 20 134.80 ± 3.92 c 145.51 ± 9.89 bns 5 : 30 5.27 ± 0.06 a 4.24 ± 3.27 a*** Solubility (%) 10 : 25 4.14 ± 0.17 b 3.27 ± 0.16 b** 15 : 20 3.13 ± 0.14 c 2.30 ± 0.16 c** 5 : 30 2.77 ± 0.06 a 2.73 ± 0.05 ans Swelling power (%) 10 : 25 2.24 ± 0.07 b 2.29 ± 0.15 bns 15 : 20 1.98 ± 0.14 c 1.70 ± 0.24 cns ** p<0.01, *** p<0.001 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test. a-c Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (4) 35 g 200 g Table 6-6. (appearance) 5 g (5:30) 4.2 4.3 (p<0.05), (p>0.05). (nutty) 5 g (5:30) 4.0 3.1 (p<0.01), (sweetness) 5 g (5:30) 3.1 2.1. (flavor) (35:0), (feeling of throat) 5 g (5:30).,,, 5 g 14 30 g. - 115 -
Table 6-6. The sensory evaluations of germinated rough rice Shindongjin and Ilpum Sunsik powders with different sub-material ratios. Items / Ratios Shindongjin Ilpum 35 : 0 1.7 c 1.6 cns Appearance 5 : 30 4.2 a 4.3 ans 10 : 25 3.9 ab 3.2 b* 15 : 20 3.6 b 3.4 bns 35 : 0 1.3 c 1.1 cns Nutty 5 : 30 4.0 a 3.1 a** 10 : 25 3.3 b 2.4 b** 15 : 20 3.1 b 2.1 b* 35 : 0 1.1 c 1.0 bns Sweetness 5 : 30 3.1 a 2.1 a* 10 : 25 2.1 b 1.7 ans 15 : 20 1.9 b 1.8 ans 35 : 0 1.7 b 1.4 cns Flavor 5 : 30 4.1 a 3.9 ans 10 : 25 4.2 a 2.8 b** 15 : 20 4.0 a 2.8 b** 35 : 0 1.3 d 1.1 bns Feeling of throat 5 : 30 3.3 b 2.9 ans 10 : 25 2.6 c 2.9 ans 15 : 20 4.1 a 2.5 a** 35 : 0 1.2 c 1.2 cns Overall acceptance 5 : 30 4.2 a 3.5 a* 10 : 25 3.8 a 3.3 ans 15 : 20 3.1 b 2.4 bns * p<0.05, ** p<0.01 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test. a-d Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. (5) Table 6-7. 37 3 caffeic acid 7.45 8.76 μg/g, p-coumaric acid 14.21 15.02 μg/g, ferulic acid 10.44 12.71 μg/g, trans-cinnamic acid 53.76 64.91 μg/g.. - 116 -
Table 6-7. The contents of major phenolic compounds of germinated rough rice Shindongjin and Ilpum Sunsik powders with different sub-material ratios. Phenolic compounds Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Ratio Shindongjin Ilpum 35 : 0 7.45 a 8.76 a 5 : 30 1.06 c 1.25 d 10 : 25 2.13 c 2.50 c 15 : 20 3.19 b 3.75 b 35 : 0 14.21 a 15.02 a 5 : 30 2.03 d 2.15 d 10 : 25 4.06 c 4.29 c 15 : 20 6.09 b 6.44 b 35 : 0 10.44 a 12.71 a 5 : 30 1.49 d 1.82 d 10 : 25 2.98 c 3.63 c 15 : 20 4.47 b 5.45 b 35 : 0 53.76 a 64.91 a 5 : 30 7.68 d 9.27 d 10 : 25 15.36 c 18.55 c 15 : 20 23.04 b 27.82 b a-d Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test.. 37 3, 50 15. 200g 2 L 1. 30 1 L, 1. 100%, 80% 60% 0.2% (Fig. 6-7, 6-8). - 117 -
Shindongjin Ilpum 100% 80% 60% 100% 80% 60% Fig. 6-7. The beverages containing water extracts of germinated rough rice Shindongjin and Ilpum with different concentrations. Concentration (%) Shindongjin Ilpum 100 80 60 Fig. 6-8. The prototype of the beverages containing water extracts of germinated rough rice Shindongjin and Ilpum with different concentrations. - 118 -
(1) ph, 100%, 80% 60% ph, Table 6-8. ph 6.07 5.80 (p<0.001), ph 60% 6.44 6.38 (p<0.05). (L) 96.95 97.30 (p<0.05), 60% 98.47 98.38. (a) -5.29-4.84 (p<0.001), -4.97-4.63 (p<0.001). (b) 11.15 7.14 (p<0.001), 7.67 4.94 (p<0.001). 10.30 cp 10.50 cp (p>0.05), 9.20 cp (p<0.05). Table 6-8. The ph, color and viscosity of the beverages containing water extracts of germinated rough rice Shindongjin and Ilpum with different concentrations. Items / Ratios Shindongjin Ilpum 100 6.07 ± 0.02 c 5.80 ± 0.02 c*** ph 80 6.24 ± 0.03 b 6.03 ± 0.02 b*** 60 6.44 ± 0.02 a 6.38 ± 0.02 a* 100 96.95 ± 0.07 c 97.30 ± 0.20 c* Lightness 80 97.92 ± 0.01 b 97.75 ± 0.03 b*** (L) 60 98.47 ± 0.23 a 98.38 ± 0.02 ans Color 100-5.29 ± 0.04 b -4.84 ± 0.03 c*** Redness 80-5.28 ± 0.02 b -4.76 ± 0.02 b*** (a) 60-4.97 ± 0.01 a -4.63 ± 0.01 a*** 100 11.15 ± 0.02 a 7.14 ± 0.08 a*** Yellowness 80 10.30 ± 0.01 b 6.20 ± 0.01 b*** (b) 60 7.67 ± 0.02 c 4.94 ± 0.01 c*** 100 10.30 ± 0.10 a 10.50 ± 0.10 ans Viscosity (cp) 80 9.40 ± 0.10 b 10.40 ± 0.10 a*** 60 9.20 ± 0.10 c 9.20 ± 0.10 bns * p<0.05, ** p<0.01, *** p<0.001 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test. a-c Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 119 -
(2) 100%, 80% 60% Table 6-9. (apprearance) (p>0.05), (p<0.05). (nutty) (p>0.05), (p<0.05). (sweetness) (p>0.05), (p<0.05). (flavor) (p>0.05).,,. Table 6-9. The sensory evaluations of the beverages containing water extracts of germinated rough rice Shindongjin and Ilpum with different concentrations. Items / Ratios Shindongjin Ilpum 100 4.4 a 3.0 ans Appearance 80 4.0 a 2.6 a* 60 4.2 a 1.9 b** 100 3.5 a 3.0 ans Nutty 80 3.7 a 2.3 b* 60 3.3 a 2.0 b* 100 2.9 a 2.4 ans Sweetness 80 3.0 a 2.2 a* 60 2.2 b 1.9 ans 100 3.4 a 2.6 ans Flavor 80 3.6 a 2.6 ans 60 3.2 a 2.3 ans 100 4.0 a 3.1 ans Overall acceptance 80 4.1 a 2.6 a* 60 3.9 a 1.7 b* * p<0.05, ** p<0.01 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test. a-d Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 120 -
. 37 3, 50 15. (Micro hammer cutter mill type-3, Culatti AG, Zurich, Swtzerland) 80 mesh.,,,,,,, 14 5 : 30 (w/w), 10 : 25, 15 : 20 SiO 2 (1%), (1.5%), 500 mg (52A/52A) (Fig. 6-9). Shindongjin Ilpum Fig. 6-9. The prototype capsules containing powders of germinated rough rice Shindongjin and Ilpum. - 121 -
,, Table 6-10. (loose bulk density) 0.427 g/cm 3 0.439 g/cm 3 (p>0.05). (tapped bulk density), 0.625 g/cm 3 0.641 g/cm 3 (p>0.05). (angle of repose) 37.24 38.79 (p>0.05).,,. (disintegration). (ph 1.2) 2 5.52, (ph 6.8) 2, 6.75 (p<0.05). (52A/52A) 4~5. Table 6-10. The density, angle of repose and disintegration test of the capsule containing powders of germinated rough rice Shindongjin and Ilpum. Test items Shindongjin Ilpum Loose bulk density (g/cm 3 ) 0.427 ± 0.012 0.439 ± 0.023 NS Tapped bulk density (g/cm 3 ) 0.625 ± 0.021 0.641 ± 0.019 NS Angle of repose ( ) 37.24 ± 2.42 38.79 ± 2.11 NS Disintegration Gastric juice (ph 1.2) Intestinal juice (ph 6.8) 5.52 ± 0.24 b 6.75 ± 0.36 a * p<0.05, ** p<0.01 indicates significant differences between groups by Student's t-test. NS indicates no significant differences between groups by Student's t-test. a-d Any means in the same column followed by the different superscripts are significantly different(p<0.05) by Duncan's multiple test. - 122 -
