[논문]Laccase Fermentation of Clove Extract Increases Content of Dehydrodieugenol, Which Has Neuroprotective Activity against Glutamate Toxicity in HT22 Cells

Lee, Han-Saem; Yang, Eun-Ju; Lee, Taeho; Song, Kyung-Sik

doi:10.4014/jmb.1709.09052

Laccase Fermentation of Clove Extract Increases Content of Dehydrodieugenol, Which Has Neuroprotective Activity against Glutamate Toxicity in HT22 Cells 원문보기

Journal of microbiology and biotechnology, v.28 no.2, 2018년, pp.246 - 254

Lee, Han-Saem (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University) , Yang, Eun-Ju (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University) , Lee, Taeho (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University) , Song, Kyung-Sik (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University)

Abstract ▼ AI-Helper

Enzyme fermentation is a type of food processing technique generally used to improve the biological activities of food and herbal medicines. In this study, a Syzygii Flos (clove) extract was fermented using laccase derived from Trametes versicolor (LTV). The fermented clove extract showed greater neuroprotective effects against glutamate toxicity on HT22 than the non-fermented extract did. HPLC analysis revealed that the eugenol (1) and dehydrodieugenol (2) contents had decreased and increased, respectively, after fermentation. The content of 2 peaked at 1 h after fermentation to $103.50{\pm}8.20mg/g_{ex}$ (not detected at zero time), while that of 1 decreased to $79.54{\pm}4.77mg/g_{ex}$ ($185.41{\pm}10.16mg/g_{ex}$ at zero time). Compound 2 demonstrated promising HT22 neuroprotective properties with inhibition of $Ca^{2+}$ influx, the overproduction of intracellular reactive oxygen species, and lipid peroxidation. In addition, LTV showed the best fermentation efficacy compared with laccases derived from Pleurotus ostreatus and Rhus vernicifera.

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가설 설정

This phenomenon promotes the cell death signaling cascades, involving the Ca²⁺ influx, intracellular reactive oxygen species (ROS) generation, and lipid peroxidation [18, 19]. Thus, we hypothesized that fermentation of plant extracts may induce an increase in the content of antioxidative constituents that can protect neuronal cells against glutamate-mediated damage.

제안 방법

All experiments were performed at least in triplicate, and the data were expressed as the mean ± standard deviation (SD).
Effects of 1 and 2 on glutamate-induced HT22 cell death. To evaluate the HT22 protective effects of 1 (EU) and 2 (DE), (A) cell morphology, (B) MTT cell viability assay, and lactase dehydrogenase (LDH) leakage were assessed. (C) Effects of 1 and 2 on glutamate-mediated Ca²⁺ influx, production of intracellular reactive oxygen species (ROS), and lipid peroxidation in HT22 cells.
To identify the compounds that had increased or decreased in content after fermentation, column chromatography and NMR analysis were performed. The chemical structures of 1 and 2 are shown in Fig.
Nuclear magnetic resonance (NMR) analysis for compound identification was performed on a Bruker Avance400 Digital NMR spectrometer (Bruker, Germany), and the chemical shifts (δ) were expressed in parts per million (ppm) relative to the tetramethylsilane internal standard. To monitor the elution pattern, thin-layer chromatography was performed using RP-18 F254S and silica gel 60 F254 plates (Merck, Germany) for reverse and normal phase chromatography, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco, USA) was used to estimate cell viability.

대상 데이터

As a preliminary screening, extracts of 10 different plants (i.e., Aralia cordata (root), Syzygium aromaticum (bud), Citrus unshiu (peel), Glycyrrhiza uralensis (root), Melia azedarach (fruit), Piper nigrum (fruit), Rehmannia glutinosa (root), Rubia akane (leaf), Salvia miltiorrhiza (root), and Schizandra chinensis (fruit)) were prepared for experimentation. These extracts were subjected to fermentation with laccase derived from Trametes versicolor (LTV) to induce changes in their molecular constituents and to improve their neuroprotective effects.
Comparison of the fermentation efficacy of various laccases by (A) HPLC analysis and (B) MTT cell viability assay. Laccases derived from Trametes versicolor (LTV), Pleurotus ostreatus (LPO), and Rhus vernicifera (LRV) were tested. # p < 0.
Plants were purchased from a local market in Daegu, Korea, and voucher specimens were deposited at the Laboratory of Natural Products Medicine, College of Pharmacy, Kyungpook National University. The plants were refluxed with 95% EtOH for 3 h, and the extracted solutions were filtered and evaporated for fermentation and examination of their neuroprotective effects.
45 μm membrane filter (Millipore, USA) and the filtrates were analyzed by HPLC (Jasco, Japan) to compare the chromatographic pattern with non LTV-treated extracts (controls). The HPLC system was equipped with a PU-2080 Plus Intelligent HPLC pump and a MD-2010 Plus Multiwavelength detector with the wavelength set at 254 nm. An AXTerra RP18 column (4.

데이터처리

All experiments were performed at least in triplicate, and the data were expressed as the mean ± standard deviation (SD). Statistical significance was determined by one-way analysis of variance. A value of p < 0.

성능/효과

The HPLC profiles of LTV-treated plant extracts were compared with those of non LTV-treated ones. The HPLC chromatograms of Syzygium aromaticum (clove), Glycyrrhiza uralensis, Piper nigrum, and Rehmannia glutinosa were significantly changed after fermentation, whereas those of Aralia cordata, Citrus unshiu, Melia azedarach, and Rubia akane were slightly altered. On the other hand, the HPLC profiles of Salvia miltiorrhiza and Schizandra chinensis were unaffected (data not shown).
The results presented in this study suggested that the neuroprotective action of clove extract was significantly improved by simple laccase fermentation that led to an increase in 2 content. These findings can be helpful in the development of functional foods for the treatment of various neurodegenerative diseases.
These extracts were subjected to fermentation with laccase derived from Trametes versicolor (LTV) to induce changes in their molecular constituents and to improve their neuroprotective effects. To our best knowledge, this study is the first to report that the fermented Syzygii Flos (clove) extract showed a significant change in high-performance liquid chromatography (HPLC) profile and enhanced protection of mouse hippocampal neuronal cells (HT22) against glutamateinduced cell death. In addition, we optimized the LTV-catalyzed clove fermentation condition, isolated and quantified the modified constituents, and compared the HT22 cell protective effects of the isolated compounds.

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