Optimization of polysaccharide extraction method of Citri Unshiu Pericarpium Immaturus by design of experiment and response surface methodology
- 주제(키워드) Citri Unshiu Pericarpium Immaturus , polysaccharide , antiviral activity , response surface methodology , box-behnken design
- 주제(DDC) 615.1
- 발행기관 아주대학교 일반대학원
- 지도교수 Seung-Hoon Baek
- 발행년도 2025
- 학위수여년월 2025. 2
- 학위명 석사
- 학과 및 전공 일반대학원 약학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034464
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This study aimed to establish the efficient extraction method of polysaccharides in Citri Unshiu Pericarpium Immaturus (CPs) using design of experiment (DOE) and response surface methodology (RSM). Ultrasonic extraction method was optimized for the factors of extraction time (30−90 min), temperature (50−70°C), NaOH concentration (1−3 M), and solvent to material ratio (10−30). Total 27 CPs were prepared from various extraction conditions generated by Box-Behnken Design. Their properties as responses were evaluated in terms of yield, antioxidant activity, total carbohydrate, protein, uronic acid, and phenolic compound. Statistical analysis showed that significant model fitting was found in between extraction factors and yields. Optimum extraction conditions and predicted yield were determined using response surface methodology with mathematical modeling. Real yield of CPs extracted in optimum condition were in accordance with predicted one. Chemical analysis data showed that optimum CPs were mainly composed of carbohydtate and uronic acid with high molecular weight over 100 kDa. Monosaccharide composition data showed that CPs consisted with arabinose (13.0%), galactose (7.1%), and glucose (5.5%). CPs inhibited EV71 infection on Vero cells (IC50, 90.6 ± 33.2 μg/mL). In conclusion, optimum ultrasonic extraction conditions for the maximum yields were found using DOE and RSM. Chemical and pharmacological properties of CPs prepared in this condition demonstrated that polysaccharides as major constituent may be responsible for anti-virus activity in vitro. The approach in this study can be applied to the development of efficient extraction methods for useful substances from natural products. Keywords: Citri Unshiu Immaturus peel; polysaccharide; antiviral activity; response surface methodology; Box-Benhken Design
more목차
1. Introduction 1
1.1. Bioactive natural polysaccharide 1
1.2. Polysaccharides of Citri Unshiu Pericarpium Immaturus 2
1.3. Polysaccharide extraction with ultrasound and alkaline solvent 3
1.4. Response surface methodology and design of experiment 4
1.5. Aim of this study 5
2. Material and methods 6
2.1. Plant materials and chemical 6
2.2. Optimization of extraction method using DOE and RSM 6
2.2.1. Design of experiments using Box-Behnken Design 6
2.2.2. Preparation of CPs from diverse extraction conditions 9
2.2.3. Analysis of main and interaction effects 9
2.2.4. Statistical analysis and model fitting 10
2.3. Evaluation of chemical and antioxidant properties of CPs 11
2.3.1. Chemical analysis 11
2.3.2. Superoxide anion radical scavenging assay 11
2.4. Chemical characterization of optimized CPs 12
2.4.1. Preparation of optimized CPs 12
2.4.2. Determination of molecular weight distribution using HP-GFC 12
2.4.3. Monosaccharide composition analysis 13
2.5. Anti-enterovirus 71 activity assay 14
2.5.1. Cell culture 14
2.5.2. Cytotoxicity and anti-virus assay using SRB staining 14
3. Results and discussion 16
3.1. Optimization of extraction method of CP 16
3.1.1. Extraction experiments and responses of CPs 16
3.1.2. Model selection using analysis of variance 19
3.2. Effect of independent variables on yield of CPs 27
3.2.1. Effect of extraction time on yield 27
3.2.2. Effect of extraction temperature on yield 27
3.2.3. Effect of NaOH concentration on yield 27
3.2.4. Effect of solvent to material ratio on yield 28
3.2.5. Interaction of independent variables on yield 28
3.3. Model fitting and validation using RSM 30
3.3.1. Multiple regression analysis for model fitting 30
3.3.2. Optimum extraction conditions and model validation 34
3.4. Chemical characterization of optimized CPs 36
3.4.1. Chemical analysis of optimized CPs 36
3.4.2. Molecular weight distribution of optimized CPs 38
3.4.3. Determination of monosaccharide composition 42
3.5. Antiviral activity of CPs against EV-71 46
4. Conclusion 48
Acknowledgements 49
Reference 50
국문초록 55
