대추 잎 유래 알파-, 베타-글루코시데이즈 저해제의 특성 규명과 식품에의 적용에 관한 연구

학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 식품공학과, 2019. 2. 최영진.대추 잎은 항산화, 혈압조절, 등의 효능이 있는 것으로 알려져 있어서 고대에는 한국, 중국 등 아시아 지역에서 약으로 이용되어왔다. 하지만 잎에는 열매에 비해 더욱 풍부한 플라보노이드와 폴리페놀이 존재함에도 불구하고 최근 대부분의 연구가 열매로 집중되면서 잎은 부산물로 여겨져 버려지고 있다. 플라보노이드와 폴리페놀은 녹차를 포함한 잎을 이용한 차류에 풍부하다고 알려져 있으며, 이들은 α-글루코시데이즈의 활성을 저해하는 것으로 알려져 있다. 이러한 α-글루코시데이즈 저해제들은 당뇨와 비만의 치료제로 활용 가능성이 있어 꾸준히 관심을 받고 있다. 최근 아카보스와 같이 α-글루코시데이즈 저해활성을 이용한 시판약들이 가진 단점들 때문에 천연의 α-글루코시데이즈 저해제를 찾는 연구가 활발히 진행되고 있다. 하지만 대추 잎에는 알려지지 않은 물질들이 다수 존재하며, 다양한 플라보노이드와 폴리페놀이 존재함에도 불구하고, 대추 잎에 대한 α-글루코시데이즈 저해활성에 관한 연구가 진행되어 있지 않았다. 그래서 이번 연구에서 대추 잎의 α-글루코시데이즈 저해활성에 대해 알아보았다. 기존에 알려진 대추 잎에 존재하는 α-글루코시데이즈 저해제의 α-글루코시데이즈 저해활성은 아카보스에 비해 약할 것으로 예상되었지만, 예비 실험 결과 아카보스보다 약 2배 강한 저해를 나타냈다. 이와 같이 예상보다 강한 저해가 나타나게 된 원인에 대해 알아보는 연구를 진행하였다. 가장 저해가 강한 물질을 분리 정제해 본 결과 3',5'-di-C-β-D-glucosyl phloretin이라는 물질에 의해 강한 저해가 나타나는 것으로 확인되었다. 이 물질은 기존에 α-글루코시데이즈 저해능력에 관하여 보고된 바가 없는 물질이었으며, α-글루코시데이즈 저해 능력을 확인해 본 결과 아카보스에 비해 약 13.5배 강한 저해를 하였다. 그리고 이 저해제는 α-아밀레이즈는 저해하지 않고, 선택적으로 α-글루코시데이즈만을 저해하여 아카보스보다 부작용이 적을 것으로 기대되었다. 게다가 모방 체외 위장관 환경하에서 실험한 결과 통계적으로 유의미한 차이 없이 -글루코시데이즈를 저해하는 것을 확인하였다. 대추 잎의 α-글루코시데이즈 저해활성을 이용하여 식후 혈당 조절에 도움을 줄 수 있는 기능성 밥의 제조를 해 보았다. 밥은 한국, 인도, 중국 등 여러 나라에서 주식으로 하고 있다. 하지만 혈당지수가 매우 높은 식품에 속하여 식후 혈당 조절이 필요한 당뇨환자들은 백미보다 현미를 권하고 있다. 하지만 현미의 식감 때문에 많은 환자들이 현미를 기피하고 있다. 그래서 이번 연구에서 대추 잎의 α-글루코시데이즈 저해활성을 이용하여 식후 혈당 조절에 도움을 줄 수 있고, 식감은 백미와 유사한 기능성 밥을 제조하는 것을 목표로 하였다. 대추 잎 추출물의 양이 0, 5, 10 mg/mL 첨가되어도 밥의 하드니스와 스티키니스에는 영향이 없는 것을 관찰하였다. 대추 잎 추출물의 양이 0, 5, 10 mg/mL로 증가할수록 당 분해가 최대치에 도달하는 시간이 180, 210, 360 분으로 증가하였다. 향과 색은 있었지만 기호도에 의한 관능평가를 하였을 때 기존의 즉석밥과 통계적으로 유의미한 차이가 없었다. 이를 통해 대추 잎의 α-글루코시데이즈 저해 활성을 이용하여 기능성 밥 제조의 가능성을 확인할 수 있었다. 대추 잎에 풍부한 루틴을 아이소쿼시틴으로 전환하는 과정에서 β-글루코시데이즈 저해제의 존재 가능성에 대해 연구하기 시작하였고, β-글루코시데이즈 저해제의 존재를 증명하였다. 대추 잎에 존재하는 β-글루코시데이즈 저해제를 분리 정제하였다. 그 저해제의 예상 분자량은 392 g/mol이었으며, 분자식은 C17H23O13N으로 예상되었다. 그리고 이 저해제는 혼합 비경쟁적 저해의 양상을 나타내는 것으로 확인되었다. 이 물질은 기존에 알려진 저해제가 아닌 새로운 저해제로 구조, 저해 기작에 대한 추가 연구가 필요하다. 대추 잎의 β-글루코시데이즈 저해활성을 이용하여 기능성이 강화된 자몽주스를 제조하였다. 쓴맛 성분인 나린진은 시트러스계 과일 주스 제조 시 품질저하의 주요한 원인이 된다. 이를 제거하기 위해 식품산업에서 나린지네이즈라는 효소를 사용하게 된다. 이때 나린지네이즈에 의해 나린진은 나린제닌으로 전환이되고, 이때 생성된 나린제닌은 아무런 맛이 없고, 다양한 기능성을 가지고 있는 것으로 알려져 있다. 하지만 이 성분은 다양한 기능성에도 불구하고 물에 대한 용해도가 낮아 생체내 이용율이 낮다는 단점이 있다. 여기서 대추 잎의 β-글루코시데이즈 저해 활성을 이용하여 나린진의 중간생성물이며, 다양한 기능성을 가지고 있고, 물에 대한 용해도가 나린제닌 보다 높은 푸르닌으로 전환이 되어 기능성이 강화된 자몽주스를 제조하는 것을 목표로 하였다. 나린지네이즈 처리 시 쓴맛 성분인 나린진은 효과적으로 제거되었고, 푸르닌의 함량이 대추 잎을 처리하지 않은 자몽주스에 비해 약 1.31배 많은 2.47 mmol/mL가 생성되었다. 게다가 더 낮은 온도, 더 적은 양의 효소로 최적점에 도달하는 것이 관찰되었고, 이 결과를 통해 식품산업에 적용 시 이점이 될 것으로 기대된다. 요컨대, 본 연구에서 대추 잎 유래의 새로운 -, β- 글루코시데이즈 저해제들을 얻을 수 있었고, 이들의 특성을 규명하였으며, 식품산업에 적용가능성도 확인할 수 있었다.The jujube leaf has been used for medicinal purposes throughout the ancient history of several Asian countries due to its antioxidant and sedative properties and regulatory effects on blood pressure. Although the leaf is more abundant in flavonoids and polyphenols as compared to the fruit, the majority of recent research efforts have focused on the fruit and the leaf is regarded as a by-product or waste. Flavonoids and polyphenols are known to inhibit the activity of α-glucosidase. Inhibitors of α-glucosidase have received growing interest