• Journal of Biomedical and Translational Research
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• 제19권4호
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• pp.130-139
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• 2018

This study aimed to analyze a high-performance liquid chromatography (HPLC) separation using a pentafluorophenyl column of parent drug hydroxychloroquine (HCQ) and its active metabolite, desethylhydroxchloroquine (DHCQ) applying to determine bioequivalence of two different formulations administered to patients. A rapid, simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for bioanalysis of HCQ and its metabolite DHCQ in human whole blood using deuterium derivative $hydroxychloroquine-D_4$ as an internal standard (IS). A triple-quadrupole mass spectrometer was operated using electrospray ionization in multiple reaction monitoring (MRM) mode. Sample preparation involves a two-step precipitation of protein techniques. The removed protein blood samples were chromatographed on a pentafluorophenyl (PFP) column ($50mm{\times}4.6mm$, $2.6{\mu}m$) with a mobile phase (ammonium formate solution containing dilute formic acid) in an isocratic mode at a flow rate of 0.45 mL/min. The standard curves were found to be linear in the range of 2 - 500 ng/mL for HCQ; 2 - 2,000 ng/mL for DHCQ in spite of lacking a highly sensitive MS spectrometry system. Results of intra- and inter-day precision and accuracy were within acceptable limits. A run time of 2.2 min for HCQ and 2.03 min for DHCQ in blood sample facilitated the analysis of more than 300 human whole blood samples per day. Taken together, we concluded that the assay developed herein represents a highly qualified technology for the quantification of HCQ in human whole blood for a parallel design bioequivalence study in a healthy male.

• Journal of Ginseng Research
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• 제43권1호
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• pp.154-160
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• 2019

Background: Compound K (CK) is an active metabolite of ginseng saponin, ginsenoside Rb1, that has been shown to have ameliorative properties in various diseases. However, its role in inflammation and the underlying mechanisms are poorly understood. In this report, the antiinflammatory role of CK was investigated in macrophage-like cells. Methods: The CK-mediated antiinflammatory mechanism was explored in RAW264.7 and HEK293 cells that were activated by lipopolysaccharide (LPS) or exhibited overexpression of known activation proteins. The mRNA levels of inflammatory genes and the activation levels of target proteins were identified by quantitative and semiquantitative reverse transcription polymerase chain reaction and Western blot analysis. Results: CK significantly inhibited the mRNA expression of inducible nitric oxide synthase and tumor necrosis factor-${\alpha}$ and morphological changes in LPS-activated RAW264.7 cells under noncytotoxic concentrations. CK downregulated the phosphorylation of AKT1, but not AKT2, in LPS-activated RAW264.7 cells. Similarly, CK reduced the AKT1 overexpression-induced expression of aldehyde oxidase 1, interleukin-$1{\beta}$, interferon-${\beta}$, and tumor necrosis factor-${\alpha}$ in a dose-dependent manner. Conclusion: Our results suggest that CK plays an antiinflammatory role during macrophage-mediated inflammatory actions by specifically targeting the AKT1-mediated signaling pathway.

• 한국자기공명학회논문지
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• 제22권4호
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• pp.91-100
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• 2018

Benzisothiazolinone (BIT) is the preservative that is widely used in industrial and household products. In this study, zebrafish (Danio rerio) was exposed to BIT at different concentrations (control, 0.5 g/L, 1.0 g/L and 2.0 g/L) for 72 hours. The techniques of nuclear magnetic resonance (NMR) spectroscopy were applied to analyze the effects of BIT on zebrafish. The advantages of NMR are the minimal sample preparation and high reproducibility of experimental results. With the multivariate statistical analysis, dimethylamine, N-acetylaspartate, glycine and histidine were identified as an important metabolite in differentiating between the control and BIT-exposed group. This study will improve the understanding the metabolite changes in the zebrafish in response to BIT exposure.