.., ( ), ( 14%) ( 50%),.,,, 3,. - 123 -
1.. (,, ) in vitro., 20 g 80% 400 ml 1. 400 ml 2. 40, -20.. α-glucosidase α-glucosidase Tibbot Skadsen (1996)., α-glucosidase ρ-nitrophenyl-α-d-glucopyranoside (ρnpg) 0.1 M sodium phosphate buffer (ph 7.0), 50 μl 0.35 unit/ml α-glucosidase 100 μl 37 10 1.5 mm ρnpg 50 μl 3 7 20. 1 M Na 2 CO 3 1 ml ELISA 405 nm (%), positive control acarbose (Sigma-Aldrich, St. Louis, MO, USA).. α-amylase α-amylase Lim (2005)., 125 μl 12 unit/ml pancreatin α-amylase 62.5 μl, 200 mm potassium phosphate buffer (ph 6.8) 62.5 μl 37 10 1% starch 125 μl 37 5. 48 mm DNS (3,5-dinitrosalicylic acid and 30% sodium potassium tartrate in 0.5 M NaOH) 125 μl 100 15, 3 ELISA 540 nm (%).. Dipeptidyl peptidase-4 Dipeptidyl peptidase-4 (DPP-4) DPP-4 Inhibitor Screening Kit (Cat. K780-100, Biovision Co., Milpitas, CA, USA)., DPP-4 enzyme 1 μl enzyme buffer 49 μl enzyme buffer DPP-4 Substrate 2 μl enzyme buffer 23 μl substrate. 96-Well - 124 -
plate 25 μl DPP-4 Enzyme buffer 50 μl 37 10 Substrate 25 μl 0 30 37 460 nm (%). kinetic 360 nm. 3T3-L1 (1) 3T3-L1 3T3-L1 ATCC (American Type Culture Collection, Manassas, VA, USA), DMEM (Dulbecco's modified Eagle's medium) 10% BCS (bovine calf serum) 1% penicillin/streptomycin, 37, 5% CO 2 2 3. 3T3-L1 80 90% confluence 0.25% trypsin-edta suspension 6-well plate confluent 48. post-confluent IBMX (0.5 mm) dexamethasone (0.25 μm), insulin (5 μg/ml), 10% FBS differentiation medium. 2 5 μg/ml insulin 10% FBS DMEM. 4, 6 10% FBS DMEM. (2) 3T3-L1 2 3 2 10% FBS DMEM 7 10. PBS Serum-free DMEM 37 6 Krebs Ringer Phospate Hepes buffer (KRPH buffer; 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 1.3 mm CaCl 2, 118 mm NaCl, 5 mm KCl, 30 mm Hepes, ph 7.5) 2% BSA KRPH buffer 3T3-L1 2-Deoxyglucose Uptake Measurement kit (COSMO BIO CO., LTD, Tokyo, Japan)., 2% BSA KRPH buffer 1 um insulin, 1 mm 2-Deoxyglucose 37 20 200 um phloretin cooled PBS., 10 mm Tris-HCl buffer (ph 8.0) well 3 ml. Cell lyssate tube 80 15 4 15,000 g 20 tube 1 sample diluent buffer (1:4) kit solution 2-Deoxyglucose Uptake Measurement kit 420 nm, 25 30-125 -
30 5 kinetic 2005). (Zou, Wang & Shen,. SPSS (Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, USA) one-way ANOVA (Analysis of variation) Duncan s multiple range test (p=0.05). 2.. α-glucosidase α-glucosidase 20 mg/ml Table 7-1. 42.04% 37.47%, 18.91~23.07% 15.37~18.10%. 19.91% 15.14%., acarbose 10 mg/ml 64.89% (data not shown). Table 7-1. The α-glucosidase inhibition activities of prototypes containing the germinated rough rice with different cultivar. Prototypes Shindongjin Ilpum Powder 42.04 ± 0.11 Aa 37.47 ± 0.09 Ba 5 18.91 ± 0.08 Af 15.37 ± 0.07 Bde Sunsik 10 20.63 ± 0.04 Ad 15.75 ± 0.03 Bd 15 23.07 ± 0.07 Ac 18.10 ± 0.04 Bc Capsule 24.17 ± 0.05 Ab 22.07 ± 0.04 Bb Beverage 19.91 ± 0.04 Ae 15.14 ± 0.03 Be Results are expressed as the mean ± SD of triplicate samples (n=3). The concentration of sample was 20 mg/ml. Different capital and small letters in the same row indicate a significant difference (p<0.05) among cultivar and prototypes by Duncan's multiple range test, respectively. - 126 -
. α-amylase α-amylase 0.5 mg/ml Table 7-2. 45.25% 62.74%, 19.24~38.24% 28.66~45.78%. 39.78% 46.54% 14.92% 25.76%. Table 7-2. The α-amylase inhibition activities of prototypes containing the germinated rough rice with different cultivar. Prototypes Shindongjin Ilpum Powder 45.25 ± 0.12 Ba 62.74 ± 0.11 Aa 5 19.24 ± 0.09 Be 28.66 ± 0.05 Ae Sunsik 10 27.52 ± 0.07 Bd 33.21 ± 0.08 Ad 15 38.24 ± 0.10 Bc 45.78 ± 0.12 Ac Capsule 39.78 ± 0.09 Bb 46.54 ± 0.11 Ab Beverage 14.92 ± 0.06 Bf 25.76 ± 0.07 Af See the legend in Table 7-1. The concentration of sample was 0.5 mg/ml.. Dipeptidyl peptidase-4 (DPP-4) DPP-4 2 mg/ml Table 7-3. 52.78% 56.24%, 15.72~ 26.97% 17.56~31.52%. 28.22% 33.14% 9.25% 11.26%. Table 7-3. The dipeptidyl peptidase-4 inhibition activities of prototypes containing the germinated rough rice with different cultivar. Prototypes Shindongjin Ilpum Powder 52.78 ± 0.09 Ba 56.24 ± 0.10 Aa Sunsik 5 15.72 ± 0.05 Be 17.56 ± 0.05 Ae 10 20.11 ± 0.03 Bd 24.56 ± 0.02 Ad 15 26.97 ± 0.05 Bc 31.52 ± 0.05 Ac Capsule 28.22 ± 0.04 Bb 33.14 ± 0.07 Ab Beverage 9.25 ± 0.01 Bf 11.26 ± 0.02 Af See the legend in Table 7-1. The concentration of sample was 2 mg/ml. - 127 -
. 3T3-L1 100 μg/ml Fig. 7-1. 137.5% 157.4%, 101.4~124.2% 111.1~138.4%. 124.2% 84.2% 135.5% 101.%. [ Shindongjin ] [ Ilpum ] Fig. 7-1. Effects of prototypes containing the germinated rough rice powder of Shindongjin and Ilpum on the glucose uptake by 3T3-L1 adipocytes. The small letters on the error bars means significant difference at p<0.05 by Duncan s multiple range test. - 128 -
1.. (GIP-HS) 60 kg 1 100 g,. 37 3 (HS ) (BR ), (Pin mill, Gyeyangsa, Incheon, Korea) 80 mesh. HS 25 g 10 g 10 12 cm, 8.5 10.5 cm Retort Pouch (Laminated aluminum foil film) 35 g.,,,,,,, 14, 1 4 (35 g 1 4 ).. 100 g 60, 100, 120 140 mesh 250>, 150, 125, <106 μm. (Colorimeter CR-300, Minolta, Tokyo, Japan) (L), (a) (b). 2 g 20 ml 1 200 rpm 4,000 rpm 15. 2 g 30 ml 80 shaking water bath 200 rpm 30 (4,000 rpm, 15 min) 80. 35 g 200 g (20~50, ) 20..,,. (Dynamic angle, ), Adobe photoshop CS2 (Adobe Systems Inc., San Jose, CA, USA) H D - 129 -
, (1) (Park, Kim & Shin, 2005). Θ d, H, D. tan (1) 30 g 100 ml (loosed bulk density, g/cm 3 ), 5 cm 60 (tapped bulk density, g/cm 3 ), (Hausner s ratio) (Kang et al., 2012; Oh et al., 2013). (2) (2) (3).. (4) r n n, n, V 0 (ml), V n n (ml), H R, ρ 0 (g/cm 3 ), ρ T (g/cm 3 ) a, b.. SPSS (Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, USA) one-way ANOVA (Analysis of variation) Duncan s multiple range test (p=0.05). 2.. HS 25 g 10 g 10 12 cm, 8.5 10.5 cm Retort Pouch (Laminated aluminum foil film) 35 g.,,,,,,, 14, 1 4 (35 g 1 4 ) - 130 -
