Elsevier

Phytochemistry Letters

Volume 35, February 2020, Pages 78-83
Phytochemistry Letters

New coumarins isolated from the stem bark of Fraxinus rhynchophylla inhibit human neutrophil elastase and LPS-induced inflammation in RAW 264.7 cells

https://doi.org/10.1016/j.phytol.2019.11.005Get rights and content

Highlights

  • Two new coumarins (1 and 2) were established by extensive spectroscopic studies and chemical evidence.

  • Compounds 2 and 8 exhibited considerable HNE inhibitory activity, with IC50 values of 3.6 and 9.6 μM.

  • Compounds 2 and 8 inhibited NO production as well as iNOS and COX-2 protein expression in RAW 264.7 cells.

  • Active coumarins from F. rhynchophylla are promising therapeutic agents for treating inflammation-related diseases.

Abstract

Two new coumarins (1 and 2), together with six known compounds (38), were isolated from the stem bark of Fraxinus rhynchophylla. The structures of these compounds were established by extensive spectroscopic studies and chemical evidence. The inhibitory activities of the isolated compounds (18) against human neutrophil elastase (HNE) were evaluated in vitro; all compounds tested exhibited inhibitory effects against HNE activity, with IC50 values ranging from 3.6–74.3 μM. Among the compounds tested, compounds 2 and 8 exhibited considerable HNE inhibitory activity, with IC50 values of 3.6 and 9.6 μM, respectively. The mechanism by which the two active compounds (2 and 8) inhibited HNE was noncompetitive inhibition, with inhibition constant (Ki) values of 2.2 and 6.8 μM, respectively. Furthermore, compounds 2 and 8 dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. Consistent with these findings, they also suppressed LPS-induced expression of iNOS and COX-2 at the protein level in RAW 264.7 cells.

Introduction

The genus Fraxinus is one of the largest in the Oleaceae family, consisting of about 60 species worldwide, which are widely distributed in temperate regions and the subtropics of the Northern hemisphere (Wallander and Albert, 2000). Many Fraxinus species have received considerable attention due to their medicinal properties and have been used in folk medicine for the treatment of constipation, dropsy, arthritis, rheumatic pain, cystitis, and scalp itching in various parts of the world (Kostova and Iossifova, 2007). Among them, Fraxinus rhynchophylla Hance is a source of the traditional Chinese herbal drug “Qinpi.” The stem bark of this plant has been used in China as an antibacterial, analgesic, and anti-inflammatory agent and has been shown to possess various pharmacological activities, including diuretic, anticoagulant, and anti-allergic properties (Shin et al., 2010). Numerous bioactive substances, such as secoiridoid glucosides, coumarins, lignans, flavonoids, and simple phenolic compounds, have been isolated from F. rhynchophylla (Ahn et al., 2013; Kostova and Iossifova, 2007; Kwon and Kim, 1996; Shin et al., 2010; Si et al., 2008, 2009).

Human neutrophil elastase (HNE, EC 3.4.21.37) is a serine protease primarily found in the azurophilic granules of neutrophils. It has broad substrate specificity and can degrade collagen, elastin, fibronectin, and other extracellular matrix proteins (Steinbrecher et al., 2008). Under normal physiological conditions, HNE activity is tightly regulated by endogenous protease inhibitors, such as secretory leukocyte protease and α1-protease inhibitors (Williams et al., 2006). However, an imbalance between HNE and its endogenous inhibitors can lead to destruction of healthy tissues and contribute to inflammatory processes (Witko-Sarsat et al., 2000). Therefore, the development and investigation of HNE inhibitors, particularly natural anti-HNE agents without side effects, may constitute a therapeutic approach for the prevention and treatment of inflammation-related diseases.

In the course of searching for novel and naturally occurring inhibitors of HNE, we found that an ethanol extract of F. rhynchophylla stem bark exhibited considerable HNE inhibitory activity. To date, however, there have been no reports of HNE inhibitory activity of this plant or its secondary metabolites. Therefore, the present study was performed to investigate the active compounds in F. rhynchophylla stem bark. Here, we isolated two new coumarins (1 and 2), together with six known compounds (38). This report describes the isolation and structural determination of these compounds, as well as the characterization of their HNE inhibitory effects. We further investigated the effects of two active compounds (2 and 8) on NO (nitric oxide) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells.

Section snippets

Results and discussion

The ethanol extract of F. rhynchophylla stem bark that significantly inhibited HNE [IC50 = 5.0 μg/mL] was subjected to Diaion HP-20 column chromatography and divided into four fractions (A–D) based on the results of TLC. The HNE inhibitory activity of these fractions was evaluated; fraction C exhibited the strongest activity, with a 50 % inhibitory concentration (IC50) of 1.6 μg/mL. Fraction C was further subjected to a series of chromatographic separation steps guided by HNE inhibitory

General experimental procedures

Optical rotations were measured using a JASCO P-2000 digital polarimeter. UV spectra were recorded using a JASCO V-550 UV/VIS spectrometer. IR spectra were recorded on a JASCO 100 IR spectrometer. 1H (400 MHz) and 13C NMR (100 MHz) spectra were obtained using a Bruker DRX-400 spectrometer with tetramethylsilane as an internal standard. 2D-NMR experiments (HMQC and HMBC) were performed on a Bruker Avance 500 NMR spectrometer. HRESIMS was performed using a Shimadzu LCMS-IT-TOF spectrometer.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by a grant (KSN1119300) from the Korea Institute of Oriental Medicine. The NMR and MS experiments were performed by the Korea Basic Science Institute (KBSI).

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These two authors contributed equally to this work.

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