Isolation and quantitation of neuroprotective compounds in Dendropanax morbifera leaves inhibiting glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells
- 주제(키워드) Dendropanax morbifera , glutamate oxidative toxicity , HT22 , neuroprotection , AIF
- 발행기관 아주대학교
- 지도교수 백승훈
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 박사
- 학과 및 전공 일반대학원 약학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029881
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Dendropanax morbifera (DM) has long been used as a traditional herbal medicine for migraines. Glutamate toxicity and oxidative stress have emerged as the possible triggers implicated in migraine pathogenesis. We aimed to examine the neuroprotective effects of DM leaves (DML) and its principal constituents on glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells. Molecular authentication of DML was assessed using DNA barcoding analysis. Four different solvent extracts of DML were prepared and subjected to antioxidant activity and phytochemical assays. Neuroprotective effects of DML extracts and its principal compounds were evaluated using relevant biochemical and imaging assays that measure cell viability/death, ROS generation, Ca2+ levels, mitochondrial dysfunction, and AIF nuclear translocation. The sequences of matK, rbcL, atpF-H, and psbK-I in DML were identical with those in voucher specimens, confirming that DML was indeed D. morbifera. The ethyl acetate extract of DML (DMLE) showed the highest flavonoid and phenolic content, and prominent DPPH/superoxide radical scavenging and reducing power activities. Five compounds were isolated from DMLE and characterized as quercetin, isoquercitrin, hyperoside, rutin, and chlorogenic acid. The content of these compounds was quantitated in DMLE and DMLB using validated HPLC-UV method which is validated following the ICH Q2(R1) guideline in terms of specificity, linearity, LOD, LOQ, accuracy and precision. In the HT22 cell model, glutamate was shown to be the causative agent for apoptotic cell death via elevation of intracellular ROS and Ca2+ levels, induction of mitochondrial depolarization and membrane permeabilization, and translocation of AIF to the nucleus. Of note, N-acetyl-L-cysteine and necrostatin-1, but not z-VAD-fmk, completely prevented glutamate-induced cell death, implying that oxidative stress and AIF translocation were pivotal in glutamate cytotoxicity. DMLE, quercetin and isoquercitrin significantly recovered glutamate-induced apoptotic cell death in a concentration-dependent manner. They completely inhibited intracellular/mitochondrial ROS generation, the elevation of Ca2+ levels, and mitochondrial dysfunction induced by glutamate during early exposure within 8 h. It significantly reversed subsequent AIF nuclear translocation after 12 h of treatment. Antioxidant activities of DMLE may be the protective mechanism that regulates homeostatic balance of ROS and Ca2+ as well as maintains mitochondrial function. Quercetin and isoquercitrin were confirmed as principals in DMLE, and isobologram study showed mild synergism between them. DMLE, quercetin and isoquercitrin show significant neuroprotective effects against glutamate-induced oxidative neuronal cell death. Therefore, DM could be a potential therapeutic candidate for neurological disorders propagated by glutamate toxicity. Keywords Dendropanax morbifera; glutamate oxidative toxicity; HT22; neuroprotection; AIF
more목차
1. Introduction 1
1.1. Natural products: On-going sources of new drugs 1
1.2. Dendropanax mobifera 3
1.3. Migraine 6
1.4. Glutamatergic mechanism: as therapeutic targets for migraine 7
1.5. Glutamate toxicity 10
1.6. Specific aims of this study 13
2. Materials and methods 14
2.1. Plant material and reagents 14
2.2. Molecular authentication of the leaves of Dendropanax morbifera using DNA barcoding analysis 15
2.2.1. Plant materials and voucher specimens 15
2.2.2. DNA extraction, polymerase chain reaction, and sequencing 17
2.2.3. Data analysis 20
2.3. Extraction and isolation of compounds 21
2.4. Structure determination of isolated compounds 21
2.5. Development of HPLC analytical method for quantitation of isolated compounds 22
2.5.1. Preparation of standard solutions and samples 22
2.5.2. HPLC analytical conditions 23
2.5.3. Method validation 26
2.6. Total flavonoid and phenolic content analysis 26
2.7. Antioxidant activity assays 27
2.7.1. DPPH radical scavenging assay 27
2.7.2. Superoxide radical scavenging assay 28
2.7.3. Reducing power assay 28
2.8. Cell culture and treatments 29
2.9. Cell viability assay 30
2.10. Cell death assay 30
2.11. PI/Annexin V apoptosis analysis 30
2.12. Measurement of intracellular ROS generation 31
2.13. Measurement of mitochondrial ROS generation 31
2.14. Measurement of intracellular calcium levels 32
2.15. Measurement of mitochondrial membrane potential 32
2.16. Measurement of mitochondrial membrane permeabilization 33
2.17. Immunofluorescence for AIF translocation 33
2.18. Subcellular fractionation 34
2.19. Western blotting 34
2.20. Analysis of synergistic effect with isobologram 35
2.21. Statistical analysis 35
3. Results and Discussion 36
3.1. Molecular authentication of DML extracts 36
3.2. Extraction and chemical analysis of DML extracts 38
3.3. Antioxidant activities of DML extracts 40
3.4. Glutamate induces oxidative cell death in HT22 cells via intracellular ROS generation, Ca2+ increase, mitochondrial dysfunction, and AIF-dependent apoptosis 42
3.4.1. Glutamate induces HT22 cell death 42
3.4.2. Glutamate induces intracellular ROS generation, Ca2+ increase and mitochondrial dysfunction in HT22 cells 45
3.4.3. Glutamate induces apoptosis in HT22 cells 48
3.4.4. Glutamate induces AIF-dependent apoptosis 50
3.5. DMLE inhibits glutamate-induced apoptotic cell death in HT22 cells 53
3.6. DMLE alleviates glutamate-induced intracellular/mitochondrial ROS generation and Ca2+ dysregulation 56
3.7. DMLE attenuates glutamate-induced mitochondrial dysfunction 60
3.8. DMLE inhibits glutamate-induced AIF translocation to the nucleus 64
3.9. Isolation of active compounds from DMLE 68
3.10. Characterization of isolated compounds from DMLE 71
3.10.1. Compound 1 71
3.10.2. Compound 2 75
3.10.3. Compound 3 79
3.10.4. Compound 4 83
3.10.5. Compound 5 87
3.11. Quantitation of isolated compounds in DMLE 92
3.11.1. Method validation 92
3.11.2. Quantitative analysis of the isolated compounds 95
3.12. Antioxidant activities of isolated compounds 98
3.13. I and Q inhibit glutamate-induced apoptotic cell death in HT22 cells 100
3.14. I and Q attenuate intracellular ROS and calcium levels 102
3.15. I and Q prevent glutamate-induced mitochondrial dysfunction. 105
3.16. I and Q inhibit AIF translocation to the nucleus 109
3.17. Combined effects of I and Q on glutamate-induced HT-22 cell death 112
4. Conclusion 115
Reference 117
국문초록 128