.. (original) HS (renewal) Fig. 8-1. 150~125 μm 51.20% 48.23%, 150~125 μm 33.61% 27.21%.,, 250 μm.,. Fig. 8-1. The size distributions of germinated Ilpum Sunsik powders. Original : germinated rough rice powder + sub-material, Renewal : germinated rough rice hull and sprout powder + sub-material. *, **, *** and ns are indicates significant differences among factors by t-test and there are means p<0.05, p<0.01, p<0.001 and not significant, respectively.. (original) HS (renewal) Table 8-1. L 99.88 52.32 (p<0.001). HS. a b -3.81-1.02 11.98 11.78. - 131 -
Table 8-1. The color of germinated Ilpum Sunsik powders. Original Renewal Lightness (L) 99.88 ± 0.05 52.32 ± 1.25 *** Redness (a) -3.81 ± 0.01-1.02 ± 0.16 * Yellowness (b) 11.98 ± 0.01 11.78 ± 0.18 ns Original : germinated rough rice powder + sub-material, Renewal : germinated rough rice hull and sprout powder + sub-material. *, **, *** and ns are indicates significant differences among factors by t-test and there are means p<0.05, p<0.01, p<0.001 and not significant, respectively.., (original) HS (renewal), Table 8-2. 295.23% 148.33% (p<0.001). 4.26% 2.29%, 3.27% 1.48%. HS,. Table 8-2. The water binding capacity, solubility and swelling power of germinated Ilpum Sunsik powders. Original Renewal Water binding capacity (%) 148.33 ± 4.98 295.23 ± 5.26 *** Solubility (%) 3.27 ± 0.16 1.48 ± 0.06 ** Swelling power (%) 2.29 ± 0.15 4.26 ± 0.07 ** Original : germinated rough rice powder + sub-material, Renewal : germinated rough rice hull and sprout powder + sub-material. *, **, *** and ns are indicates significant differences among factors by t-test and there are means p<0.05, p<0.01, p<0.001 and not significant, respectively. - 132 -
. (original) HS (renewal) Table 8-3.,,,.,. Table 8-3. The sensory evaluation of germinated Ilpum Sunsik powders. Original Renewal Appearance 3.2 2.4 * Nutty flavor 2.4 2.6 ns Sweetness 1.7 2.0 ns Smell 2.8 2.0 * Feeling of throat 2.9 1.6* Overall feeling 3.3 2.2* Original : germinated rough rice powder + sub-material, Renewal : germinated rough rice hull and sprout powder + sub-material. *, **, *** and ns are indicates significant differences among factors by t-test and there are means p<0.05, p<0.01, p<0.001 and not significant, respectively.. 0.25~0.64 g/cm 3.. a, b, a a., a (Table 8-4). cellulose HS, a (Table 8-5).,. - 133 -
Table 8-4. Physical characteristics of hull &sprout fractions from germinated rough rice with different germination condition and sub-materials. Germination Constant of equation Loosed bulk Tapped bulk Dynamic angle ( ) density (g/cm 3 ) density (g/cm 3 Hausner ratio Temp. Period ) a b Cereal powder 39.07 ± 3.10 0.321 ± 0.012 0.601 ± 0.000 1.873 ± 0.070 0.564 ± 0.035 0.085 ± 0.016 Cellulose 27.95 ± 1.94 0.683 ± 0.016 0.827 ± 0.013 1.211 ± 0.015 0.192 ± 0.022 0.164 ± 0.076 Hull &sprout 37.32 ± 1.74 0.411 ± 0.027 0.578 ± 0.000 1.410 ± 0.091 0.318 ± 0.055 0.203 ± 0.044 1 36.56 ± 3.05 0.389 ± 0.003 0.605 ± 0.007 1.557 ± 0.016 0.417 ± 0.023 0.117 ± 0.032 32 2 39.44 ± 2.98 0.371 ± 0.026 0.613 ± 0.000 1.660 ± 0.112 0.547 ± 0.034 0.058 ± 0.032 3 39.49 ± 2.57 0.309 ± 0.007 0.557 ± 0.010 1.803 ± 0.027 0.608 ± 0.009 0.047 ± 0.004 4 40.79 ± 1.89 0.304 ± 0.003 0.550 ± 0.006 1.811 ± 0.036 0.622 ± 0.023 0.043 ± 0.004 1 37.07 ± 3.25 0.389 ± 0.008 0.609 ± 0.007 1.567 ± 0.015 0.415 ± 0.011 0.129 ± 0.019 35 2 41.09 ± 2.13 0.347 ± 0.006 0.613 ± 0.000 1.769 ± 0.031 0.527 ± 0.026 0.083 ± 0.015 3 34.23 ± 2.68 0.309 ± 0.004 0.557 ± 0.000 1.802 ± 0.021 0.609 ± 0.025 0.048 ± 0.006 4 38.22 ± 2.87 0.301 ± 0.000 0.530 ± 0.005 1.765 ± 0.018 0.633 ± 0.023 0.036 ± 0.004 1 35.29 ± 2.85 0.365 ± 0.009 0.622 ± 0.007 1.704 ± 0.063 0.505 ± 0.046 0.079 ± 0.018 37 2 33.06 ± 2.12 0.334 ± 0.010 0.593 ± 0.007 1.776 ± 0.049 0.571 ± 0.026 0.058 ± 0.006 3 36.19 ± 3.24 0.322 ± 0.002 0.582 ± 0.007 1.807 ± 0.018 0.611 ± 0.035 0.049 ± 0.009 4 42.99 ± 0.57 0.301 ± 0.000 0.537 ± 0.010 1.786 ± 0.032 0.629 ± 0.019 0.039 ± 0.003 Table 8-5. Physical characteristics of hull &sprout prototypes from germinated rough rice with different germination condition and sub-materials. Germination Constant of equation Loosed bulk Tapped bulk Dynamic angle ( ) Hausner ratio Temp. Period density (g/cm3) density(g/cm3) a b Hull &sprout 39.94 ± 0.38 0.438 ± 0.011 0.647 ± 0.008 1.479 ± 0.049 0.365 ± 0.024 0.137 ± 0.046 1 36.69 ± 0.39 0.429 ± 0.009 0.647 ± 0.008 1.507 ± 0.034 0.405 ± 0.017 0.092 ± 0.015 32 2 39.90 ± 0.71 0.394 ± 0.012 0.661 ± 0.008 1.679 ± 0.064 0.549 ± 0.023 0.049 ± 0.004 3 37.72 ± 3.12 0.345 ± 0.008 0.608 ± 0.007 1.765 ± 0.041 0.597 ± 0.037 0.047 ± 0.007 4 43.02 ± 2.71 0.334 ± 0.009 0.588 ± 0.007 1.760 ± 0.032 0.634 ± 0.030 0.039 ± 0.007 1 39.53 ± 0.57 0.423 ± 0.007 0.638 ± 0.008 1.507 ± 0.022 0.427 ± 0.017 0.071 ± 0.013 35 2 42.15 ± 3.97 0.376 ± 0.010 0.638 ± 0.008 1.698 ± 0.064 0.571 ± 0.039 0.048 ± 0.006 3 42.47 ± 3.17 0.337 ± 0.008 0.596 ± 0.007 1.770 ± 0.040 0.613 ± 0.043 0.044 ± 0.012 4 40.18 ± 1.57 0.326 ± 0.007 0.577 ± 0.006 1.771 ± 0.021 0.613 ± 0.010 0.042 ± 0.005 1 39.74 ± 3.26 0.392 ± 0.005 0.642 ± 0.000 1.638 ± 0.021 0.488 ± 0.012 0.071 ± 0.005 37 2 39.14 ± 3.28 0.368 ± 0.009 0.629 ± 0.000 1.708 ± 0.042 0.541 ± 0.014 0.062 ± 0.016 3 39.46 ± 0.56 0.351 ± 0.004 0.625 ± 0.007 1.779 ± 0.030 0.578 ± 0.010 0.054 ± 0.004 4 39.05 ± 1.05 0.327 ± 0.002 0.593 ± 0.013 1.823 ± 0.048 0.679 ± 0.022 0.034 ± 0.004-134 -
