속단의 항당뇨 활성 및 활성 성분 품질평가 = Antidiabetic activity and quality evaluation of active components in Dipsaci Radix

국문 초록 (Abstract)

Dipsacaceae 과의 일종인 Dipsacus Asperoides C. Y. Cheng et T. M. A. (천속단)은 "Dipsaci Radix"로 알려진 뿌리 때문에 전통적으로 중국에서 약용 식물로 사용되었다. Dipsaci Radix는 AGE 형성에 대한 하향 조절 효과와 함께 항혈당, 저지질혈증 및 항산화 특성을 나타낸다고 제안된다. 그러나 현재 당뇨병에 대한 Dipsaci Radix에서 파생된 효과적인 성분에 대한 신뢰할 수 있는 보고는 없다. 이 연구에서는 DPPH, ABTS, FRAP, BSA-glucose, BSA-MGO, α- 글루코시다아제 체외 방법으로 Dipsaci Radix의 메탄올 추출물과 에틸 아세테이트 분획을 항당뇨제 및 항산화제의 잠재적인 천연 공급원으로 조사하였다. 추출물과 분획물에 페놀 성분이 존재하면 항산화 및 항당뇨 특성이 향상된다. 이는 분리된 모든 페놀 성분이 그러한 특성을 나타내었고, 그 중 절반(화합물 2, 4, 5, 8, 10)이 다방향 억제 효과를 나타냄에 의해 입증된다. 회색 관계 분석 (GRA)과 결합된 HPLC-UV 기술을 사용하여 활성 성분의 품질을 평가하고 활성 성분과 활성 사이의 스펙트럼 효과 관계를 더욱 명확히 하였다. 그중에서 '5-O-caffeoylquinic acid'이라는 활성 성분의 존재는 Dipsaci Radix의 항당뇨 및 항산화 특성에 크게 기여하였다. 이것은 전통적인 건강 제품 및 화장품 분야에서 광범위한 응용 전망과 의의를 가지고 있다.

다국어 초록 (Multilingual Abstract)

The plant Dipsacus asperoides C. Y. Cheng et T. M. A. (川续断), a member of the Dipsacaceae family, has been traditionally utilized as a medicinal plant in China due to its roots known as “Dipsaci Radix”. It is suggested that Dipsaci Radix exhibits antihyperglycemic, hypolipidemic, and antioxidant properties, along with down-regulating effects on AGEs formation. However, there are currently no reliable reports on the effective components derived from Dipsaci Radix for anti-diabetes. In this study, the methanol extract and its corresponding EA fraction of Dipsaci Radix were investigated as potential natural sources of antidiabetic agents and antioxidants by DPPH, ABTS, FRAP, BSA-glucose, BSA-MGO, α-glucosidase in vivo methods. The existence of phenolic components in extract and fraction enhances their antioxidant and antidiabetic properties. This is evidenced by the fact that all isolated phenolic compounds exhibited such properties, with half of them (Compound 2, 4, 5, 8, 10) displaying multidirectional inhibitory effects. Quality evaluation of active components using HPLC-UV technology combined with grey relational analysis provides further information regarding the spectrum-effect relationship between active components and their activities. Among them, the presence of the active ingredient ‘5-O-caffeoylquinic acid’ significantly contributes to the antidiabetic and antioxidant properties of Dipsaci Radix. This holds promising applications in traditional health products and cosmeceuticals.

목차 (Table of Contents)

