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Subcritical Water Extraction and Hydrolysis of Citrus Flavonoids

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Abstract
감귤 껍질에는 flavanone으로서 hesperidin과 narirutin이 다량 함유되어 있으며, polymethoxyflavones(PMFs)로서 sinensetin, nobiletin, tangeretin이 소량 함유되어 있 는데, 이들 성분은 항산화, 항염, 항암, 심장 보호 등의 기능성을 나타낸다. 제1부 에서는 아임계수를 이용하여 온주밀감 껍질로부터 플라보노이드를 여러 온도(120 −180 °C)와 유속(1.0−2.0 mL/min)에서 추출하여 추출기작을 밝혀내었다. Hesperidin, narirutin, PMFs의 추출수율은 120 °C에서 40.9%, 69.0%, 67.4%이었으 며, 추출온도를 160 °C로 증가시키면 79.6%, 81.9%, 89.0%로 증가하였으나, 추출 온도 180 °C에서는 열분해되어 감소하였다. 물의 유속 1.0−2.0 mL/min에서의 추 출 곡선을 thermodynamic partitioning과 kinetic desorption 모델에 적용하여 추출 기작을 예측하였다. 추출 속도는 추출 초기 단계에서는 빨랐지만 후기 단계에서 는 느렸다. Thermodynamic partitioning 모델은 추출 초기에는 추출속도를 잘 예측 할 수 있었으나 추출 후반부에는 잘 예측하지 못하였다. 반면 two-site kinetic desorption 모델은 추출 전반부 뿐만 아니라 후반부에서도 추출 속도를 매우 예측 할 수 있었으며, 이로부터 감귤 껍질로부터 플라보노이드의 추출은 주로 입자 내 확산에 의해 이루어진다는 것을 알 수 있었다. 또한 two-site kinetic desorption 모 델은 열분해되는 온도(180 °C)에서도 추출속도를 잘 예측할 수 있었던 것으로 보 아, 이 모델은 아임계수를 이용한 감귤 플라보노이드의 추출은 물론 분해 기작을 모두 잘 설명 할 수 있었다. Hesperidin의 확산계수는 120 °C, 1 mL/min에서보다 160 °C, 2 mL/min에서 약 9.8배 높았다. Hesperidin의 확산에 필요한 활성화 에너 지(37.2−43.8 kJ/mol)는 narirutin과 PMFs의 활성화 에너지(8.2−36.8 kJ/mol)보다 높았다. 따라서 감귤 껍질로부터 플라보노이드의 추출은 주로 입자 내 확산에 의 해 이루어졌으며, 환경 친화적인 용매인 물을 사용하여 단시간 내에 추출할 수 있음을 보여주었다. 제2부에서는 아임계수를 사용하여 감귤 껍질로부터 플라보노이드를 추출과 가 수분해하였고, 아임계수 추출물의 개별 플라보노이드의 수율, 항산화 활성 및 효 소 억제 활성을 측정하였다. Hesperidin과 narirutin의 추출수율은 추출온도가 145 °C에서 165 °C로 증가함에 따라 증가하였다. 제1배당체(hesperetin-7-O-glucoside, prunin), 비배당체(hesperetin, naringenin)와 같은 가수분해물은 160 °C 이상의 온도 에서 생성되었다. 아임계수 추출물에서 hesperidin, hesperetin-7-O-glucoside, hesperetin 함량은 항산화 활성과 밀접한 관련이 있는 반면, hesperetin과 naringenin 의 함량은 효소 억제 활성과 밀접한 관련이 있었다. Hesperetin은 가장 높은 항산 화 활성을 보인 반면, hesperetin-7-O-glucoside는 angiotensin-І converting enzyme(ACE)과 pancreatic lipase(PL)에 대한 강한 저해 활성을 나타내었다. Naringenin은 xanthine oxidase와 α-glucosidase에 대한 강한 저해 활성을 나타내었 다. PMFs도 ACE와 PL에 대해 상대적으로 높은 저해 활성을 나타내었다. 따라서 친환경 용매인 물을 이용하여 짧은 시간 내에 감귤 껍질로부터 생리횔성이 높은 플라보노이드를 추출 및 가수 분해할 수 있음을 확인하였다. 제3부에서는 감귤 미숙과 착즙박을 대상으로 반연속식 아임계수 추출 및 가수 분해 장치을 이용하여 아임계수 추출물 중의 hesperidin을 생리활성이 높은 hesperetin-7-O-glucoside와 hesperetin으로 가수분해하였다. Hesperidin의 손실을 최 소화하면서 동시에 hesperetin-7-O-glucoside와 hesperetin의 생성을 극대화하기 위한 최적조건은 가수분해 온도 182.8 °C와 체류시간 3.2분이었으며, 위 조건에서 hesperetin-7-O-glucoside와 hesperetin의 생성량은 각각 11,499와 8,030 μg/g dry sample이었다. 처리 온도는 hesperidin의 아임계수 가수분해에 있어서 가장 중요한 요소였다. 체류시간이 가수분해에 미치는 영향은 온도에 따라 달랐으며, 고온의 가수분해 조건에서는 짧은 체류 시간이 요구되었다. 따라서 환경 친화적 용매인 물을 사용하여 짧은 시간 내에 hesperidin을 기능성이 높은 물질로 연속적으로 전 환할 수 있는 가능성을 확인하였다.
Citrus peels are the main source of many important flavonoids, flavanones (hesperidin and narirutin) and polymethoxyflavones (PMFs; sinensetin, nobiletin, and tangeretin), which have antioxidant, anti-inflammatory, anticancer, and cardioprotective properties. In part 1, the mechanisms controlling the extraction rates of flavonoids from Citrus unshiu peel using subcritical water (SW) were studied at different temperatures (120−180 °C) and flow rates (1.0−2.0 mL/min). The extraction yields increased from 40.9, 69.0, and 67.4% at 120 °C to 79.6, 81.9, and 89.0% at 160 °C for hesperidin, narirutin, and PMFs, respectively, while decomposition occurred at 180 °C. The extraction rate curves at different flow rates were