Yuzu Bioactive Components: Optimization of Sample Pre-treatment, Extraction, and Antiplatelet Activity = 유자 중 플라보노이드의 반응표면분석을 통한 추출법 검증 및 생리 활성

국문 초록 (Abstract)

건강 기능성의 이해도 증진과 함께 유자의 생산소비량이 급증하고 있다. 일례로 최근 유자 재배 면적 및 생산량은 2,237 ha / 19,127 ton으로 집계된 바 있다. 한국, 일본 및 중국 등지에서 재배되고 있는 유자는 특유의 향미, 적절한 산도 등으로 다양한 용도의 식용 자원으로 사용되고 있다. 한편 다른 감귤류와 비교하여, 유자는 과육뿐만 아니라 과피 역시 다양한 용도로 사용되고 있다. 그러나 이와 같은 소비성향의 변화에도 불구하고 유자의 활성 성분 조성, 효용성 있는 추출/정제법 등은 세부 연구가 미진하다.
본 연구에서는 우선적으로 유자 및 동속 식물의 과실 추출물의 화학적 분석을 실시하여 지방산, 토코페롤류, 및 기타 대사물과 항산화능간의 상관관계를 연구, 제시하였다. 대사물 분석결과 유자 과피 및 과육에는 naringin이 주요한 플라보노이드로 밝혀졌으며, 동속 식물의 조직에서는 해당 대사물이 부재하거나 미량이 검출되어 유자의 항산화 효과 및 관련 생리활성에 naringin이 큰 기여를 함을 알 수 있었다.
본 연구에서는 상업적 이용 가능성이 높은 유자 과피, 과실 및 종실 중 플라보노이드, 페놀류 등의 기능성 성분의 저장, 가공 및 추출법에 따른 변이를 연구하였다. 그 결과 다양한 유기용매 (아세톤, 메타놀, 에타놀 등)와 물 혼합물이 높은 기능성 성분 추출효율을 나타내었다. 열풍건조 및 동결건조 등, 건조 방법에 따른 연구 결과, 동결 건조 유자에서 가장 높은 항산화 성분의 유지가 가능하였다. 이상의 결과, 유자 가공과 관련, 동결건조시료의 유기용매/물 혼합물 추출이 가장 적절한 처리법으로 밝혀졌다.
유자 중, 주요 활성 성분으로서 플라보노이드의 효과적 추출법 개발을 위하여 반응표면분석기법 (response surface methodology, RSM)을 활용하여 최적 추출법을 확립하고자 하였다. 이를 위하여 총 29회, 5종의 변수조건하에서 최적화를 수행하였다. 표면 분석에 사용한 인자로는 에타놀 (EtOH), 용매/시료 총량비(L/S), 추출온도 (T), 및 처리시간 (t)를 설정하였다. 그리고 해당 조건하에서 총페놀함량(TPC), 총플라보노이드함량(TFC), 2종의 방법에 따른 항산화능(FRAP 및 DPPH), 3종의 생리활성 플라보노이드의 추출 효율을 검토하였다. 표면분석의 통계적 처리에 따라 산출된 다수의 예측모델의 실험값과 적절히 일치하여 해당 모델식의 효과를 입증하였다. 한편 이와 같은 분석 결과, 최적의 추출 조건은 EtOH = 65.5%, T = 43.9oC, t = 119.7 min, 및 L/S = 37.2 ml/g로 나타났으며, 해당 조건에 따른 주요 활성 성분의 회수율은 95%이었다. 본 연구에서는 상기와 같은 이론적 최적화 방법을 통하여 다수 활성 성분의 효과적 동시 추출체계를 확립할 수 있었다.
한편 유자와 관련 감귤류의 대사체를 비교 분석한 결과, 다양한 차이점이 발견되었으며, 심혈관계 질환 개선효과, 비만 억제 등의 효과가 보고된 naringin의 경우, 유자피에서 다량 발견된 반면, 다른 감귤류 과피에서는 소량 존재하거나 발견되지 않았다. 또한 높은 항산화성을 지닌 ascorbic acid의 경우에도 유자과피에서 다량 존재함이 확인된 반면, 기타 감귤류에는 과육에서만 존재하는 것으로 나타났다.
상기의 최적화된 방법에 따라 분리된 조추출물의 심혈관계 질환 개선효과를 검토하였다. 메타놀, 헥산, 에틸아세테이트 등의 상이한 용매 추출물의 항혈전형성능을 비교한 결과, 에틸아세테이트 분획의 혈전형성 저해효과가 가장 탁월하였다. 한편 동일 추출법을 기타 감귤 재배 품종에 적용하여 오렌지 및 한라봉 등을 대상으로 동일 실험을 수행하여, 유자와 비교한 바, 유자 조추출물의 효과를 확인할 수 있었다.