. HS,, Table 8-6. HS 202.21%, 251~300%. HS 17.16%, 20~31%. HS 0.905% 0.918~0.956%. Table 8-6. The water binding capacity, solubility and swelling power of hull &sprout prototypes from germinated rough rice with different germination condition and sub-materials. Germination Temp. Period Water binding capacity (%) Water solubility (%) Swelling power (%) Hull & sprout 202.21 ± 17.29 17.16 ± 0.50 0.905 ± 0.003 1 253.81 ± 7.73 19.98 ± 0.79 0.919 ± 0.007 32 2 255.79 ± 6.35 23.68 ± 0.96 0.919 ± 0.015 3 296.12 ± 5.04 26.69 ± 1.36 0.949 ± 0.000 4 299.91 ± 3.23 26.27 ± 1.82 0.941 ± 0.017 1 258.78 ± 6.22 21.66 ± 0.41 0.918 ± 0.033 35 2 251.26 ± 4.73 24.98 ± 1.24 0.946 ± 0.009 3 284.67 ± 3.08 25.41 ± 0.39 0.942 ± 0.004 4 273.39 ± 7.20 30.19 ± 1.01 0.954 ± 0.014 1 289.69 ± 11.85 22.18 ± 1.28 0.942 ± 0.007 37 2 279.95 ± 11.80 22.94 ± 0.26 0.939 ± 0.018 3 294.77 ± 4.18 24.33 ± 0.47 0.934 ± 0.020 4 272.41 ± 10.39 30.69 ± 2.13 0.956 ± 0.008-135 -
1.. 2013 (Oryza sativa L.; Ilpum). Lee (2008)., 1 2 3. (37 ) (85%) (WGC-450, Dahan Inc, Seoul, Korea) 6, 1 2. ( ), 50 (WFO-459PD, EYELA, Tokyo, Japan) 2.. (FC2K, Kett Inc., Tokyo, Japan) (hull & sprout; HS) (Brown rice; BR), (Micro hammer cutter mill type-3, Culatti AG, Zurich, Switzerland) 80 mesh -20.. 10 80% (v/v) 80 3,, -20.. Sun (2002)., 0.5 g 50 ml acidic methanol (95% methanol : 1 M HCl = 85 : 15) (10,000 rpm, 5 min). 4,000 rpm 10 45. acidic methanol 10 ml. Sun (2002)., 40 ml 2 M NaOH 1. hexane HCl ph 7.0. 50 ml ethyla acetate 5 ethyl acetate 10 ml. - 136 -
. 0.45 μm membrane filter. HPLC system(acme 9100 series)., ODS column (5 μm, 46 250 mm, Agilent Eclipse XDB-C18, Agilent Technologies, USA), 0.1% acetic acid acetonitrile (A), 0.1% acetic acid water (B) A B 8 : 92, 2 10 : 90, 27 30 : 70, 50 90 : 10, 51 100 : 0, 60 100 : 0, 70 8 : 92. 10 μl 1.0 ml/min, 280 nm (Ti et al., 2014).. (C5), ph ferulic acid. -70, -18, 4 37 30. (25 ) 30. ph ph 2, 4, 6, 8 10 (25 ) 30.. ferulic acid. polyethylene (PE), aluminum laminated polyethylene (PEL), aluminum (Al) polyehylene tetraphthalate (PET) 25 37 60.. SPSS (Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, USA) one-way ANOVA (Analysis of variation) Duncan s multiple range test (p=0.05). - 137 -
2.,,,.. Table 9-1. 4.44 mg/g, 20.86 mg/g,. 25.30 mg/g 29.41 mg/g,.. Table 9-1. Free, bound and total polyphenol content of rough rice before and after germination. Germination Before After Part Polyphenol content (mg/g) Free Bound Total Brown rice 0.37 ± 0.01 4.07 ± 0.13 4.44 ± 0.08 Hull 1.49 ± 0.08 19.37 ± 0.24 20.86 ± 0.11 Whole grain 1.86 ± 0.07 23.44 ± 0.18 25.30 ± 0.21 Brown rice 0.73 ± 0.05 3.15 ± 0.04 3.88 ± 0.09 Hull &sprout 4.31 ± 0.12 21.22 ± 0.15 25.53 ± 0.14 Whole grain 5.04 ± 0.09 24.37 ± 0.13 29.41 ± 0.11. Table 9-2. coumaric acid, ferulic acid, cinnamic acid 47.06 μg/g, 117.71 μg/g 144.90 μg/g, 65.65 μg/g, 157.19 μg/g 205.62μg/g.. phenylalanine cinnamic acid benzoic acid p-coumaric acid shikimic acid pathway. - 138 -
Table 9-2. Free, bound and total phenolic acid content of rough rice before and after germination. Germination Part Form Before After Brown rice Hull Whole grain Brown rice Hull &sprout Whole grain Gallic acid Catechin Caffeic acid Phenolic acid content (μg/g) p-coumaric acid Ferulic acid Cinnamic acid Kaempferol Free 0.15 0.75 0.09 0.57 0.21 1.76 1.32 4.84 Total Bound 1.47 7.37 0.84 5.60 29.72 17.23 12.96 75.19 Total 1.62 8.12 0.93 6.17 29.93 18.99 14.28 80.03 Free 0.74 2.31 0.48 3.23 2.47 9.95 7.48 26.66 Bound 10.04 31.35 6.57 43.83 115.24 134.95 101.50 443.49 Total 0.66 33.66 7.06 47.06 117.71 144.90 108.98 460.03 Free 0.89 3.06 0.57 3.80 2.68 11.70 8.80 31.50 Bound 11.51 38.71 7.41 49.43 144.96 152.19 114.47 518.68 Total 2.28 41.77 7.98 53.23 147.64 163.89 123.27 540.06 Free 0.58 1.47 0.38 2.00 2.10 6.87 1.59 14.99 Bound 1.44 7.22 0.82 5.49 29.13 16.89 12.70 73.69 Total 2.02 8.68 1.20 7.48 31.23 23.76 14.29 88.67 Free 2.81 7.16 1.86 9.76 10.26 33.56 7.76 73.17 Bound 12.80 39.97 8.38 55.88 146.93 172.07 129.42 565.45 Total 15.62 47.13 10.24 65.65 157.19 205.62 137.18 638.62 Free 3.39 8.63 2.24 11.76 12.36 40.43 9.35 88.16 Bound 14.25 47.18 9.21 61.37 176.06 188.96 142.12 639.14 Total 17.64 55.81 11.45 73.13 188.42 229.39 151.47 727.30. Table 9-3. ferulic acid, trans-cinnamic acid kaempferol 157.19 μg/g, 205.62 μg/g 137.18 μg/g. gallic acid,. C18 C5 160.42 μg (66.7%) (Table 9-4). C5 Diaion HP-20 ion exchange resin D3 50% (Table 9-5). D3 C18 column preparative HPLC P3 52% (Table 9-6). P3 gel filtration column recycling HPLC R1 76.6% (Table 9-7). ferulic acid, cinnamic acid kaempferol. - 139 -
Table 9-3. Free, bound and total phenolic acid content of solvent fractions from germinated rough rice extract. Phenolic acid content (μg/g) Fractions Form Gallic acid Catechin Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Kaempferol Total Free 2.81 7.16 1.86 9.76 10.26 33.56 7.76 73.17 GIP-HS_E Bound 12.80 39.97 8.38 55.88 146.93 172.07 129.42 565.45 Total 15.62 47.13 10.24 65.65 157.19 205.62 137.18 638.62 Free 0.90 2.29 0.59 3.12 3.28 10.74 2.48 23.42 GIP-HS_E_H Bound 4.10 12.79 2.68 17.88 47.02 55.06 41.41 180.94 Total 5.00 15.08 3.28 21.01 50.30 65.80 43.90 204.36 Free 0.59 1.50 0.39 2.05 2.15 7.05 1.63 15.37 GIP-HS_E_C Bound 2.69 8.39 1.76 11.74 30.86 36.13 27.18 118.74 Total 3.28 9.90 2.15 13.79 33.01 43.18 28.81 134.11 Free 0.79 2.01 0.52 2.73 2.87 9.40 2.17 20.49 GIP-HS_E_E Bound 3.58 11.19 2.35 15.65 41.14 48.18 36.24 158.33 Total 4.37 13.20 2.87 18.38 44.01 57.57 38.41 178.81 Free 0.31 0.79 0.20 1.07 1.13 3.69 0.85 8.05 GIP-HS_E_B Bound 1.41 4.40 0.92 6.15 16.16 18.93 14.24 62.20 Total 1.72 5.18 1.13 7.22 17.29 22.62 15.09 70.25 Free 0.23 0.57 0.15 0.78 0.82 2.68 0.62 5.85 GIP-HS_E_W Bound 1.02 3.20 0.67 4.47 11.75 13.77 10.35 45.24 Total 1.25 3.77 0.82 5.25 12.58 16.45 10.97 51.09 Table 9-4. Free, bound and total phenolic acid content of C18 column chromatography fractions from germinated rough rice extract. Phenolic acid content (μg/g) Fractions Form Gallic acid Catechin Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Kaempferol Total Free 0.90 2.29 0.59 3.12 3.28 10.74 2.48 23.42 GIP-HS_E_H Bound 4.10 12.79 2.68 17.88 47.02 55.06 41.41 180.94 Total 5.00 15.08 3.28 21.01 50.30 65.80 43.90 204.36 Free 0.19 0.49 0.13 0.67 0.71 2.31 0.53 5.03 GIP-HS_E_H_C1~4 Bound 0.88 2.75 0.58 3.84 10.11 11.84 8.90 38.90 Total 1.07 3.24 0.70 4.52 10.81 14.15 9.44 43.94 Free 0.71 1.80 0.47 2.45 2.58 8.43 1.95 18.38 GIP-HS_E_H_C5 Bound 3.22 10.04 2.11 14.04 36.91 43.22 32.51 142.04 Total 3.92 11.84 2.57 16.49 39.49 51.65 34.46 160.42-140 -