used to determine whether the extraction was best described by a thermodynamic partitioning or kinetic desorption model. The extraction rate curves showed that the initial extraction phase is fast, while the subsequent phase is slow. The thermodynamic partitioning model did not match with the experimental data for the latter part of the extraction period. The two-site kinetic desorption model fits the entire extraction period very well, suggesting that the extraction of citrus flavonoids was mainly controlled by intra-particle diffusion. Interestingly, this model fits well even at the pyrolysis temperature (180 °C). Therefore, the two-site kinetic model well described both the decomposition and extraction mechanism of citrus flavonoids when using SW. The diffusion coefficient of hesperidin increased about 9.8-fold at 160 °C and 2 mL/min relative to 120 °C and 1 mL/min. The activation energy of hesperidin (37.2−43.8 kJ/mol) was higher than those of narirutin and PMFs (8.2−36.8 kJ/mol). The use of small amounts of SW, an environmentally friendly solvent, promotes good recovery of flavonoids from citrus peel in a short time. In part 2, citrus flavonoids were extracted and hydrolyzed from Citrus unshiu peel using SW. The individual flavonoid yields, antioxidant and enzyme inhibitory activities of the SW extracts were analyzed. The extraction yields of hesperidin and narirutin increased with increasing temperature from 145 °C to 165 °C. Hydrothermal hydrolysis products (HHP), such as monoglucosides (hesperetin-7-O-glucoside and prunin) and aglycones (hesperetin and naringenin) were obtained in the SW extracts at temperatures above 160 °C. The sum of hesperidin and its HHP in the SW extracts was strongly correlated with antioxidant activities, whereas the contents of hesperetin and naringenin were strongly correlated with enzyme inhibitory activities. Hesperetin exhibited the highest antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, ferric-reducing antioxidant power, and oxygen radical absorbance capacity), whereas hesperetin-7-O-glucoside exhibited the highest enzyme inhibitory activities (angiotensin-Іconverting enzyme (ACE) and pancreatic lipase (PL)). Naringenin exhibited the highest enzyme inhibitory activities (xanthine oxidase and α-glucosidase). PMFs (sinensetin, nobiletin, and tangeretin) also exhibited relatively high inhibitory activities against ACE and PL. This result confirms the potential of SW for extracting and hydrolyzing bioactive flavonoids from Citrus unshiu peel. In part 3, hesperidin was converted into hesperetin-7-O-glucoside and hesperetin with high biological activity in a semi-continuous mode. The optimum condition for maximum production of hesperetin-7-O-glucoside and hesperetin with minimal loss of hesperidin was temperature of 182.8 °C, and residence time of 3.2 min, where the predicted maximum yields of hesperetin-7-O-glucoside and hesperetin were 11,499.5 and 8,030.1 μg/g dry sample, respectively. Temperature was an important factor in SW hydrolysis of hesperidin. The effect of the residence time on hydrolysis was dependent on the temperature, where the short residence time was required at high temperature. This result confirmed the possibility of the conversion of hesperidin to valuable compounds with short time using an environmentally friendly solvent (water).