because of their potential of being used as treatment for diabetes and obesity. Many research groups are trying to find α-glucosidase inhibitors from nαatural products because of the disadvantages of commercial drugs that possess α-glucosidase inhibitory activity. However, despite the many unknown compounds and various flavonoids in the jujube leaf, no studies have reported on an α-glucosidase inhibitory activity from the jujube leaf. Therefore, in this study, I investigated α-glucosidase inhibitory activity of the jujube leaf. The inhibitory activity of jujube leaf on α-glucosidase was expected to be low compared to acarbose, but preliminary experiments showed that jujube leaf α-glucosidase inhibition was approximately twice as strong as acarbose. Therefore, this strong inhibition was further investigated in this work, which ultimately revealed that the strong inhibition was caused by 3',5'-di-C-β-D-glucosyl phloretin. This substance was not previously reported to inhibit α-glucosidasethus, the α-glucosidase inhibitory activity of this inhibitor was characterized. It was confirmed that the α-glucosidase inhibitory activity of this substance was approximately 13.5-times stronger than that of acarbose. This inhibitor can selectively inhibit α-glucosidase, as it does not inhibit α-amylase. Because of this inhibition specificity, 3',5'-di-C-β-D-glucosyl phloretin is expected to have less side effects than acarbose. In addition, in vitro experiments showed that the rate of α-glucosidase inhibition was not statistically significantly different. The functional rice that can control postprandial blood glucose through the use of jujube leaf inhibitory activity on α-glucosidase was produced. Rice is a staple food in many Asian countries. However, some people who require control of postprandial blood glucose are more likely to eat brown rice than white rice, but many patients avoid brown rice due to the texture. Therefore, in this study, I aimed to produce functional rice with similar texture to white rice that can use α-glucosidase inhibitory activity of jujube leaf to control postprandial blood glucose. It was observed that the addition of jujube leaf extract did not affect the texture of rice including hardness and stickiness. As the amount of jujube leaf extract increased from 0 to 10 mg/mL, the time required for glucose degradation reached its maximum value, increasing from 180 to 360 min. Although the rice possessed the flavor and color of jujube leaf, there was no statistically significant difference from conventional instant rice when sensory evaluation was performed by preference degree. Thus, the possibility of producing functional rice by using the α-glucosidase inhibitory activity of jujube leaf was confirmed. I demonstrated the presence of a β-glucosidase inhibitor in the process of converting the rutin present in jujube leaf into isoquercitrine. The expected molecular weight of this β-glucosidase inhibitor was 392 g/mol and the predicted molecular formula was C17H23O13N. It was confirmed that this inhibitor showed mixed non-competitive inhibition. This substance is a new inhibitor, so further studies of its structure and inhibition mechanism analysis are needed. A functionally enhanced grapefruit