Two pyrrole acids isolated from Phyllanthus emblica L. and their bioactivities

Abstract

An undescribed pyrrole acid, 1-(4'-methoxy-4'-oxobutyl)-1 H-pyrrole-2,5-dicarboxylic acid (1) and one known pyrrole acid (2) were isolated from the fruits of Phyllanthus emblica. The structures of these compounds were elucidated via the comprehensive analyses of IR, HRESIMS, 1D and 2D spectroscopic data. A series of biological assays revealed that compounds 1 and 2 could inhibit LPS-induced over-production of nitric oxide (NO), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor-α (TNF-α) by reducing the phosphorylation of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinases (JNK) in RAW 264.7 cells. Additionally, compounds 1 and 2 were found to reduce lipid deposition and increase the mRNA expression of ATP-binding cassette transporter A1 in oxidized low-density lipoprotein-treated RAW264.7 macrophages.

Keywords: ATP-binding cassette transporter A1; Lipid deposition; Phyllanthus emblica L.; Pyrrole acid; RAW264.7 macrophages.

Fig. 1

Structures of compounds 1 and 2. Key ¹ H-¹ H COSY (bold bonds in red), HMBC (arrows in blue) correlations of 1

Fig. 2

Effect of the two pyrrole acids 1 and 2 and P. emblica extract (PE) on LPS-induced NO (A), IL-6 (B), TNF-α (C) and MCP-1 (D) production in RAW264.7 cells. Macrophages were pre-treated with/without compounds 1, 2 (100 µM) or PE (500 µg/mL) for 2 h and then stimulated with 500 ng/mL LPS for 24 h. The production of these markers was measured by Griess/ELISA. Dexamethasone (DXM) was set as a control (100 µM). Control was cultured without samples and LPS. The data presented are the means ± SD, #p < 0.05 significant compared with the control group, and *p < 0.05, **p < 0.01 significant compared with LPS-treated group

Fig. 3

Effects of the two pyrrole acids 1 and 2 and PE on the LPS-induced phosphorylation of the MAPK signaling pathway in RAW264.7 macrophages. Cells were pretreated with/without compounds 1, 2 (100 µM) or PE (500 µg/mL) for 2 h and then stimulated with LPS (500 ng/mL) for 24 h. A–B) Whole cell lysates were analyzed by western blotting for detection of phosphorylated p-ERK, ERK, p-JNK, and JNK proteins. Control was cultured without samples and LPS. The data presented are the means ± SD, #p < 0.05 significant compared with the control group, and *p < 0.05, **p < 0.01

Fig. 4

Effects of two pyrrole acids 1 and 2 and PE on lipid accumulation. (A) RAW264.7 cells were treated with ox-LDL (50 µg/mL) in the presence or absence of compounds 1, 2 (100 µM) or PE (500 µg/mL). (B) After treatment, cultured cells were treated with Ox-LDL (50 µg/mL) for an additional 24 h. Then, macrophages were lysed and the intracellular TC content and FC content were quantified according to the protocol by the manufacturers. (C) After treatment, cultured cells were treated with Ox-LDL (50 µg/mL) for an additional 24 h. The mRNA expression of ABCA1 was assessed by real-time PCR, and expressed as fold induction relative to control. The date presented are the means ± SD, #p < 0.05 significant compared with the control group, and *p < 0.05, **p < 0.01