• Biomolecules & Therapeutics
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• 제29권4호
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• pp.399-409
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• 2021

1,2-Diacetylbenzene (DAB) is a metabolite of 1,2-diethylbenzene, which is commonly used in the manufacture of plastics and gasoline. We examined the neurotoxic effects of DAB in young and old rats, particularly its effects on hippocampus. Previously, we reported DAB impairs hippocampal neurogenesis but that the underlying mechanism remained unclear. In this study, we evaluate the toxicities exhibited by DAB in the hippocampi of 6-month-old (young) and 20-month-old (old) male SD rats by treating animals intraperitoneally with DAB at 3 mg/kg/day for 1 week. Hippocampal areas were dissected from brains and RNA was extracted and subjected to RNA-seq analysis. RNA results showed animals exhibited age-dependent sensitivity to the neurotoxic effects of DAB. We observed that inflammatory pathways were up-regulated in old rats but that metabolism- and detoxification-related pathways were up-regulated in young rats. This result in old rats, especially upregulation of the TREM1 signaling pathway (an inflammatory response involved in Alzheimer's disease (AD)) was confirmed by RT-PCR. Our study results provide a better understanding of age-dependent responses to DAB and new insight into the association between DAB and AD.

• Animal Bioscience
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• 제36권7호
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• pp.1130-1142
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• 2023

Objective: Cow urine possesses several bioactive properties but the responsible components behind these bioactivities are still far from identified. In our study, we tried to identify the possible components behind the antimicrobial activity of cow urine by exploring the peptidome and metabolome. Methods: We extracted peptides from the urine of Sahiwal cows belonging to three different physiological states viz heifer, lactation, and pregnant, each group consisting of 10 different animals. The peptides were extracted using the solid phase extraction technique followed by further extraction using ethyl acetate. The antimicrobial activity of the aqueous extract was evaluated against different pathogenic strains like Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae. The safety of urinary aqueous extract was evaluated by hemolysis and cytotoxicity assay on the BuMEC cell line. The urinary peptides were further fractionated using high-performance liquid chromatography (HPLC) to identify the fraction(s) containing the antimicrobial activity. The HPLC fractions and ethyl acetate extract were analyzed using nLC-MS/MS for the identification of the peptides and metabolites. Results: A total of three fractions were identified with antimicrobial activity, and nLC-MS/MS analysis of fractions resulted in the identification of 511 sequences. While 46 compounds were identified in the metabolite profiling of organic extract. The urinary aqueous extract showed significant activity against E. coli as compared to S. aureus and S. agalactiae and was relatively safe against mammalian cells. Conclusion: The antimicrobial activity of cow urine is a consequence of the feeding habit. The metabolites of plant origin with several bioactivities are eliminated through urine and are responsible for their antimicrobial nature. Secondly, the plethora of peptides generated from the activity of endogenous proteases on protein shed from different parts of tissues also find their way to urine. Some of these sequences possess antimicrobial activity due to their amino acid composition.

• 한국산학기술학회논문지
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• 제21권2호
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• pp.432-439
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• 2020

이 연구는 급여하는 조사료와 농후사료 비율을 달리하였을 때, 우분 내 대사체 비교를 핵자기공명장치로 확인하기 위해 실시되었다. 착유중인 젖소 6마리를 두 그룹으로 나누어 조사료와 농후사료 급여 비율(HR 그룹=8:2, HC 그룹=2:8)을 달리 하였다. 우분은 직장을 통해 마리별 1회 채취 하였으며 대사체 분석은 SPE-800 MHz NMR-MS system을 이용하였다. 대사체의 정성 및 정량은 Chenomx NMR suite 8.4을 이용하였으며, 확인된 대사체들을 Metaboanalyst 4.0을 이용하여 다변량 분석, 대사경로 분석을 진행하였다. 통계분석은 SAS program을 이용하여 Dunnett's test로 그룹 간 대사체 평균값을 비교 하였다. 분석 결과, 다변량 분석에서는 그룹간의 분리가 확인 되었으며 확인된 대사체 중 통계분석에는 77개의 대사체가 이용되었다. HC 그룹에서 유의적으로 높았던 대사체는 succinate를 포함한 dimethylamine, histamine, homovanillate, thymol, acetate, propionate, butyrate, isovalerate, valerate, imidazole, N-Nitrosodimethylamine 그리고 O-Acetylcholine 이다. HC 그룹에서 분석된 대사체들의 평균 농도가 HR 그룹에 비해 높았으며, 분석된 대사경로 또한 HR 그룹에 비해 다양했다. 연구결과, 핵자기공명분광장치를 통해 분석한 우분 내 대사체는 장내 미생물 뿐 만 아니라 체내 대사 그리고 향후 축산 관련 다양한 연구에 유용하게 사용될 것으로 사료된다.

