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Memoirs of The Faculty of B. O. S. T. of Kindai University No. 47 （2022）
英文抄録
Elucidation of the molecular mechanism of anti-inflammatory effect of mango leaf extract
by a SWATH acquisition method
Kento Yoshimoto1, Mone Kurokawa2, Hibiki Oomi2, Rika Kawahata2, Yuki Minamoto2, Kunihiro Kishida1,3,
Tetsuya Matsukawa1,4, Kouhei Nagai1, 2
It is often difficult to fully elucidate the molecular mechanism of functional foods, since foods generally contain
multiple functional substances, which may act on different targets. Therefore, there is a need to develop a method
that comprehensively captures the effects of food ingredients on cells. In this study, we first analyzed the details of
the anti-inflammatory effect of mango leaf ethanol extract (MLE) using macrophage-like Raw264.7 cells activated
with lipopolysaccharide (LPS). Secondary, we attempted to elucidate the molecular mechanism of the
anti-inflammatory effect of MLE by analyzing intracellular protein fluctuations by SWATH mass analysis. Addition
of MLE to Raw264.7 cells activated with 1.0 ng/mL LPS significantly reduced the concentrations of NO and IL-6 in
the culture supernatant at 0.56 μg/mL, and that of TNFα at 2.3 μg/mL. All of them decreased to below the detection
limit at 18 μg/mL of MLE. Similarly, the expression level of iNOS was significantly reduced at 2.5 μg/mL of MLE,
and that of TNFα, IL-6, and IL-1β was at 10 μg/mL. At 40 µg/mL of MLE, the expression level of all genes was
reduced to the level of LPS-untreated cells. When MLE was fractionated by normal phase chromatography, the NO
production inhibitory effect was not concentrated, and weak effects were detected on all fractions. This indicated that
multiple components in MLE may act on multiple receptors to exert a strong anti-inflammatory effect. When the
quantitative data obtained by SWATH-MS was analyzed by PC-DA, it was clearly distinguished into three groups,
untreated cells (LPS -), those treated with 10 ng/mL LPS (LPS+), and those treated with LPS and 10 µg/mL MLE
(MLE), indicating that the three groups possess distinctive proteome profiles. Among them, Annexin A1 and
Annexin A2, which have been reported to maintain cell homeostasis and contribute to the termination of
inflammation, were increased in the MLE group. Focusing on the factors related to the LPS-activated MAPK
signaling pathway, SQSTM1 involved in anti-inflammation increased, and p38MAPK and ERK1/2, which are the
central factors of the pathway, decreased. These factors were thought to be involved in the anti-inflammatory effect
of MLE.
Keywords: SWATH mass spectrometry, quantitative proteomics, anti-inflammatory effect, mango
Received 20 December 2021, Accepted 16 February 2022.
1. Graduate School of Biology-Oriented Science and Technology, Kindai University, Wakayama 649-6493, Japan
2. Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology, Kindai University, Wakayama 649-6493, Japan
3. Department of Science and Technology on Food Safety, Faculty of Biology-Oriented Science and Technology, Kindai University,
Wakayama 649-6493, Japan
4. Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kindai University, Wakayama 649-6493,
Japan
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