juice was produced using the β-glucosidase inhibitory activity of jujube leaf. Naringin, a bitter compound in citrus fruit juice, is one of the main reasons for a deterioration in the production quality of citrus fruit juice. In the food industry, naringinase is used to remove naringin and then naringenin is produced from naringin. Naringenin is tasteless and has various functionalities. The main drawback of naringenin, despite its different functionalities, is its low bioavailability due to low water solubility. The aim of the present study was to produce grapefruit juice with enhanced functionality by degrading naringin and enhancing the production of prunin using the β-glucosidase inhibitory activity of jujube leaf. Naringin was effectively removed and 2.47 mmol/mL of prunin was produced, which was approximately 1.31 times higher than that of grapefruit juice without jujube leaf extract. Moreover, it has been observed that the optimum condition of prunin production was reached with lower temperature, lower amount of enzyme, and this result is expected to be an advantage when applied to the food industry. In summary, in this study, novel α- and β-glucosidase inhibitors derived from jujube leaf were isolated and characterized and their applicability to the food industry was confirmed.Contents Abstract ………………………………………………………………… I Contents ……………………………………………………………...... V List of Tables ……………………………………………………….. XI List of Figures …………………………………………………..... XIII Chapter I. Literature review……………………………………1 I-1. Introduction ……………………………….…..………………... 2 I-2. Glucosidase Inhibitors …….……………..…...…...….…….... 4 I-2-1. α-Glucosidase inhibitors…………………..……..….………... 4 I-2-1-1. α-Glucosidase inhibitors present in jujube leaf ………......10 I-2-1-1-1. Rutin …………………………………...…………...…10 I-2-1-1-1. Catechin ………..……………………...………………13 I-2-1-1-1. Isoquercitrin …………..…….………...………………15 I-2-2. β-Glucosidase inhibitors ….....………………………...…….. 17 I-3. Application of Glucosidase Inhibitors …..……………... 19 I-3. References …....…………………………...……………………..21 Chapter II. A novel α-glucosidase inhibitor from jujube leaf extract ………..…………………………………….… 30 II-1. Introduction ………………………………………………….. 31 II-2. Materials and Methods …………………......……………... 35 II-2-1. Chemicals …..……………………………………………….. 35 II-2-2. Sample preparation ……………………..………………….. 36 II-2-3. α-Glucosidase inhibition assay …………...……..……..…... 37 II-2-4. α-Amylase inhibition assay …………….………………….. 38 II-2-5. Isolation of α-glucosidase inhibitor ……………………….. 39 II-2-6. Structure analysis …………..………….……..…………….. 40 II-2-7. Enzyme kinetic study ……...……………………………...…42 II-2-8. In vitro digestion …………..………………………………...43 II-2-9. Statistical analysis …………………………………………...46 II-3. Results and Discussion ………………….………………..... 47 II-3-1. α-Glucosidase inhibition ………………………….…………47 II-3-2. Isolation and purification of novel α-glucosidase inhibitor ……………………………………...…………………………………51 II-3-3. Structure analysis of novel α-glucosidase inhibitor ………54 II-3-4. Characterization of novel α-glucosidase inhibitor ……..…57 II-3-4-1. α-Glucosidase inhibitory activity …………….…….....57 II-3-4-1. α-Amylase inhibitory activity …………..……...……...60 II-3-5. Kinetic studies …………………………………..…...………64 II-3-6. In vitro digestion ………………………………..