Table 9-5. Free, bound and total phenolic acid content of Diaion HP-20 ion exchange chromatography fractions from germinated rough rice extract. Phenolic acid content (μg/g) Fractions Form Gallic acid Catechin Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Kaempferol Total Free 0.71 1.80 0.47 2.45 2.58 8.43 1.95 18.38 GIP-HS_E_H_C5 Bound 3.22 10.04 2.11 14.04 36.91 43.22 32.51 142.04 Total 3.92 11.84 2.57 16.49 39.49 51.65 34.46 160.42 Free 0.16 0.40 0.10 0.54 0.57 1.87 0.43 4.07 GIP-HS_E_H_C5_D1 Bound 0.71 2.22 0.47 3.11 8.17 9.57 7.19 31.43 Total 0.87 2.62 0.57 3.65 8.74 11.43 7.63 35.50 Free 0.06 0.16 0.04 0.22 0.24 0.77 0.18 1.68 GIP-HS_E_H_C5_D2 Bound 0.29 0.92 0.19 1.28 3.38 3.95 2.97 13.00 Total 0.36 1.08 0.24 1.51 3.61 4.73 3.15 14.68 Free 0.36 0.91 0.24 1.24 1.30 4.26 0.99 9.30 GIP-HS_E_H_C5_D3 Bound 1.63 5.08 1.06 7.10 18.66 21.86 16.44 71.83 Total 1.98 5.99 1.30 8.34 19.97 26.12 17.43 81.13 Free 0.06 0.16 0.04 0.22 0.24 0.77 0.18 1.68 GIP-HS_E_H_C5_D4 Bound 0.29 0.92 0.19 1.28 3.38 3.95 2.97 13.00 Total 0.36 1.08 0.24 1.51 3.61 4.73 3.15 14.68 Table 9-6. Free, bound and total phenolic acid content of Prep. HPLC fractions from germinated rough rice extract. Phenolic acid content (μg/g) Fractions Form Gallic acid Catechin Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Kaempferol Total Free 0.36 0.91 0.24 1.24 1.30 4.26 0.99 9.30 GIP-HS_E_H_C5_D3 Bound 1.63 5.08 1.06 7.10 18.66 21.86 16.44 71.83 Total 1.98 5.99 1.30 8.34 19.97 26.12 17.43 81.13 Free 0.02 0.07 0.01 0.09 0.22 0.29 0.19 0.89 GIP-HS_E_H_C5_D3_P1 Bound 0.07 0.21 0.05 0.29 0.69 0.91 0.61 2.82 Total 0.09 0.27 0.06 0.38 0.91 1.19 0.80 3.71 Free 0.13 0.40 0.09 0.56 1.33 1.74 1.16 5.40 GIP-HS_E_H_C5_D3_P2 Bound 0.17 0.51 0.11 0.71 1.69 2.21 1.48 6.88 Total 0.30 0.91 0.20 1.26 3.02 3.95 2.64 12.28 Free 0.78 2.36 0.51 3.29 7.88 10.31 6.88 32.03 GIP-HS_E_H_C5_D3_P3 Bound 0.29 0.87 0.19 1.22 2.92 3.81 2.54 11.85 Total 1.07 3.24 0.70 4.51 10.80 14.13 9.42 43.87 Free 0.08 0.24 0.05 0.33 0.79 1.03 0.69 3.19 GIP-HS_E_H_C5_D3_P4 Bound 0.44 1.33 0.29 1.86 4.45 5.82 3.88 18.07 Total 0.52 1.57 0.34 2.19 5.23 6.85 4.57 21.26-141 -
Table 9-7. Free, bound and total phenolic acid content of Re. HPLC fractions from germinated rough rice extract. Phenolic acid content (μg/g) Fractions Form Gallic acid Catechin Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Kaempferol Total Free 0.78 2.36 0.51 3.29 7.88 10.31 6.88 32.03 GIP-HS_E_H_C5_D3_P3 Bound 0.29 0.87 0.19 1.22 2.92 3.81 2.54 11.85 Total 1.07 3.24 0.70 4.51 10.80 14.13 9.42 43.87 Free 0.60 1.81 0.39 2.52 6.04 7.90 5.27 24.53 GIP-HS_E_H_C5_D3_P3_R1 Bound 0.22 0.67 0.15 0.93 2.23 2.92 1.95 9.07 Total 0.82 2.48 0.54 3.45 8.27 10.82 7.22 33.60 Free 0.18 0.55 0.12 0.77 1.85 2.42 1.61 7.50 GIP-HS_E_H_C5_D3_P3_R2 Bound 0.07 0.20 0.04 0.29 0.68 0.89 0.60 2.77 Total 0.25 0.76 0.16 1.06 2.53 3.31 2.21 10.27. 3T3-L1,. Table 9-8., gallic acid ferulic acid. ferulic acid 0.956 (p<0.01), 0.942 (p<0.01). ferulic acid 3T3-L1. Table 9-8. Correlation analysis among glucose uptake activity, free, bound and total phenolic acid content of germinated rough rice extract. Phenolic acid content (μg/g) Form Gallic acid Catechin Caffeic acid Coumaric acid Ferulic acid Cinnamic acid Kaempferol Total Free 0.928** 0.412 0.385 0.451 0.956** 0.515 0.498 0.854** Bound 0.518 0.305 0.312 0.322 0.624 0.367 0.361 0.441 Total 0.912* 0.408 0.362 0.415 0.942** 0.492 0.412 0675* * : P<0.05, **: P<0.01-142 -
. C5,, ph. C5 70, -18, 4 3 7 30 70 18 30 94.58% 92.14% (ferulic acid), 37 20 90.73% (p<0.05), 25 82.93% (p<0.001), 30 75.22% (p<0.001) (Fig. 9-1 [A]). C5 30 5 92.75% 20 51.75%, 30 47.42% (Fig. 9-1 [B]). ph 2~10 C5 30 ph ph 6 25 90% ph 8 15 85.45%. ph 4 10 74.45% 30 68.54%, ph 2 0 84.25% (Fig. 9-1 [C]). 3T3-L1 (Fig. 9-1 [D]). [A] [B] [C] [D] Fig. 9-1. Changes in ferulic acid content and glucose uptake activity of active fraction (GIP-HS_E_H_C5) from germinated rough rice with different storage conditions. [A] with different storage temperatures and periods, [B] with exposed a light or kept in dark, [C] with different ph values, [D] glucose uptake assay - 143 -
. polyethylene (PE), aluminum laminated polyethylene (PEL) aluminum foil (Al) 35 g 1, polyethylene tetraphthalate (PET) 500 g 1 60 ferulic acid (Fig. 9-2). ferulic acid 11.35 μg/g. Al 25 60 11.12 μg/g, 37 60 10.57 μg/g. PE PEL 25 60 10.11 μg/g 9.46 μg/g, 37 8.41 μg/g 8.98 μg/g. PET. 25 15 9.78 μg/g 60 7.45 μg/g. 37 60 6.54 μg/g. ferulic acid Al PET. Fig. 9-2. Changes in ferulic acid content of Sunsik prototypes from germinated rough rice with different storage temperatures and periods. PE : polyethylene; PEL : aluminum laminated polyethylene, Al : aluminum foil, PET : polyethylene tetraphthalate, storage temperatures are 25 and 37. - 144 -
C5 ( / /C18 ) (Fig. 9-3). C5 control 136.4% 243.3%. (sub materials) / 212.1%. PE 25 15, 30 37 30 157.1%. PEL, Al PET 37 30 153.3%, 181.9% 160.0%. Al. Fig. 9-3. Effects of Sunsik prototypes from germinated rough rice on the glucose uptake by 3T3-L1 adipocytes with different storage conditions and package materials. PE : polyethylene; PEL : aluminum laminated polyethylene, Al : aluminum foil, PET : polyethylene tetraphthalate, storage temperatures are 25 and 37. - 145 -