한편 최근 식품을 포함한 다양한 산업분야에 활용도가 기대되고 있는 대기압-플라즈마 제트 (direct atmospheric pressure plasma jet, APPJ) 처리의 생리활성성분 함량에 대한 영향을 연구하였다. APPJ 처리는 유자의 대표적 기능성 성분으로서 총 페놀 함량 및 항산화 활성에 영향이 없는 것으로 나타났다. 특기할 점으로 APPJ 처리 시료의 경우, hesperidin, naringin, 및 quercetin의 경우, 해당 처리에 의하여 그 함량이 감소함을 알 수 있었다 . 표준물질 용액의 경우 해당 성분의 편균적 감소량은 hesperidin, naringin, 및 quercetin에 대하여 각 50.0, 36.4, and 32.0%로 나타났다.

다국어 초록 (Multilingual Abstract)

Due to growing of interest in health and well-being, demands for plants with medicinal importance have been increasing time to time. The production of yuzu fruit has increased greatly, with a cultivation area of 2,237 ha and annual production of 19,127 metric tons in 2005. Its market price has soared since. Yuzu, which is mainly cultivated in Korea, Japan, and China, is also becoming popular in some other parts of the world. Its distinct flavor and refreshing aroma which is less tart than other citrus counterparts along the wide applicability in different dishes and beverages boosts its popularity. Moreover, unlike the other citrus fruits, the peel of yuzu fruit is also widely used to flavor and color dishes, making tea and marmalade. Phytochemicals isolated from different citrus fruits have proven a significant effect for prevention of cardiovascular related diseases. Hesperedin and naringin, which are the main components of yuzu, along with their derivatives has shown beneficial effects in the treatment of heart failure and cardiac remodeling, hypertension, myocardial ischemia, and infarction. Despite the growing interest of yuzu fruit by consumers as well as its medicinal property, it is not a well-researched like its citrus counterparts.
The main objectives of this thesis was to investigate the conditions that affect the recovery of biologically active phytochemicals from yuzu samples, develop an optimum extraction conditions by using a statistical model, evaluate the phytochemical compositions, and asses the biological activities of yuzu fruit. Along with, we have evaluated the phytochemical compositions and biological activity of other citrus fruits consumed in Korea.
In this study, the effects of various drying methods and extraction solvents were evaluated on the content of phenolics, flavonoids and antioxidant activities of peel, pulp, and seeds of yuzu fruit. Organic solvents (acetone, methanol, and ethanol) mixed with water exhibited better results relative to their corresponding absolute solvents. Drying methods also significantly affected the polyphenol content and antioxidant potential of yuzu fruits. Freeze dried samples showed the highest percent retentions of polyphenols and antioxidants. In overall, the extraction of secondary metabolites and antioxidants was highly dependent on the extraction solvent, type of sample and sample processing methods.
Extraction of antioxidants and flavonoids from the peels of yuzu fruit was studied using a single factor experiment and a response surface methodology (RSM) based on central composite design. Four independent variables were evaluated at five levels with total 29 experimental runs, including ethanol concentration (EtOH), ratio of liquid to material (L/S), extraction temperature (T), and extraction time (t). The responses considered were total phenolic content (TPC), total flavonoids content (TFC), two indicators of antioxidant capacity (FRAP and DPPH), and three individual major flavonoids in yuzu (hesperidin, naringin, and phloretin). Quadratic polynomial equations were obtained by multiple regression analysis to predict the optimal extraction conditions. The regression analysis showed that > 95 % of variations were explained by the models of different responses considered. The responses were significantly influenced by all studied factors. The Multiresponse optimized conditions targeted at maximizing all the responses were found to be EtOH = 65.550 %; T = 43.864 °C; t = 119.673 min; and L/S = 37.168 ml/g, with a desirability of 0.950. The experimental values at the optimized conditions were in a reasonable agreement with the predicted values. The extraction method was applied successfully to extract antioxidants and flavonoids from yuzu peels. It also allows a fast and cost-saving process for extraction of the studied phytochemicals, in addition to improvement of the quantity of the targeted extract.
Comparative metabolite profiling (total phenolics, total flavonoids, fatty acids, tocopherols, hesperedin, naringin, ascorbic acid) and antioxidant potential was performed with peels and pulp of yuzu and other citrus fruits, which are commonly consumed in Korea. Naringin, which is known to exhibit several pharmacological effects, including, inhibition of vascular endothelial growth factor, reduction of diabetes-induced neuropathy, and protection against cognitive dysfunction and oxidative damage, was found in appreciable amount in both pulp and peel of yuzu but not in all other citrus fruits. Yuzu peel was found to be a useful source of ascorbic acid than the pulp. Unlike yuzu, it is the pulp of other fruits which exhibited higher ascorbic acid.