……………66 II-4. Conclusion ……………………..…………………...……….... 69 II-5. References …………………………………………..………… 70 Chapter III. Application of α-glucosidase inhibitory activity to produce functionalized rice…………………… 75 III-1. Introduction ……………………………………………….... 76 III-2. Materials and Methods ……………………….………….. 78 III-2-1. Chemicals ………………………………………………….. 78 III-2-2. Sample preparation …………………………….…………. 79 III-2-3. Texture analysis …………………..……...………………... 80 III-2-4. Preparation of rat intestinal enzyme solution ………..…. 81 III-2-5. Measurement of degree of carbohydrate hydrolysis…….. 82 III-2-6. Sensory analysis ……………………………………...…… 83 III-2-6. Statistical analysis ………………………………………… 84 III-3. Results and Discussion …………………………………….85 III-3-1. Texture of rice ……………………………………………... 85 III-3-2. Degree of carbohydrate hydrolysis ……………..………... 89 III-3-2. Sensory test …………………………..…………..………… 91 III-4. Conclusion ……………………………..…………….. 94 III-5. References …………………………………………………… 95 Chapter IV. A natural β-glucosidase inhibitor from jujube leaf extract………………………….………………..…...... 97 IV-1. Introduction ………………………………………..………... 98 IV-2. Materials and Methods …………………………….……. 101 IV-2-1. Chemicals ……………….…………..………...……………101 IV-2-2. Preparation of jujube leaf extract ………………………..102 IV-2-3. β-Glucosidase inhibition assay ………………..…………..103 IV-2-4. Isolation of β-glucosidase inhibitor ……….…..……...…..104 IV-2-4-1. Size exclusion chromatography…..…….…...……....104 IV-2-4-2. Semi-preparative HPLC………….…..……...……....105 IV-2-5. Characteristic analysis …………………….…..……….....107 IV-2-5-1. Prediction of the empirical formula….……...……...107 IV-2-5-2. Enzyme kinetic study………………….……..……....108 IV-2-6. Statistical analysis ………………………….…..…...……..109 IV-3. Results and Discussion ……………………………...…… 110 IV-3-1. Isolation of β-glucosidase inhibitor ………………………110 IV-3-1-1. Size exclusion chromatography…….………………...110 IV-3-1-2. Semi-preparative LC……….……..…………………..114 IV-3-2. Characteristic of β-glucosidase inhibito?r from jujube leaf ………………………………………………………………………116 IV-3-2-1. Prediction of empirical formula ……...…………...…116 IV-3-2-2. Enzyme kinetic study………………..………………..123 IV-4. Conclusion …………………..…………………………….... 127 IV-4. References …………………………………….………...….. 128 Chapter V. Application of β-glucosidase inhibitory activity of jujube leaf to produce functionalized grapefruit juice …………………………….…….……………... 132 V-1. Introduction ………………………..……………………..... 133 V-2. Materials and Methods …….……………….…………… 136 V-2-1. Chemicals ………………………………………………….. 136 V-2-2. Enzymatic biotransformation ……………...….…………. 137 V-2-3. HPLC analysis ………..…………..……..……………….... 138 V-2-4. Response surface methodology ………………….………...140 V-2-5. Statistical analysis……………………………...……….…. 144 V-3. Results and Discussion …………………..……..………….145 V-3-1. Enzymatic biotransformation ………………...………….. 145 V-3-2. Response surface methodology ………...………..……..… 147 V-4. Conclusion ……………………………..…………….. 153 V-5. References …………………………………………………… 154 국문 초록 ………………………………………………….………..156Docto

한글機械化(打字機)를 위한 構造의 硏究

解放後 우리 한글의 풀어쓰기 문제가 論議되고 檢討된 적이 있다. 이 풀어 쓰기의 長點中에 한글의 機械化 特히 打字機의 製造, 使用에 있어 그 효율적이고 편리한 點이 지적 되었다. 그러나 當時 한글을 이미 읽을 수 있고 쓸수 있었던 많은 국민들로 부터 그 익숙하지 않은 점, 읽기에 不便한 點 等이 지적되어 結局 풀어쓰기는 施行되지 않고 現行의 한글 使用法이 施行되어 오늘에 이르렀다.