Author(s)
Kim, Dong Shin
Issued Date
2020
Awarded Date
2020. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009653
Alternative Author(s)
金東信
Affiliation
제주대학교 대학원
Department
대학원 식품공학과
Advisor
Lim, Sang Bin
Table Of Contents
Abstract 1
Introduction 6
Part. Ⅰ. Kinetic Study of Subcritical Water Extraction of Flavonoids from
Citrus unshiu peel
1. Materials and Methods
1.1. Samples and chemicals 11
1.2. Subcritical water extraction 11
1.3. Conventional organic solvent extractions 14
1.4. HPLC analysis 14
1.5. Kinetic modeling 15
1.6. Statistic analysis 16
2. Results and Discussion
2.1. Composition of flavonoids in Citrus unshiu peel 17
2.2. Effect of extraction temperature 18
2.3. Effect of water flow rate 21
2.4. Kinetic modeling 24
2.4.1. Thermodynamic partitioning model 24
2.4.2. Two-site kinetic desorption model 27
2.4.3. Adjustment quality between the experimental data and each model 29
2.5. Diffusion coefficient 31
2.6. Activation energy 34
2.7. Comparison with organic solvent extraction 37
3. Conclusion 38
Part. Ⅱ. Subcritical Water Extraction of Flavonoids from Citrus unshiu peel:
Their Biological Activity
1. Materials and Methods
1.1. Sample preparation 40
1.2. Chemicals 40
1.3. Subcritical water extraction 41
1.4. Acid and base hydrolysis 41
1.5. HPLC analysis 42
1.6. Response surface design 42
1.7. Antioxidant activity measurement 43
1.8. Biological activity measurement 44
1.9. Statistical analysis 45
2. Results and Discussion
2.1. Optimization of subcritical water extraction process 46
2.2. Effect of extraction parameters on individual flavonoid yields 52
2.3.
Comparison of flavonoid profiles between SW extract and acid and
base hydrolysis
54
2.4. Biological activities of subcritical water extracts 57
2.5.
Correlations between flavonoid yields and biological properties of
subcritical water extracts
59
2.6. Antioxidant activities of individual citrus flavonoids 61
2.7. Biological activities of individual citrus flavonoids 64
3. Conclusion 66
Part. Ⅲ. Subcritical Water Extraction and Hydrolysis of Flavonoids from
Immature Citrus unshiu pomace
1. Materials and Methods
1.1. Sample preparation 68
1.2. Chemicals 68
1.3. Subcritical water extraction and hydrolysis 68
1.4. HPLC analysis 71
1.5. Calculation of hydrolysis yield and loss of hesperidin 71
1.6. Response surface design 72
2. Results and Discussion
2.1. Composition of flavonoids in immature Citrus unshiu pomace 73
2.2. Optimization of subcritical water extraction 74
2.3. Effect of extraction parameters 78
2.4. Optimization of subcritical water hydrolysis 79
2.5. Effect of hydrolysis parameters 85
3. Conclusion 87
국문요약 88
References 90
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Kim, Dong Shin. (2020). Subcritical Water Extraction and Hydrolysis of Citrus Flavonoids
Appears in Collections:
General Graduate School > Food science and Engineering
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