Antiplatelet activity of crude extracts of aqueous methanol fractionated in to hexane, ethyl acetate, and methanol extracts of yuzu and other two citrus varieties (orange and hallabong) was studied. In addition, selected metabolites and antioxidant potential of the fractions were evaluated. The results of the antiplatelet activity test showed that ethyl acetate fraction has the highest inhibition relative to other solvent fractions. Ethyl acetate fraction also contained the highest total flavonoid and total phenolic content and exhibited the highest antioxidant activities.
Finally, we have studied the effect of plasma gas treated water and direct application of atmospheric plasma jet on extraction of secondary metabolites and antioxidants from yuzu fruits. The total flavonoid content of yuzu extracts of plasma treated water was higher than the untreated water. However, plasma treated and untreated water sample didn’t show a significant difference on the concentration of hesperidin, naringin, total phenolic content, and antioxidant potential of yuzu extracts. Direct application of atmospheric pressure plasma jet (APPJ) to the extract solution caused degradation of metabolites and antioxidants in water suspensions of powdered yuzu. The APPJ application to standards of hesperidin, naringin and quercetin also resulted in degradation of the standard to the extent of 49.98, 36.42, and 32.03% respectively.

목차 (Table of Contents)

• Chapter 1: Introduction and review of literature 1
• 1.1. Origin, history, cultivation, and distribution of yuzu 1
• 1.2. Uses, nutritional content, and health benefits of yuzu 2
• 1.3. Chemical composition of citrus fruit 4
• 1.3.1. Phenolic compounds 5
• 1.3.2. Limonoids 8
• 1.3.3. Fatty acids 8
• 1.3.4. Carotenoids and tocols 9
• 1.4. Antioxidant and antiplatelet activities of citrus phytochemicals 11
• 1.4.1. Antioxidant activity 11
• 1.4.2. Antiplatelet activity 16
• 1.5. Sample preservation methods 18
• 1.6. Extraction 20
• 1.6.1. Conventional extraction 20
• 1.6.2. Advanced extraction techniques 22
• 1.7. Aims and objectives of the project 24
• Chapter 2: Effect of various drying methods and solvent on polyphenol content and antioxidant activity of yuzu 26
• 2.1. Abstract 26
• 2.2. Introduction 27
• 2.3. Materials and methods 29
• 2.3.1. Chemicals and reagents 29
• 2.3.2. Plant material and drying process 30
• 2.3.3. Extraction of phenolics and antioxidants 31
• 2.3.4. Determination of total phenolic content (TPC) 32
• 2.3.5. Determination of total flavonoid content (TFC) 32
• 2.3.6. Ferric reducing antioxidant power (FRAP) assay 33
• 2.3.7. DPPH free radical-scavenging assay 33
• 2.3.8. Cupric reducing antioxidant capacity (CUPRAC) 34
• 2.3.9. Weight, size, soluble solids and moisture content 34
• 2.3.10. Calculation of percentage true retention 35
• 2.3.11. Statistical analysis 36
• 2.4. Results and discussion 36
• 2.4.1. Selected physical properties of different parts of yuzu 36
• 2.4.2. Effect of drying methods on the TPC and TFC of peel, pulp, and
• seeds of yuzu 37
• 2.4.3. Effect of drying methods on the antioxidant activity (AOA) of peel,
• pulp, and seeds of yuzu 41
• 2.4.4. Effect of extraction solvent on the antioxidant activity (AOA) of peel,
• pulp, and seeds of yuzu 44
• 2.4.5. Effect of extraction solvent on the TPC and TFC of peel, pulp, and
• seeds of yuzu 49
• 2.5. Conclusion 52
• Chapter 3: Optimization of extraction conditions of antioxidants and secondary metabolites from yuzu peels using response surface methodology 53
• 3.1. Abstract 53
• 3.2. Introduction 54
• 3.3. Materials and methods 56
• 3.3.1. Chemicals and reagents 56
• 3.3.2. Extraction and sample preparation 56
• 3.3.3. Experimental design 57
• 3.3.4. Determination of TPC and TFC 59
• 3.3.5. Antioxidant activity (FRAP and DPPH assay) 59
• 3.3.6. Determination of hesperidin, naringin, and phloretin 59
• 3.3.7. Statistical analysis 60
• 3.4. Results and discussion 60