1.. 2014 (Oryza sativa L.). 20 1 2 3. 85% (WGC 450, Daihan Scientific Co., Ltd., Seoul, Korea) 32, 35 37, 1, 2, 3 4. 50 (WFO-459PD, Eyela, Tokyo, Japan) 2 (FC2K, Kett Inc., Tokyo, Japan).,.. 20 12 air filter polyethylene bag 1 kg 121 120. Monascus pilosus 305-9 (KFCC 11410P) 100 ml 29±1, 75±5% 20. 100 30 2 55 10% 60 mesh.. 5 g 75% (v/v) 50 ml 1 3 (Whatman No. 42, GE Healthcare, Buckinghamshire, UK). (N-1000, Eyela, Tokyo, Japan) 40 (Freeze dryer, FD5508, Ilshin Lab Co., Ltd., Dongducheon, Korea) -18.. (CR-200, Minolta, Tokyo, Japan) L (lightness), a (redness), b (yellowness). (Monascus pigment) (Whatman No. 42) spectrophotometer(uv-1601, Shimadzu, Kyoto, Japan) 500 nm - 146 -
(red color group), 470 nm (orange color group) 400 nm (yellow color group) (Johns & Stuart, 1991).. Monacolin K citrinin Monacolin K K (III.3.6.6 K) (KFDA, 2014). 4 g 50 ml 75% 40 ml 1. 3,500 rpm 10 50 ml 0.45 μm,. monacolin K (Mevinolin, Sigma-Aldrich, St. Louis, MO, USA) 10 mg 75% 50 ml, monacolin K 2 ml 0.05 N 0.5 ml 30. Monacolin K Avula (2014). HPLC (Acme 9000 system, Younglin Inst., Anyang, Korea) Mightysil RP-18 GP column (250 4.6 mm, 5 μm pore size, Kanto Chemical, Tokyo, Japan) acetonitrile A 0.2% phosphoric acid B 20 μl 1 ml/min 237 nm. Citrinin 1 (III.2.5.4.1 ( 1 )) (KFDA, 2014). 2.5 g 50 ml 20 ml 30. 3,500 rpm 10 1 ml 0.45 μm. citrinin (Sigma-Aldrich) 10 mg 100% 100 ml. Citrinin Avula (2014). HPLC (Agilent 1200 Series, Agilent, Santa Clara, CA, USA) Mightysil RP-18 GP column acetonitrile : 0.1% trifluoroacetic acid = 40 : 60 1 ml/min 335 nm, 502 nm.. Folin-Ciocalteu phenol reagent (Dewanto, Xianzhong & Liu, 2002). 100 μl 2% Na 2 CO 3 2 ml 3 50% Folin-Ciocalteu reagent 100 μl 30 750 nm. gallic acid (Sigma-Aldrich Co., St. Louis, MO, USA) - 147 -
, g mg gallic acid. Dewanto (2002). 250 μl 1 ml 5% NaNO2 75 μl, 5 10% AlCl 3 6H 2 O 150 μl 6 1 N NaOH 500 μl. 11 510 nm. catechin (Sigma-Aldrich) g mg catechin.. DPPH (Blois, 1958). 0.2 ml 0.2 mm 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich) 0.8 ml 60 520 nm, TEAC (trolox equivalent antioxidant capacity, mg trolox eq/g). ABTS cation decolorization assay (Blois, 1958). ABTS (2,2'-azino-bis-3-ethylbenzo-thiazoline- 6-sulfonic acid) 7.4 mm potassium persulphate 2.6 mm ABTS 735 nm 1.4~1.5. ABTS 1 ml 50 μl 60, L-ascorbic acid AEAC (ascorbic acid equivalent antioxidant capacity).. SPSS (Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, IL, USA) one-way ANOVA(Analysis of variation) Duncan s multiple range test (P=0.05). Pearson s correlation analysis (P=0.05). - 148 -
2.. Fig. 10-1... ( ). Germination temperature ( ) Germination periods (day) 1 2 3 4 32 35 37 Fig. 10-1. Photographs of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. - 149 -
. Table 10-1 L a b.,. Monascus, monascin ankaflavin, rubropunctatin monascorubrin, rubropunctamine monascorubramine 10. UV-absorbance Table 10-2. (A 500 ), (A 470 ) (A 400 ),. (Table 10-1).,.. α-amylase glucoamylase glucose,. Table 10-1. Changes in the color of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. Germination Whiteness Redness Yellowness Temp. Period (L-value) (a-value) (b-value) ( ) (day) White rice 45.11±0.03 b 1.68±0.04 m 0.15±0.01 n Brown rice 44.26±0.03 c 2.88±0.03 l 0.91±0.01 m 1 37.67±0.03 n 9.25±0.12 a 5.99±0.02 g 32 2 38.88±0.04 m 8.33±0.04 b 7.25±0.02 c 3 42.88±0.03 e 4.17±0.01 j 4.30±0.03 k 4 43.54±0.01 d 3.47±0.02 k 4.44±0.01 j 1 41.05±0.02 h 5.61±0.04 g 7.07±0.02 d 35 2 39.73±0.02 j 7.06±0.06 e 7.35±0.03 b 3 41.71±0.02 g 5.11±0.03 h 5.57±0.01 h 4 45.27±0.01 a 1.16±0.01 n 1.03±0.02 l 1 39.27±0.01 l 7.43±0.03 d 7.43±0.01 a 37 2 39.83±0.01 i 6.87±0.01 f 6.76±0.02 f 3 39.39±0.04 k 7.59±0.05 c 6.94±0.03 e 4 42.04±0.01 f 4.82±0.04 i 5.43±0.03 i Different small letters in the same column indicate a significant difference at P<0.05 by Duncan's multiple range test. - 150 -
Table 10-2. Changes in the Monascus pigment of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. Germination Red color group Orange color group Yellow color group Temp. Period (500 nm) (470 nm) (400 nm) ( ) (day) White rice 0.122±0.001 l 0.099±0.001 l 0.181±0.001 j Brown rice 0.235±0.007 k 0.178±0.004 j 0.353±0.010 i 1 2.320±0.024 a 1.909±0.017 a 4.663±0.026 a 32 2 1.669±0.038 b 1.398±0.033 b 3.760±0.075 b 3 0.494±0.017 i 0.420±0.014 h 1.169±0.041 h 4 0.432±0.003 j 0.383±0.002 i 1.225±0.010 h 1 0.936±0.004 f 0.815±0.004 f 2.178±0.010 f 35 2 1.289±0.017 d 1.113±0.014 d 3.074±0.048 d 3 0.701±0.007 g 0.597±0.005 g 1.738±0.016 g 4 0.129±0.002 l 0.131±0.001 k 0.415±0.004 i 1 1.425±0.008 c 1.230±0.007 c 3.580±0.017 c 37 2 1.189±0.014 e 1.015±0.013 e 2.705±0.034 e 3 1.323±0.061 d 1.111±0.051 d 3.117±0.140 d 4 0.664±0.009 h 0.577±0.007 g 1.744±0.022 g Different small letters in the same column indicate a significant difference at P<0.05 by Duncan's multiple range test.. Monacolin K citrinin monacolin K Table 10-3. monacolin K 215.85 mg/kg 40.41 mg/kg 5.3. 32 1 1263.04 mg/kg 5.9. monacolin K, 35 4 165 mg/kg. monacolin K 75% 53%, 50%. monacolin K acid form lactone form 6:5 monacolin K. (KFDA) Monascus anka, M. purpureus, M. pilosus M. ruber 35,,, monacolin K / monacolin K. Monacolin K, glucose sucrose lactose,. - 151 -
Table 10-3. Changes in the content of activated and non-activated monacolin K of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. Germination Monacolin K content (mg/kg) Citrinin Temp. Period Activated type Non-activated type Total content ( C) (day) (Acid form) (Lactone form) monacolin K (mg/kg) White rice 30.22±1.24 j 10.19±0.12 j 40.41±1.19 k ND Brown rice 113.82±2.13 h 102.03±1.38 h 215.85±2.91 h ND 1 573.53±10.46 a 689.51±5.28 a 1263.04±15.75 a ND 32 2 424.89±8.81 b 331.55±5.27 c 756.44±14.07 b ND 3 72.95±1.14 i 62.35±1.21 i 135.30±2.35 j ND 4 73.56±1.21 i 108.79±5.82 h 182.35±7.03 i ND 1 137.14±1.43 g 204.51±2.19 f 341.66±3.62 f ND 35 2 266.07±0.07 e 269.45±0.52 d 535.52±0.59 d ND 3 135.18±0.26 g 123.21±1.57 g 258.38±1.83 g ND 4 9.96±0.13 k 14.60±0.26 j 24.57±0.39 l ND 1 309.32±8.29 d 447.02±12.25 b 756.33±20.54 b ND 37 2 204.63±2.48 f 198.67±2.28 f 403.30±4.76 e ND 3 333.67±3.10 c 248.84±3.13 e 582.51±6.23 c ND 4 116.95±0.87 h 128.04±0.51 g 244.99±1.37 g ND ND : not detected. Different small letters in the same column indicate a significant difference at P<0.05 by Duncan's multiple range test. monacolin K glucose, monacolin K. Citrinin mycotoxin (6) citrinin 0.05 mg/kg (5). citrinin (7,8). citrinin (Table 3) M. pilosus 305-9 (KFCC 11410P) citrinin monacolin K. - 152 -