• 한국작물학회지
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• 제63권4호
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• pp.360-377
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• 2018

The levels of 12 isoflavones were measured in soybean (Glycine max (L.) Merrill) sprouts of 68 genetic varieties from three countries (China, Japan, and Korea). The isoflavone profile differences were analyzed using data mining methods. A principal component analysis (PCA) revealed that the CSRV021 variety was separated from the others by the first two principal components. This variety appears to be most suited for functional food production due to its high isoflavone levels. Partial least squares discriminant analysis (PLS-DA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) showed that there are meaningful isoflavone compositional differences in samples that have different countries of origin. Hierarchical clustering analysis (HCA) of these phytochemicals resulted in clusters derived from closely related biochemical pathways. These results indicate the usefulness of metabolite profiling combined with chemometrics as a tool for assessing the quality of foods and identifying metabolic links in biological systems.

• 한국방사선학회논문지
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• 제9권1호
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• pp.47-53
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• 2015

본 연구에서 3.0 T에서 조영제 몰 농도에 따른 뇌 대사물질의 MR spectrum을 Phantom에서 PRESS 펄스 파형으로 분석하고자 하였다. 뇌 대사물질인 N-Acetyl Asparatate(NAA), Choline(Cho), Creatine(Cr)의 spectrum은 획득할 수 있었지만, Lactate(Lac)는 획득할 수 없었다. 조영제가 없는 대조군의 TR 2000 ms가 NAA, Cho에서 TR 1700 ms와 TR 1500 ms보다 높게 측정되었고, Cr에서는 TR 1500 ms에서 높게 측정되었다. 조영제가 희석된 TR 1700 ms에서 $0.1mmol/{\ell}$의 NAA 73%, Cho 249%, Cr 37%로 다른 TR 값보다 가장 높게 측정되었고, $0.3mmol/{\ell}$에서도 신호크기가 증가하였다. $0.5mmol/{\ell}$에서는 뇌 대사물질들의 신호크기가 감소하였으며, 특히 TR 1500 ms과 TR 2000 ms에서는 대조군보다도 감소하였다. NAA/Cr, Cho/Cr에서도 조영제 농도가 $0.1mmol/{\ell}$, $0.3mmol/{\ell}$, $0.5mmol/{\ell}$로 증가할수록 신호크기가 감소하였다. 조영제에 의한 MRS PRESS 펄스파형의 적정화를 위하여 3.0T에서 TR 2000 ms 보다 조영제 농도가 낮은 $0.1mmol/{\ell}$와 $0.3mmol/{\ell}$에서 신호크기가 가장 높고, 조영제 농도가 높은 $0.5mmol/{\ell}$에서는 신호크기가 가장 적게 감소한 TR 1700 ms로 반복시간을 단축시켜 사용하는 것이 유용할 것으로 사료된다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 춘계학술발표회
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• pp.14-14
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• 2018

