Andrograpanin, isolated from Andrographis paniculata, exhibits anti-inflammatory property in lipopolysaccharide-induced macrophage cells through down-regulating the p38 MAPKs signaling pathways

doi:10.1016/j.intimp.2007.12.014

International Immunopharmacology

Volume 8, Issue 7, July 2008, Pages 951-958

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https://doi.org/10.1016/j.intimp.2007.12.014 Get rights and content

Abstract

Andrographis paniculata Nees is an official herbal medicine for treatment of infection and inflammation in China. Andrograpanin, the one of diterpene lactones in A. paniculata, is a hydrolysate from neoandrographolide in vivo and in vitro. The goal of the present study was to investigate andrograpanin which effects on over production of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-12p70) and the key signaling pathways involved in lipopolysaccharide (LPS)-activated macrophage cells. The results showed that NO and all three pro-inflammatory cytokines were inhibited by andrograpanin (15–90 μM) in a dose-dependent manner. The RT-PCR and western blotting assays showed that andrograpanin inhibited productions of NO and pro-inflammatory cytokines through down-regulating iNOS and pro-inflammatory cytokines gene expression levels. Further studies suggested that down-regulation of p38 mitogen-activated protein kinase (MAPKs) signaling pathways were involved in the anti-inflammatory activities of andrograpanin. This study provided evidences that andrograpanin might be useful as a potential anti-inflammatory leading compound for inflammatory drug development.

Introduction

Endotoxin (LPS, lipopolysaccharide)-induced pro-inflammatory mediators and cytokines production contribute to the sequence of events in activated macrophage cells, which lead to kill of microorganisms and causing tissue damage during the inflammatory process. Over production of nitric oxide (NO) has been implicated in the pathogenesis of inflammation via inducible nitric oxide synthase (iNOS), which reflects the degree of inflammation and provide a measure for assessing the effect of inflammatory process. In addition, pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukins appear to play an active role in inflammation. Production and release of pro-inflammatory mediators and cytokines by LPS depends on inducible gene expression mediated by the activation of different transcription factors, such as mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-κB) [1], [2], [3], [4]. Particular, MAPKs is a group of serine/threonine protein kinases comprising three subfamilies: the p42/p44 extracellular regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. A major consequence of MAPKs phosphorylation is the activation of these transcription factors, which serve as immediate or downstream substrates of these kinases [1]. Therefore, MAPKs and NF-κB activation have been used as potential target for the anti-inflammatory drug.

Natural products have been used as potentially important sources of anti-inflammatory drugs [5]. Andrographis paniculata Nees. (Acanthaceae) has long been used in Chinese official herbal medicine and the extracts of the herb have displayed a wide spectrum of bioactivities, including anti-inflammatory and anti-infective activities [6], [7]. Neoandrographolide is a bicyclic diterpenoid lactone isolated from leaves of A. paniculata [8] and we have reported that anti-inflammatory effects previous [9], [10]. However, andrograpanin is a hydrolysate from neoandrographolide in vivo and in vitro and it is also a minor compound of A. paniculata. Our recent study has indicated that andrograpanin has the potential to modulate the chemokine pathway and the effect of andrograpanin might contribute to the anti-infectious function of A. paniculata [11]. In order to extend the understanding of its mechanism of anti-inflammatory, we investigated the effect of andrograpanin on production of NO and pro-inflammatory cytokines TNF-α, IL-6 and IL-12p70 in LPS-activated macrophage cells by interfering both MAPKs and NF-κB signaling pathways. We used primary bone marrow-derived macrophages as cellular model, since they represent a homogenous, non-transformed population of macrophages that can be stimulated in vitro to proliferate by macrophage colony-stimulating factor (M-CSF) or activated by LPS. The present results showed that andrograpanin might down-regulate iNOS and pro-inflammatory cytokines expressions particular through down-regulation of p38 MAPKs signaling pathway. It suggests that andrograpanin had potential as one of leading compounds for inflammatory diseases.

Section snippets

Preparation of andrograpanin

The leaves of A. paniculata were collected from Linquan, Anhui province, China and authenticated by Prof. Zheng Tao Wang. A voucher specimen was deposited in the Herbarium of Shanghai University of Traditional Chinese Medicine, Shanghai, China. Andrograpanin was isolated from A. paniculata leaves as described in previous publication [11]. Briefly, the dried leaves (1 Kg) were macerated in 95% ethanol and kept at room temperature for three days. After filtration, the ethanol solution was

Effect of the andrograpanin on NO production and cell viability in LPS-stimulated macrophage cells

The induction of macrophage cells into inflammatory state by treatment with LPS caused synthesis and secretion of NO. Macrophage cells were pretreated with andrograpanin (15–90 μM) prior to stimulate with LPS (5 μg/ml) for 24 h. As shown in Fig. 2C, andrograpanin showed a dose-related inhibition of NO production in which significant inhibition was evident at 30 μM. At 75 μM, andrograpanin almost completely inhibited NO production. Based on results of cell viability test, andrograpanin showed no

Discussion

Alternative therapy has been becoming more popular and attractive and there are increasing interest arisen on elucidating the efficacy, safety, and functional mechanism of herbal medicine. Bicyclic diterpenoid lactones isolated from A. paniculata (such as andrographolide and neoandrographolide), which are mainly responsible for the anti-inflammatory effects in vitro and in vivo. Andrograpanin, the minor compound of A. paniculata is also a hydrolysate from neoandrographolide. Andrograpanin

Acknowledgements

This study was supported by the Shanghai Leading Discipline Foundation and Science and Technology Development Found (no. 03DZ19546). We also thank Dr. Q.J. Tang, Shanghai Academy of Agricultural Sciences Shanghai for technical support with L-929 cell culture.

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Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes.
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Andrograpanin, isolated from Andrographis paniculata, exhibits anti-inflammatory property in lipopolysaccharide-induced macrophage cells through down-regulating the p38 MAPKs signaling pathways

Abstract

Introduction

Section snippets

Preparation of andrograpanin

Effect of the andrograpanin on NO production and cell viability in LPS-stimulated macrophage cells

Discussion

Acknowledgements

Eur J Pharmacol

Mol Immunol

Trends Biotechnol

Tetrahedron Lett

Anal. Biochem

Cell Signal

Cell

FEBS Lett

Arch Biochem Biophys

Free Radic Biol Med

Cell Signal

Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases

Science

A protein kinase involved in the regulation of inflammatory cytokine biosynthesis

Nature

Pharmacology and application of Chinese material medica