. Table 10-4. 3.71 0.35 mg/g 0.31 0.08 mg/g. 1.40 0.19 mg/g (data not shown).. 4.62~13.80 0.43~1.30 mg/g.,,,. Table 10-4. Changes in the total polyphenol and flavonoid contents of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. Germination Total polyphenol content Total flavonoid content Temp. Period (mg/g) (mg/g) ( C) (day) White rice 0.31±0.01 m 0.08±0.00 m Brown rice 3.71±0.14 l 0.35±0.00 l 1 4.62±0.08 k 0.43±0.01 k 32 2 5.21±0.11 j 0.49±0.01 j 3 7.32±0.09 h 0.69±0.01 h 4 8.84±1.24 f 0.83±0.02 f 1 5.86±0.08 i 0.55±0.01 i 35 2 7.33±0.11 h 0.69±0.00 h 3 9.14±0.12 e 0.86±0.01 e 4 11.53±0.08 b 1.08±0.01 b 1 8.26±0.14 g 0.78±0.00 g 37 2 10.30±0.09 d 0.97±0.01 d 3 10.84±0.08 c 1.02±0.02 c 4 13.80±0.09 a 1.30±0.02 a Different small letters in the same column indicate a significant difference at P<0.05 by Duncan's multiple range test. - 153 -
. Table 10-5. 1.14 mg TE/g 2.85 mg AAE/g (data not shown), 3.73 mg TE/g 10.47 mg AAE/g., 32 /1 22.16 mg TE/g 62.27 mg AAE/g. DPPH ABTS,.. 3~4 6.,. Table 10-5. Changes in the free radical scavenging activities of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. Germination DPPH radical scavenging activity ABTS radical scavenging activity Temp. Period (mg TE/g) (mg AAE/g) ( C) (day) White rice 0.56±0.01 m 1.56±0.03 m Brown rice 3.73±0.09 i 10.47±0.13 i 1 22.16±0.12 a 62.27±0.21 a 32 2 13.23±0.08 b 37.16±0.14 b 3 2.31±0.08 k 6.49±0.11 k 4 3.14±0.11 j 8.83±0.10 j 1 5.94±0.09 f 16.69±0.15 f 35 2 9.35±0.07 d 26.27±0.21 d 3 4.48±0.12 g 12.58±0.14 g 4 1.37±0.14 l 3.03±0.09 l 1 13.24±0.17 b 37.21±0.12 b 37 2 7.03±0.08 e 19.75±0.08 e 3 10.18±0.15 c 28.59±0.11 c 4 4.25±0.09 h 11.94±0.10 h Different small letters in the same column indicate a significant difference at P<0.05 by Duncan's multiple range test. - 154 -
.,, monacolin K Table 10-6.,., 0.769~0.968, -0.967~-0.983. Monacolin K,, (0.633~0.969). (0.742~0.830),,,, monacolin K., monacolin K,.,. Table 10-6. Correlation coefficients among Monascus pigment, color, monacolin K content and antioxidant activity of Monascus pilosus (305-9) cultivated brown rice from germinated rough rice with different germination temperatures and periods. A 500 A 470 A 400 L a b MK TPC TFC DPPH ABTS Temp 0.470 ** 0.493 ** 0.559 *** -0.558 *** 0.525 *** 0.759 *** 0.325 * 0.742 *** 0.747 *** 0.336 * 0.333 * Period -0.193-0.180-0.116 0.134-0.140 0.126-0.289 0.830 *** 0.827 *** -0.275-0.279 A 500 1.000 0.999 *** 0.988 *** -0.967 *** 0.965 *** 0.769 *** 0.969 *** -0.026-0.017 0.966 *** 0.967 *** A 470 1.000 0.992 *** -0.972 *** 0.968 *** 0.788*** 0.963 *** -0.007 0.002 0.960 *** 0.961 *** A 400 1.000-0.983 *** 0.978 *** 0.841 *** 0.940 *** 0.070 0.078 0.937 *** 0.938 *** L 1.000-0.996 *** -0.887 *** -0.897 *** -0.092-0.101-0.889 *** -0.892 *** a 1.000 0.877 *** 0.899 *** 0.064 0.074 0.890 *** 0.893 *** b 1.000 0.633 *** 0.329 * 0.337 * 0.622 *** 0.625 *** MK 1.000-0.128-0.119 0.997 *** 0.998 *** TPC 1.000 0.979 *** -0.115-0.119 TFC 1.000-0.106-0.109 DPPH 1.000 0.991 *** * : P<0.05, ** : P<0.01, *** : P<0.001, A 500 : red color groups, A 470 : orange color groups, A 400 : yellow color groups, L : lightness, a : redness, b : yellowness, MK : total monacolin K content (mg/kg), TPC: total polyphenol content (mg/g), TFC : total flavonoid content (mg/g), DPPH : DPPH radical scavenging activity (mg TE/g), ABTS : ABTS radical scavenging activity (mg AAE/g). - 155 -
. 0.25~0.64 g/cm 3.. a, b, a a. cellulose (27.95 ), (1.211) a (0.192). 39.07, (1.873) a (0.564).. (Table 10-7). 25 g 10 g ( : cellulose = 1 : 1) Table 10-8., a 37.14, 1.558 0.384, 32 37.45 35.84.., cellulose HS, a.,. - 156 -
Table 10-7. Physical characteristics of Monascus pilosus (305-9) cultivated brown rough rice from germinated rough rice with different germination condition and sub-materials. Germination Constant of equation Loosed bulk Tapped bulk Dynamic angle ( ) density (g/cm 3 ) density (g/cm 3 Hausner ratio Temp. Period ) a b Cereal powder 39.07 ± 3.10 0.321 ± 0.012 0.601 ± 0.000 1.873 ± 0.070 0.564 ± 0.035 0.085 ± 0.016 Cellulose 27.95 ± 1.94 0.683 ± 0.016 0.827 ± 0.013 1.211 ± 0.015 0.192 ± 0.022 0.164 ± 0.076 White rice 30.05 ± 1.58 0.716 ± 0.017 0.959 ± 0.017 1.341 ± 0.040 0.067 ± 0.027 0.277 ± 0.044 Brown rice 32.46 ± 1.99 0.586 ± 0.007 0.893 ± 0.015 1.525 ± 0.023 0.357 ± 0.009 0.422 ± 0.086 1 37.82 ± 3.65 0.462 ± 0.007 0.777 ± 0.012 1.681 ± 0.013 0.444 ± 0.005 0.169 ± 0.010 32 2 38.65 ± 2.08 0.477 ± 0.008 0.784 ± 0.012 1.644 ± 0.036 0.430 ± 0.015 0.195 ± 0.016 3 38.46 ± 1.09 0.454 ± 0.017 0.777 ± 0.012 1.716 ± 0.090 0.452 ± 0.035 0.233 ± 0.042 4 36.75 ± 1.24 0.467 ± 0.004 0.777 ± 0.012 1.664 ± 0.022 0.415 ± 0.004 0.378 ± 0.048 1 39.62 ± 1.81 0.421 ± 0.003 0.745 ± 0.011 1.769 ± 0.011 0.484 ± 0.003 0.147 ± 0.018 35 2 38.65 ± 2.28 0.475 ± 0.011 0.771 ± 0.000 1.624 ± 0.039 0.437 ± 0.013 0.129 ± 0.013 3 37.72 ± 1.22 0.449 ± 0.018 0.784 ± 0.012 1.748 ± 0.066 0.496 ± 0.020 0.187 ± 0.053 4 35.67 ± 1.81 0.449 ± 0.000 0.777 ± 0.012 1.733 ± 0.026 0.442 ± 0.012 0.425 ± 0.210 1 41.10 ± 1.78 0.444 ± 0.010 0.854 ± 0.128 1.924 ± 0.297 0.530 ± 0.086 0.118 ± 0.053 37 2 39.08 ± 1.14 0.410 ± 0.003 0.721 ± 0.010 1.760 ± 0.021 0.530 ± 0.002 0.080 ± 0.004 3 38.62 ± 2.17 0.444 ± 0.004 0.758 ± 0.011 1.706 ± 0.035 0.463 ± 0.012 0.157 ± 0.018 4 40.12 ± 1.82 0.444 ± 0.004 0.751 ± 0.000 1.692 ± 0.014 0.452 ± 0.005 0.173 ± 0.042 Table 10-8. Physical characteristics of Monascus pilosus (305-9) cultivated brown rough rice prototypes from germinated rough rice with different germination condition and