• 3.4.1. Effect of independent variables on extraction of flavonoids and
• antioxidant potential 60
• 3.4.1.1. Effect of ethanol concentration on FRAP and TFC 61
• 3.4.1.2. Effect of extraction temperature on FRAP and TFC 62
• 3.4.1.3. Effect of extraction time on FRAP and TFC 63
• 3.4.1.4. Effect of ratio of liquid to material on FRAP and TFC 64
• 3.4.2. The model fitting and statistical analysis 66
• 3.4.2.1. Antioxidant activities 68
• 3.4.2.2. Total polyphenolic content (TPC) and Total flavonoid content
• (TPC) 70
• 3.4.2.3. Hesperidin, naringin, and phloretin 71
• 3.4.3. Influence of experimental conditions 75
• 3.4.3.1. Antioxidant activities 76
• 3.4.3.2. Total polyphenolic content (TPC) and Total flavonoid content
• (TPC) 77
• 3.4.3.3. Hesperidin, naringin, and phloretin 79
• 3.4.4. Optimization of extraction conditions and validation of the models
• 81
• 3.5. Conclusion 85
• Chapter 4: Comparative study of phytochemical compositions of yuzu and other citrus fruits: Fatty acids, tocopherols, antioxidant activities, and other selected metabolites 87
• 4.1. Abstract 87
• 4.2. Introduction 88
• 4.3. Materials and methods 92
• 4.3.1. Plant material 93
• 4.3.2. Chemicals and reagents 93
• 4.3.3. Fatty acid extraction, fatty acid methyl ester (FAME) preparation and
• analysis 93
• 4.3.4. Extraction and quantification of tocopherols 95
• 4.3.5. Extraction of phenolics and antioxidants 96
• 4.3.6. Ferric reducing antioxidant power (FRAP) 97
• 4.3.7. DPPH radical scavenging activity 97
• 4.3.8. Total Phenolic contents 97
• 4.3.9. Total Flavonoids 97
• 4.3.10. Determination of hesperidin and naringin 97
• 4.3.11. Ascorbic acid extraction and determination 97
• 4.3.12. Statistical analysis 98
• 4.4. Results and discussion 98
• 4.4.1. Fatty acid methyl esters 99
• 4.4.2. Total flavonoid and total phenolic contents 105
• 4.4.3. Antioxidant activities 107
• 4.4.4. Concentrations of ascorbic acid 109
• 4.4.5. Concentrations of hesperidin and naringin 110
• 4.5. Conclusion 112
• Chapter 5: Antiplatelet and antioxidant activity of different solvent fractions of yuzu in comparison with other fruits 113
• 5.1. Abstract 113
• 5.2. Introduction 114
• 5.3. Materials and methods 117
• 5.3.1. Plant material and sample preparation 117
• 5.3.2. Chemicals and reagents 117
• 5.3.3. Extraction procedure and preparation of dried Fractions 118
• 5.3.4. Determination of total phenolic and flavonoid contents 118
• 5.3.5. Ferric reducing power determination 119
• 5.3.6. Copper ion reducing power determination 119
• 5.3.7. DPPH radical scavenging activity 119
• 5.3.8. Preparation of platelets 119
• 5.3.9. Determination of platelet aggregation 120
• 5.3.10. Statistical analysis 120
• 5.4. Results and discussion 121
• 5.4.1. Total phenolic and flavonoid content 121
• 5.4.2. Antioxidant activity 123
• 5.4.3. Antiplatelet activity 126
• 5.4.4. Correlations 129
• 5.5. Conclusion 131
• Chapter 6: Effect of application of atmospheric pressure plasma jet (APPJ) on extraction of secondary metabolites and antioxidants from yuzu 132
• 6.1. Abstract 132
• 6.2. Introduction 133
• 6.3. Materials and methods 134
• 6.3.1. Chemicals and reagents 134
• 6.3.2. Extraction using plasma treated water and untreated water 134
• 6.3.3. Extraction by application of direct atmospheric pressure plasma jet
• (APPJ) 135
• 6.3.4. Determination of TPC 137
• 6.3.5. Determination of TFC 137
• 6.3.6. Ferric reducing antioxidant power (FRAP) assay 137
• 6.3.7. HPLC-DAD analysis of hesperidin, naringin, and quercetin 137
• 6.3.8. Statistical analysis 138
• 6.4. Results and discussion 138
• 6.4.1. Effect of plasma treated water on extraction of secondary metabolites
• and antioxidant activity 138
• 6.4.2. Effect of APPJ treatment time on extraction of secondary metabolites
• and antioxidant activity 140
• 6.5. Conclusion 143
• Chapter 7: Summary, conclusions, and future work 144
• 7.1. Summary and conclusions 144
• 7.2. Suggestion for future work 149
• References 151
• Appendix 181
• Abstract (in Korean) 199