This study aimed to establish the quantitative method to analyze the content of peroxynitrite-scavengers belonging to polyphenols in six Korean Quercus species (Quercus mongolica, Q. dentata, Q. acutissima, Q. alienta, Q. serrata, and Q. variabilis) by HPLC. The twelve peroxynitrite-scavengers, flav...
This study aimed to establish the quantitative method to analyze the content of peroxynitrite-scavengers belonging to polyphenols in six Korean Quercus species (Quercus mongolica, Q. dentata, Q. acutissima, Q. alienta, Q. serrata, and Q. variabilis) by HPLC. The twelve peroxynitrite-scavengers, flavanols (catechins: (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin), flavonols (kaempferol and quercetin), flavonol glycosides (astragalin, quercitrin, and isoquercitrin), flavonol acylated glycosides (astragalin 6''-gallate and isoquercitrin 6''-gallate), gallic acid and its dimer (ellagic acid) were analyzed by HPLC. Further, anti-Alzheimer's activity was assayed in a passive avoidance testusing mice by measuring the retention latency (sec), the concentration of acetylcholine (ACh), and acetylcholinesterase (AChE) activity. Simultaneous analysis of the extracts of the six Quercus leaves was achieved on a Capcell C18 column ($5{\mu}m$, $250mm{\times}4.6mm\;i.d.$) with a gradient elution of 0.05% HAc and 0.05% HAc in $CH_3CN$. In the extract of Q. mongolica leaves, the content of gallic acid (32.53 mg/g), (+)-catechin (28.78 mg/g), (-)-epicatehin (22.03 mg/g), astragalin 6''-gallate (20.94 mg/g), and isoquercitrin 6''-gallate (44.11 mg/g) and peroxynitrite-scavenging activity ($IC_{50}$, $0.831{\mu}g/ml$) were high. This extract delayed the retention latency and inhibited acetylcholinesterase activity in scopolamine-induced memory impairment of mice, suggesting that it has anti-Alzheimer's activity.
This study aimed to establish the quantitative method to analyze the content of peroxynitrite-scavengers belonging to polyphenols in six Korean Quercus species (Quercus mongolica, Q. dentata, Q. acutissima, Q. alienta, Q. serrata, and Q. variabilis) by HPLC. The twelve peroxynitrite-scavengers, flavanols (catechins: (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin), flavonols (kaempferol and quercetin), flavonol glycosides (astragalin, quercitrin, and isoquercitrin), flavonol acylated glycosides (astragalin 6''-gallate and isoquercitrin 6''-gallate), gallic acid and its dimer (ellagic acid) were analyzed by HPLC. Further, anti-Alzheimer's activity was assayed in a passive avoidance testusing mice by measuring the retention latency (sec), the concentration of acetylcholine (ACh), and acetylcholinesterase (AChE) activity. Simultaneous analysis of the extracts of the six Quercus leaves was achieved on a Capcell C18 column ($5{\mu}m$, $250mm{\times}4.6mm\;i.d.$) with a gradient elution of 0.05% HAc and 0.05% HAc in $CH_3CN$. In the extract of Q. mongolica leaves, the content of gallic acid (32.53 mg/g), (+)-catechin (28.78 mg/g), (-)-epicatehin (22.03 mg/g), astragalin 6''-gallate (20.94 mg/g), and isoquercitrin 6''-gallate (44.11 mg/g) and peroxynitrite-scavenging activity ($IC_{50}$, $0.831{\mu}g/ml$) were high. This extract delayed the retention latency and inhibited acetylcholinesterase activity in scopolamine-induced memory impairment of mice, suggesting that it has anti-Alzheimer's activity.
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제안 방법
mongolica leaves with the highest content of peroxynitrite scavengers will be used to prevent diabetic complications such as nephropathy, neuropathy, retinopathy, and cardiovascular disease that can be caused by the overproduction of peroxynitrites.5In addition, it was established to reliably analyze the peroxynitrite scavengers in the Korean Quercus species by the validation experiments.
Recovery tests were performed on the extract of Q. mongolica spiked with each standard compound except for (−)-epigallocatechin.
Linearity was evaluated by calculating the R2 value of each regression equation. Sensitivity was evaluated by calculating the values of LOD (limit-of-detection) and LOQ (limit-ofquantification). The LOD and LOQ values were determined by the signal-to-noise (S/N) method, where an S/N ratio of 3 was used for LOD and 10 for LOQ
Accuracy and precision of the method were investigated by the intermediate evaluation method measuring the intraday and inter-day variability. The intra-day variability was examined by injecting sample solutions repetitively onto a HPLC system on the dame day, and the inter-day variability was assessed by testing five times a day four consecutive days. Relative standard deviation (RSD) was determined by calculating the retention times and peak area produced from five different experiments.
RSD values were considered as the measure of accuracy and precision. To assess accuracy, recovery tests were carried out by adding a standard compound to the sample solution. Recovery rates (%) were determined by calculating the rate of the spiked extract solution versus the non-spiked extract solution.
대상 데이터
Each collected set of leaves was dried and pulverized for extraction. The six plants were identified by Byong-Min Song, Department of Forestry Science, Sangji University, Korea. Voucher specimens were deposited in the Laboratory of Natural Product Chemistry, Department of Pharmaceutical Engineering, Sangji University, Korea.
The six plants were identified by Byong-Min Song, Department of Forestry Science, Sangji University, Korea. Voucher specimens were deposited in the Laboratory of Natural Product Chemistry, Department of Pharmaceutical Engineering, Sangji University, Korea.
이론/모형
Validation of the HPLC method − The validation of the HPLC method was performed following ICH guideline (International Conference on Harmonization) in terms of linearity, sensitivity, precision, and accuracy.
성능/효과
The method was sufficiently precise and stable because the RSD values obtained from the intra-day and inter-day variability tests were over the ranges of 0.66 – 4.15% and 1.17 – 4.91%, respectively.
참고문헌 (24)
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