sub-materials. Germination Constant of equation Loosed bulk Tapped bulk Dynamic angle ( ) density (g/cm 3 ) density (g/cm 3 Hausner ratio Temp. Period ) a b White rice 37.82 ± 1.56 0.643 ± 0.014 0.915 ± 0.000 1.424 ± 0.030 0.313 ± 0.020 0.256 ± 0.094 Brown rice 37.13 ± 3.18 0.559 ± 0.010 0.871 ± 0.015 1.558 ± 0.039 0.384 ± 0.010 0.262 ± 0.086 1 37.45 ± 2.09 0.480 ± 0.027 0.781 ± 0.012 1.630 ± 0.104 0.425 ± 0.042 0.165 ± 0.023 32 2 34.39 ± 2.20 0.496 ± 0.022 0.795 ± 0.000 1.605 ± 0.070 0.411 ± 0.029 0.187 ± 0.023 3 38.08 ± 3.02 0.487 ± 0.013 0.768 ± 0.023 1.577 ± 0.045 0.410 ± 0.023 0.148 ± 0.020 4 35.84 ± 2.77 0.507 ± 0.010 0.788 ± 0.012 1.548 ± 0.028 0.387 ± 0.017 0.195 ± 0.080 1 36.50 ± 1.67 0.484 ± 0.004 0.736 ± 0.000 1.520 ± 0.014 0.404 ± 0.013 0.103 ± 0.028 35 2 37.77 ± 2.40 0.506 ± 0.005 0.768 ± 0.011 1.517 ± 0.020 0.400 ± 0.005 0.096 ± 0.007 3 35.46 ± 3.44 0.506 ± 0.005 0.774 ± 0.020 1.530 ± 0.027 0.399 ± 0.005 0.120 ± 0.009 4 35.36 ± 3.48 0.518 ± 0.022 0.788 ± 0.012 1.521 ± 0.045 0.373 ± 0.009 0.216 ± 0.075 1 38.17 ± 1.69 0.509 ± 0.013 0.774 ± 0.020 1.521 ± 0.007 0.394 ± 0.006 0.119 ± 0.008 37 2 35.94 ± 1.52 0.467 ± 0.008 0.731 ± 0.021 1.564 ± 0.038 0.465 ± 0.010 0.063 ± 0.009 3 34.93 ± 3.43 0.477 ± 0.024 0.755 ± 0.019 1.583 ± 0.055 0.421 ± 0.012 0.120 ± 0.026 4 38.07 ± 1.31 0.472 ± 0.007 0.749 ± 0.011 1.587 ± 0.013 0.412 ± 0.005 0.160 ± 0.016-157 -
., Table 10-9. 337.62% 344.85%,. 32 392.65~435.22%, 35 410.79~410.11%. 12.43% 13.37%.. Table 10-9. The water binding capacity, solubility and swelling power of Monascus pilosus (305-9) cultivated brown rough rice prototypes from germinated rough rice with different germination condition and sub-materilas. Germination Temp. Period Water binding capacity (%) Water solubility (%) Swelling power (%) White rice 337.62 ± 9.34 12.43 ± 1.10 0.907 ± 0.029 Brown rice 344.85 ± 6.00 13.37 ± 0.97 0.922 ± 0.007 1 392.65 ± 13.89 19.82 ± 1.19 0.936 ± 0.013 32 2 406.21 ± 11.55 20.09 ± 1.30 0.944 ± 0.003 3 421.96 ± 10.20 18.83 ± 0.56 0.920 ± 0.018 4 435.22 ± 23.16 18.98 ± 1.27 0.933 ± 0.017 1 410.79 ± 21.57 20.91 ± 0.39 0.940 ± 0.004 35 2 411.31 ± 14.26 21.40 ± 1.19 0.931 ± 0.016 3 367.19 ± 17.80 17.38 ± 2.82 0.908 ± 0.035 4 410.11 ± 10.90 16.83 ± 3.55 0.890 ± 0.081 1 399.63 ± 13.70 18.93 ± 0.37 0.944 ± 0.012 37 2 422.90 ± 23.51 20.71 ± 0.50 0.928 ± 0.027 3 387.93 ± 8.41 20.53 ± 0.13 0.939 ± 0.023 4 407.13 ± 13.55 21.39 ± 0.36 0.936 ± 0.017-158 -
in vitro - in vitro - - 100% 1 (2012) - Activity-guided purification, C5 - C5-100% -,, - 100% -,,, 4, - 100% 2 (2013) in vitro in vivo in vitro - Western blot assay (C5) in vitro - 100% - 2 (C5) - 100% - 4 in vitro - 100% -, - 100% - 159 -
in vivo - Western blot assay (C5) in vivo - 100% -,, ph - 100% 3 (2014) -,,, - 100% - - 100% ( ) -,, - 160 -
1. (3/3) ( ) 10-2014-0068297 (2014.06.05.) 10-2015-0110171 (2015.08.04.) 10-2015-0109877 (2015.08.04.) K 2. (1/2) ( ) 2015.09 3. (1/1) ( ) 2015.06.02 4. (2/2) ( 10 ) ( 3 ) 2015.08.13 2015.10.15-161 -
5. SCI (2/3) SCI (5/4) Effects of high hydrostatic pressure treatment on the Food Chemistry SCI enhancement of functional components of germinated rough rice 2015 166 (Oryza sativa L.) SCI Influence of Applied Pressure on Bioactive Compounds of Germinated Rough Rice (Oryza sativa L.) SCI (Oryza sativa L.) SCI SCI (Oryza sativa L.) SCI SCI Monacolin K Food and Bioprocess Technology 8 2015 42(6) 2013 42(11) 2013 46(1) 2014 47(2) 2015 44(8) 2015 6. (8/6) Changes of Enzyme Inhibitory Activities on the Different Part of Rough Rice (Oryza setiva L.) with Germination Periods. Effect of High Pressure (HP) Treatment on the Increase of Antioxidant Compounds and Activity of Germinated Rough Rice (Oryza setiva L.). Effect of High Pressure Treatment on the Change of Functional Components of Germinated Rough rice (Oryza sativa L.). Enzyme Inhibitory Activities of Rough Rice (Oryza sativa L.) Extracts with Germination and High Pressure Treatment. Influence of Applied Pressure on Enhancement of Functional Component of Germinated Rough Rice. Anti-diabetic Activity of Germinated Rough Rice (Oryza sativa L.) Extracts in 3T3-L1 Adipocytes. Changes of Phenolic-Acids and Vitamin E Profiles on Germinated Rough Rice (Oryza sativa L.) Treated by High Hydrostatic Pressure. Effects of High Hydrostatic Pressure Treatment on the Chemical Composition of Germinated Rough Rice (Oryza sativa L.). The International Society for Nutraceuticals and Functional Foods (ISNFF) 2013 2013 2013 2013 2014 ISNFF 2014 2015 2015-162 -
(1) (2) - 163 -
1. (Germination). DNA,. 2.., amylase, lipase glycerol, glycerol.,.,.,,,,.,,.,,. - 164 -
3.. Fatty acids,, 3. n-3 n-6. Linoleic acid (18:2 n-6, LA) docasahesanoic acid (DHA) eicosapentanoic acid (ELA) 18 3 n-3. LA elongate desaturase EPA, EPA PH3, EPA DHA. (conjugated linoleic acid, CLA) linoleic acid. Linoleic acid linoleic acid, CLA grilled ground beef CLA.. Phenolic acids phenolic acids,,. phenylpropamoid 3 hydroxycinnamic acids, hydroxybenzoic acids,, ester. phenolic acid ferulic acid,ρ-coumaric acid, caffeic acid, trans-cinamic acid, benzoic acid, isovitexin.. ferulic acid,,. 1, 2,. - 165 -
. γ-aminobutyric acid (GABA) GABA. GABA, glutamate glutamatedecarboxylase (GAD). GABA,. GABA.. Arabinoxylan (Arabinoxylan) arabinose xylose,,,, lignin hemicellulose. β-(1 4)-xylan glucuronic acid 4-methyl ether.,,,,., NK cell,.. γ-oryzanol (γ-oryzanol) 10 phytosteryl ferulate 80% cycloartenyl ferulate, 2,4-methylenecycloartanyl ferulate campesteryl ferulate. sterol ferulic acid ester plasma lipid, plasma-cholesterol, HDL-cholesterol,,,. - 166 -
1.,. 5~10%, 2014 400 100 8.3. 2025 680.,,. (β-cell).,,,. 多飮, 多食, 多尿, 10~15%, 50%. 2.,..... - 167 -
,,,,,,,,,,,,.,,,. 2010 3,760 (IDF) 2030 4,900.. 2009 263 2016 443, 2010 4,401 2016 9,300.. 2 1,., TZD, α-glucosidase, 2.,,. GLP-1, DPP-4, SGLT2. - 168 -
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