This Camptotheca acuminata Decne. (CA), belonging to Nyssaceae, is a deciduous tree. and has been used as Traditional Chinese medicine since ancient times. The CA produces camptothecin a natural indole alkaloid, and reported to have anti-cancer effects. But the studies on biological activities of CA leaves are insufficient. Therefore, this study confirmed various biological activities such as antioxidant, antidiabetic, anticancer, antiinflammatory and metabolism analysis by HPLC-MS/MS of CA leaves. The $RC_{50}$ values of DPPH radical scavenging activity of ethyl acetate fraction, n-Butanol fraction, methanol extraction, water fraction and n-Hexane fraction were $12.23{\pm}0.01$, $15.93{\pm}0.42$, $55.12{\pm}0.45$, $56.29{\pm}4.15$ and $427.29{\pm}6.13ug/mL$, respectively. The $IC_{50}$ values of ${\alpha}$-glucosidase inhibitory activity of ethyl acetate fraction, n-Butanol fraction, methanol extraction, n-Hexane fraction and water fraction were $24.29{\pm}0.14$, $47.86{\pm}0.45$, $54.23{\pm}1.21$ $466.76{\pm}2.21$ and $623.91{\pm}9.67ug/mL$, respectively. The nitric oxide release activity of n-Hexane fraction, methanol extraction, ethyl acetate fraction, water fraction and n-Butanol fraction were $31.49{\pm}5.74$, $29.79{\pm}0.71$, $26.89{\pm}0.71$, $8.24{\pm}5.83$ and $7.75{\pm}4.08%$ at 25 ug/mL, respectively. The anti-cancer activity of n-Hexane fraction, methanol extraction, ethyl acetate fraction, water fraction and n-Butanol fraction were $31.49{\pm}5.74$, $29.79{\pm}0.71$, $26.89{\pm}0.71$, $8.24{\pm}5.83$ and $7.75{\pm}4.08%$ at 25 ug/mL, respectively. The ethyl acetate fraction activities showed higher biological activities than other fractions. Thus, Additional studies were conducted using ethyl acetate fraction. Metabolite analysis was performed using a LCMS-8040 triple quadrupole mass spectrometer. As a result, Five compounds (1-5) were identified in the ethyl acetate fraction of the CA leave. The identification of these compounds was generated by the analysis of fragmentation methods of the negative and positive ion modes. Five compounds were identified as gallic acid (1), chlorogenic acid (2), isoquercetin (3), astragalin (4) and camptothecin (5). These results suggest that the CA leave can be used for functional materials.

• 한국자기공명학회논문지
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• 제15권1호
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• pp.54-68
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• 2011

$^1H$ nuclear magnetic resonance (NMR) spectroscopy of biological samples has been proven to be an effective and nondestructive approach to probe drug toxicity within an organism. In this study, ketoprofen toxicity was investigated using $^1H$-NMR spectroscopy coupled with multivariate statistical analysis. Histopathologic test of ketoprofen-induced acute gastrointestinal damage in rats demonstrated a significant dose-dependent effect. Furthermore, principal component analysis (PCA) derived from $^1H$-NMR spectra of urinary samples showed clear separation between the vehicle-treated control and ketoprofen-treated groups. Moreover, PCA derived from endogenous metabolite concentrations through targeted profiling revealed a dose-dependent metabolic shift between the vehicle-treated control, low-dose ketoprofen-treated (10 mg/kg body weight), and high-dose ketoprofen-treated (50 mg/kg) groups coinciding with their gastric damage scores after ketoprofen administration. The resultant metabolic profiles demonstrated that the ketoprofen-induced gastric damage exhibited energy metabolism perturbations that increased urinary levels of citrate, cis-aconitate, succinate, and phosphocreatine. In addition, ketoprofen administration induced an enhancement of xenobiotic activity in fatty oxidation, which caused increase levels of N-isovalerylglycine, adipate, phenylacetylglycine, dimethylamine, betaine, hippurate, 3-indoxylsulfate, N,N-dimethylglycine, trimethyl-N-oxide, and glycine. These findings demonstrate that $^1H$-NMR-based urinary metabolic profiling can be used for noninvasive and rapid way to diagnose adverse drug effects and is suitable for explaining the possible biological pathways perturbed by nonsteroidal anti-inflammatory drug toxicity.