커피추출물의 항당뇨 및 콜린에스터라제 억제 효과와 기능성 성분의 분리 및 구조규명 = Anti-Diabetic and Cholinesterase Inhibitory Effects of Coffee Extracts and Isolation of Functional Compounds

다국어 초록 (Multilingual Abstract)

Diabetes mellitus (DM) and Alzheimer’s disease (AD) are representative chronic metabolic and neurodegenerative disorders that share common pathological mechanisms, including oxidative stress, inflammation, and abnormal enzyme activity. Coffee (Coffea arabica) is a well-known natural source rich in bioactive phytochemicals, including chlorogenic acid derivatives, methylxanthines, and diterpenoids. These compounds have been reported to exhibit antioxidant, anti-diabetic, and neuroprotective properties. In this study, the antioxidant, anti-diabetic, and anti-Alzheimer’s activities of extracts, solvent fractions, and isolated compounds from Brazil Santos and Ethiopia Yirgacheffe coffee were evaluated in vitro. The H₂O decoction extracts were sequentially fractionated with CH₂Cl₂, EtOAc, n-BuOH, and H₂O, and the EtOAc and n-BuOH fractions were further purified to yield six compounds: Atractyligenin-2-O-β-D-glucoside (1), Trigonelline (2), Caffeine (3), Theobromine (4), 3-O-feruloylquinic acid (5), and 5-O-caffeoylquinic acid (6). Their structures were elucidated by ¹H and ¹³C NMR spectroscopy and confirmed by comparison with literature data. Quantitative profiling of chlorogenic acids and alkaloids was performed using UPLC-QToF/ESI-MS. Ethiopia Yirgacheffe samples exhibited generally higher contents of phenolic acids in decoction and EtOAc fractions, whereas alkaloids were predominantly enriched in decoction and CH₂Cl₂ fractions of both varieties, indicating differences in extraction efficiency according to solvent polarity. The distribution patterns revealed by UPLC were largely consistent with the biological activities of each extract and fraction. Antioxidant activity was determined by peroxynitrite (ONOO⁻) scavenging assay, anti-diabetic activity was assessed by α-glucosidase and AGEs formation inhibition assays, and anti-Alzheimer’s potential was evaluated by AChE and BChE inhibition assays. The EtOAc and n-BuOH fractions of Ethiopia Yirgacheffe showed the strongest antioxidant and α-glucosidase inhibitory effects, compared to L-penicillamine and acarbose used to a positive control, respectively. In the cholinesterase assay, the CH₂Cl₂ and EtOAc fractions of Brazil Santos exhibited the highest inhibitory effects, but showed lower activity compared to berberine used as a positive control. Among the isolated compounds, 5-O-caffeoylquinic acid (6) exhibited the most potent ONOO⁻ scavenging activity with an IC50 value of 1.86 ± 0.12 μM and inhibited AGEs formation by 79.34%, comparable to the positive controls L-penicillamine and aminoguanidine, respectively. Atractyligenin-2-O-β-D-glucoside (1) demonstrated the strongest α-glucosidase inhibitory activity (35.1%), while two alkaloids, caffeine (3) and theobromine (4) showed moderate inhibition against both AChE and BChE. Overall, the bioactivities of coffee extracts were found to be markedly influenced by solvent polarity and structural characteristics of the compounds. In particular, chlorogenic acid derivatives, atractyligenin glycosides, and methylxanthine alkaloids exhibited complementary roles across antioxidant, anti-diabetic, and neuroprotective mechanisms. Among them, 5-O-caffeoylquinic acid (6) consistently exhibited multifunctional activity, identifying it as a key bioactive constituent in coffee and supporting the potential of coffee-derived compounds as natural functional ingredients for the prevention of metabolic and neurodegenerative diseases.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• 1. Coffee (Coffea arabica L.) 1
• 2. Diabetes and diabetic complications 5
• 3. Alzheimer's disease 8
• 3. Aims of study 10
• Ⅱ. MATERIALS AND METHODS 11
• 1. General experimental procedures 11
• 2. Chemicals and reagents 12
• 3. Plant material 13
• 4. Extraction, fractionation and isolation 14
• 5. UPLC-QToF/ESI-MS analysis 22
• 6. Phytochemical contents 23
• 6-1. Determination of total phenolic contents (TPC) 23
• 6-2. Determination of total flavonoids contents (TFC) 24
• 7. Antioxidant activity 25
• 7-1. In vitro assay for DPPH radical scavenging activity 25
• 7-2. In vitro assay for ABTS radical scavenging activity 26
• 7-3. In vitro assay for peroxynitrite scavenging activity 27
• 8. In vitro assay for α-glucosidase inhibitory activity 29
• 9. In vitro assay for AGEs formation inhibitory activity 31
• 10. In vitro assay for Cholinesterase inhibitory activity 32
• 11. Statistics 34
• Ⅲ. RESULTS 35
• 1. Isolation and identification of six functional compounds and UPLC analysis of major compounds from the fractions of Brazil Santos and Ethiopia Yirgacheffe 35
• 2. TPC and TFC of the three extracts and their organic solvent fractions from Brazil Santos and Ethiopia Yirgacheffe 52
• 3. In vitro assay for DPPH, ABTS radical scavenging activity 54
• 4. In vitro assay for ONOO- scavenging activity 56
• 5. In vitro assay for α-glucosidase inhibitory activity 58
• 6. In vitro assay for AGEs formation inhibitory activity 60
• 7. In vitro assay for Cholinesterase inhibitory activity 62
• Ⅳ. DISCUSSION 67
• Ⅴ. CONCLUSION 74
• REFERENCES 75
• 국문초록 93