Toleration as basic virtue in the pluralistic society and its implications for civil education

학위논문(박사)--서울대학교 대학원 :사회교육과 일반사회전공,1998.Docto

대추잎 추출물로부터 효소를 활용한 아이소쿼시틴 생산의 최적화와 베타글루코시데이즈 저해제의 존재 가능성 제시

학위논문 (석사)-- 서울대학교 대학원 : 식품공학과, 2014. 2. 최영진.Recently, quercetin and its glucosides including isoquercetin (quercetin-3-β-D-glucoside, Q3G) and rutin (quercetin-3-rutinoside) draw attention due to healthful bioactivities such as antiproliferative, antioxidant, anti-iflammatory. However, isoquercetin exhibits the highest bioavailability, which varies according to the type of sugar moiety. Rutin is the most common flavonoid in plant resources, especially in jujube leaves which are the waste of food industry. To increase the availability of isoquercetin, the enzymatic biotransformation of isoquercetin from rutin in the jujube leaf extract using hesperidinase from Aspergillus niger was optimized. Hesperidinase is an enzyme complex containing α-L-rhamnosidase and β-D-glucosidase activities. Employing response surface methodology (RSM), the isoquercetin yield was optimized concerning the temperature (40~60°C, X1) time (24~72 h, X2) and pH (2~6, X3). The second-order polynominal model was developed by experiments based on Boc-behnken design containing 15 experimental runs with three replicates at the center point. The coefficient (R2) and p-value of response surface regression equation for isoquercetin yield were 0.93 and 0.01, respectively. The results showed the statistical significance. Consequently, the optimum condition was predicted at stationary point as to produce 2.65 mg/mL of isoquercetin by processing at 47.3°C, 52.16 h, and under pH 5.31. The verification of the model was carried out at the optimal conditions. The experimental value of 2.57 mg/mL of isoquercetin showed good agreement with the predicted one. These results might provide important information for utilization of jujube leaf as a waste in the food industry. From the optimization study, one interesting phenomenon was observed. Under various treatment conditions, the bioconversion of isoquercetin from rutin (α-L-rhamnosidase activity) was well-performed but the accumulation of quercitin (β-D-glucosidase activity) was not. A further study was carried out to ensure the cause of the loss of β-D-glucosidase activity of hesperidinase whether it is affected by enzyme kinetics or possible enzyme inhibitor. Firstly, to examine the effect of enzyme kinetics, enzymatic biotransformation was conducted at 40°C and pH 3.8 with enzyme concentration ranging from 0.03125 mg/mL to 160.0 mg/mL for 24, 48 and 72 h. Regardless of reaction time, quercetin was not produced in the concentration of enzyme below 5 mg/mL, while the activity of rhamnosidase was observed. The esculin hydrolysis test was conducted by phenolic compounds to appraise whether it is competitive inhibition or not. Secondly, to evaluate the existence of possible inhibitors, jujube leaf extracts with various concentrations (0 – 20%) were mixed with 0.5 mM of 4-nitrophenyl β-D-glucospyranoside solution containing 0.3 unit/mL of β-D-glucosidase from Almonds. The Hanes-Woolf plot showed non-competitive inhibition. Therefore, these results strongly suggested that certain components in the jujube leaf extract act like an inhibitor of β-d-glucosidase. Further study is necessary to identify and confirm the possible inhibitor.Contents..................................................................... І LIST OF TABLES........................................................ Ⅳ LIST OF FIGURES........................................................Ⅴ Abstract .....................................................................1 Part 1...........................................................................4 I. INTRODUCTION.........................................................5 II. MATERIALS AND METHODS.....................................10 2.1. Chemicals and reagents .......................................10 2.2. Jujube leaf extract ................................................10 2.3. Enzymatic biotransformation...................................11 2.3.1. Effect of enzyme concentration ...........................11 2.3.2. Effect of enzyme reacting time ............................12 2.3.3. Effect of pH .......................................................12 2.3.4. Effect of enzyme reaction temperature .................12 2.4. Analytical methods ...............................................13 2.5. Response surface methodology (RSM) ...................15 III. RESULTS AND DISCUSSION.....................................17 3.1. Effect of the concentration of enzyme ......................17 3.2. Effect of the time....................................................19 3.3. Effect of the pH .....................................................21 3.4. Effect of the reaction temperature ............................23 3.5. Response surface methodology (RSM) ...................25 Part 2 ........................................................................31 I. INTRODUCTION........................................................32 II. MATERIALS AND METHODS.....................................34 2.1. Chemicals and reagents .......................................34 2.2. Verification of existence of β-D-glucosidase inhibitor .....................................................................34 2.3. Analysis of phenolic compounds ............................34 2.4. Esculin hydrolysis test .........................................34 2.5. Enzyme kinetics ...................................................37 2.7. Analysis of metal ions............................................38 III . RESULTS AND DISCUSSION .........,,,,,,,...................39 3.1. Verification of existence of β-D-glucosidase inhibitor .....................................................................39 3.2. Phenolic compound in the jujube leaf extract ...........41 3.3. Enzyme kinetics ...................................................44 3.4. Analysis of metal ions ...........................................46 IV. CONCLUSIONS......................................................48 V. References.............................................................50 Ⅵ. 국문초록 ...............................................................54 LIST OF TABLES Table 1. Instrument and operating conditions for HPLC analysis.....................................................................14 Table 2. Central composite design for optimization of the enzyme reaction condition of isoquercetin from jujube leaf extracts .....................................................................16 Table 3. Analysis of variance for isoquercetin as linear, quadratic term and interactions on response variables ...................................................................27 Table 4. Analysis of variance of the factors for isoquercetin ...............................................................28 Table 5. Predicted maximum values of isoquercetin, the response variables of jujube leaf extracts treated by hesperidinase ............................................................30 Table 7. Operating condition for HPLC to analysis phenolic compounds ...............................................................36 Table 8. Analysis the metal ions, which is famous non-competitive inhibitors ..................................................47 LIST OF FIGURES Figure 1. Biotransformation of rutin to isoquercetin and quercetin by hesperidinase............................................7 Figure 2. The effect of hesperidinase concentration on the flavonoid yields (Alphabets represent Duncan's grouping). ..................................................................18 Figure 3. The effect of enzyme reaction time on the flavonoid yields...........................................................20 Figure 4. The effect of the pH on the enzymatic biotransformation (Alphabet is meaning Duncans grouping). .................................................................22 Figure 5. The effect of treatment temperature on the concentration of products (rutin, isoquercetin, quercetin). (Alphabet is meaning Duncans grouping)......................24 Figure 6. Response surface plot showing the effect of temperature, time and pH on amount of isoquercetin in jujube leaf extracts treated by hesperidinase ............................................................30 Figure 7. The effect of enzyme concentration of the concentration of products. The samples were taken every 24 h for 3 days. The treatment time of a, b, and c were 24, 48, and 72 h, respectively............................................40 Figure 8. Analysis of phenolic compounds for getting the fractions from jujube leaf extracts. ...............................42 Figure 9. Esxulin hydrolysis test. Peak points, which had phenolic compounds, were 9,10, 14, 15, 35, 45, 47, 49, 53, 57, 58, 60, and 76. However, the inhibition activity was occurred at 28, 29, 30, 34, 39, and 41. This section was from 5.5 min to 75 min.................................................43 Figure 10. The ratio of jujube leaf extracts were increased from 0% to 20%. The enzyme concentration was fixed on 0.3 unit/mL. (a) is Michaelis-Menten plot. (b) is Hanes-Woolf plot. ................................................................45Maste

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Analysis and Development for Organic Additives used in the Zinc Electroplating

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