• I. Introduction 1
• II. Materials and Method 8
• II-1. Plant materials and chemical reagents 8
• II-2. Equipment and instruments 10
• II-3. Methods 11
• II-3-1. Preparation of extracts and fractions 11
• II-3-2. Isolation of compounds from EA fraction 12
• II-3-3. Compound identification 14
• II-3-3-1. NMR spectroscopy 14
• III-3-4. Assays of antioxidant and antidiabetic activities of extracts, fractions, and compounds 15
• II-3-4-1. DPPH assay 16
• II-3-4-2. ABTS assay 17
• II-3-4-3. FRAP assay 19
• II-3-4-4. Inhibition assay in BSA-glucose system. 21
• II-3-4-5. Inhibition assay in BSA-MGO system 23
• II-3-4-6. α-glucosidase inhibition assay 24
• II-3-5. Quality evaluation 26
• II-3-5-1. Selection of target compounds 26
• II-3-5-2. Preparation of standard solutions 27
• II-3-5-3. Preparation of sample solutions 28
• II-3-5-4. Development of HPLC analysis assay 29
• II-3-5-5. Validation of HPLC analysis 31
• II-3-5-5-1. Calibration curves 32
• II-3-5-5-2. LOD and LOQ 33
• II-3-5-5-3. Precision and accuracy of intra-inter day 34
• II-3-5-5-4. Repeatability 35
• II-3-5-5-5. Robustness 36
• II-3-5-5-6. Stability 37
• II-3-5-5-7. Recovery 38
• II-3-5-6. Grey relationship analysis (GRA) 39
• II-3-5-6-1. Efficacy evaluation data processing 39
• II-3-5-6-2. Reference sequence settings 40
• II-3-5-6-3. Calculation of relationship coefficient 41
• II-3-5-6-4. Difference degree calculation 42
• II-3-5-6-5. Calculation of correlation degree between component difference and activity difference 43
• II-3-5-7. Content of compounds 44
• II-3-6. Statistical analysis 45
• III. Results 46
• III-1. Structural identification of compounds 46
• III-1-1. NMR spectroscopy 47
• III-1-1-1. 4,5-O-caffeoylquinic acid methyl ester (Compound 1) 47
• III-1-1-2. 3,5-O-caffeoylquinic acid methyl ester (Compound 2) 49
• III-1-1-3. Esculetin (Compound 3) 51
• III-1-1-4. Caffeic acid methyl ester (Compound 4) 52
• III-1-1-5. Protocatechuic acid (Compound 5) 53
• III-1-1-6. 5-O-caffeoylquinic acid methyl ester (Compound 6) 54
• III-1-1-7. Isoferulic acid (Compound 7) 55
• III-1-1-8. 5-O-caffeoylquinic acid (Compound 8) 56
• III-1-1-9. Caffeic acid (Compound 9) 57
• III-1-1-10. 1-O-caffeoyl-β-d-glucopyranose (Compound 10) 58
• III-2. Assessment of antioxidant and antidiabetic activities of extracts, fractions, and compounds 59
• III-2-1. Assessment of antioxidant activities of extracts 59
• III-2-2. Assessment of antioxidant activities of fractions 61
• III-2-3. Assessment of antidiabetic activities of extracts 63
• III-2-4. Assessment of antidiabetic activities of fractions 65
• III-2-5. Assessment of antioxidant activities of compounds 67
• III-2-6. Assessment of antidiabetic activities of compounds 69
• III-2-7. Discussion of antioxidant and antidiabetic activities 71
• III-3. Quality evaluation 73
• III-3-1. Analysis and selection of target compounds 73
• III-3-2. Validation of HPLC analysis 74
• III-3-2-1. Purity 74
• III-3-2-2. Specificity 75
• III-3-2-3. Linearity, LOD, and LOQ 76
• III-3-2-4. Intra-day/Inter-day precision and accuracy 77
• III-3-2-5. Repeatability 79
• III-3-2-6. Robustness 80
• III-3-2-7. Stability 83
• II-3-2-8. Recovery 85
• III-3-3. GRA 87
• III-3-3-1. Nondimensionalization in the compounds' group 88
• III-3-3-2. Absolute value of the difference sequence in the compounds' group 90
• III-3-3-3. Correlation degree and difference degree in the compounds' group 91
• III-3-3-4. Nondimensionalization in the efficacies' group 93
• III-3-3-5. Absolute value of the difference sequence in the efficacies' group 95
• III-3-3-6. Correlation degree and difference degree in the efficacies' group 96
• III-3-3-7. Grey comprehensive relationship degree 98
• III-3-4. Content of compounds 99
• III-3-4-1. Optimization of sample preparation 99
• III-3-4-2. Content 101
• III-3-5. Discussion of quality evaluation 104
• IV. Conclusion 106
• References 109
• Appendix 123
• 국문초록 143