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Isolation and quantitation of neuroprotective compounds in Dendropanax morbifera leaves inhibiting glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells
- Alternative Title
- Hye-Jin Park
- Author(s)
- 박혜진
- Alternative Author(s)
- Hye-Jin Park
- Advisor
- 백승훈
- Department
- 일반대학원 약학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2020-02
- Language
- eng
- Alternative Abstract
- 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
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- Appears in Collections:
- Graduate School of Ajou University > Department of Pharmacy > 4. Theses(Ph.D)
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