Elsevier

Food Chemistry

Volume 134, Issue 2, 15 September 2012, Pages 742-748
Food Chemistry

Anti-inflammatory activity of lipophilic epigallocatechin gallate (EGCG) derivatives in LPS-stimulated murine macrophages

https://doi.org/10.1016/j.foodchem.2012.02.172Get rights and content

Abstract

Epigallocatechin gallate (EGCG), the major polyphenol in green tea and the main bioactive compound responsible for the health benefits of tea consumption, has been proposed as a functional ingredient for food and natural health products. However, EGCG is hydrophilic with poor cellular absorption and thus compromised bioefficiency in vivo. In order to enhance the lipophilicity of EGCG for improved bioefficiency and to take advantage of the health beneficial omega 3 fatty acids, the EGCG molecule was esterified with docosapentaenoic acid (DPA), upon which a mixture of ester derivatives with different degrees of substitution was produced. The EGCG–DPA esters were evaluated for their anti-inflammatory activity in LPS (lipopolysaccharides)-stimulated murine RAW 264.7 macrophages. The production of pro-inflammatory mediators nitric oxide (NO) and prostaglandin (PGE2) was significantly inhibited by treatment of EGCG–DPA esters, and the inhibition was largely due to their down-regulatory effect on iNOS (inducible NO synthase) and COX (cyclooxygenase)-2 gene expression at transcriptional level. The EGCG–DPA esters effectively suppressed the expression of iNOS and COX -2 proteins as well as their mRNA, as observed with western blotting and RT-PCR analyses. Ester derivatives of EGCG with other fatty acids (stearic acid, SA; eicosapentaenoic acid, EPA; and docosahexaenoic acid, DHA) were also prepared in the form of pure tetraesters, which also exhibited anti-inflammatory effect in the macrophages. The results suggest that EGCG ester derivatives with anti-inflammatory potentials may be useful in preventing/treating inflammation-mediated diseases and health conditions.

Highlights

► Lipophilic ester derivatives of EGCG were prepared. ► They inhibited NO and PGE2 production in LPS-stimulated macrophage. ► They down-regulated iNOS and COX-2 gene expression at transcriptional level. ► They may be useful in preventing/treating inflammation and related diseases.

Introduction

Inflammation is a normal physiological response of the immune system to counteract pathological states such as irritation and infection caused by chemicals, microbial pathogens and/or wounding. However, unbalanced or prolonged inflammation leads to progressive tissue damage and has been implicated in the development of many chronic diseases such as cancer and neurodegenerative disorders as well as diabetes and cardiovascular disease. Inflammation is characterised by recruitment of a wide range of immune cells (e.g. neutrophils, macrophages monocytes, etc.) to the inflamed sites and the release of various pro-inflammatory cytokines and reactive oxygen/nitrogen species (ROS/RNS). Oxidative stress through continuous overproduction of ROS/RNS by activated phagocytes constitutes the major tissue-destructive force in vivo, and in turn promotes inflammation by stimulating production of inflammatory mediators and cytokines (Shahidi & Zhong, 2009). Evidence has shown that ROS/RNS are involved in the activation of a variety of kinases and transcription factors, whose regulation is dependent on the redox changes. The transcription factors nuclear factor-kappa B (NF-κB) and activator protein (AP)-1, for example, are redox-sensitive and become activated under oxidative/nitrosative stress. Once activated, NF-κB and AP-1 translocate from the cytoplasm to the nucleus, leading to the up-regulation of numerous inflammatory genes, such as those coding for inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, among others (Kamata & Hirata, 1999). Hence, ROS/RNS and pro-inflammatory cytokines work in a synergistic manner through a ROS/RNS-cytokine-transcription factor regulatory loop, thereby augmenting the inflammatory response and tissue damage (Fiocchi, 1998). The production of NO and prostaglandins by iNOS and COX-2, respectively, are considered to be the most prominent molecular mechanisms in the inflammatory processes (Moncada, 1999, Turini and DuBois, 2002), and are also involved in the multistage carcinogenesis, especially the promotion stage (Pan & Ho, 2008). Excessive and prolonged NO generation caused by overexpression of iNOS has also been implicated in inflammational tumourigenesis; while COX-2-mediated prostaglandin production stimulates cell proliferation, invasion and angiogenesis in cancer development (Mann, Backlund, & DuBois, 2005).

Anti-inflammatory agents can block ROS/RNS and cytokine-involved inflammatory cascade. Compared to steroidal or non-steroidal chemical drugs for treating inflammation, naturally derived substances are readily available at lower costs, while having limited side effects and intolerance. Polyphenols found abundantly in plant foods have been studied for their anti-inflammatory activities in suppressing the synthesis (gene expression) and action (enzyme activity) of many pro-inflammatory mediators. Tea catechins, especially epigallocatechin gallate (EGCG), have been reported to exhibit anti-inflammatory activity through their ability to scavenge NO, peroxynitrite (ONOO) and other ROS/RNS, to inhibit the translocation of NF-κB and AP-1 from the cytoplasm to nucleus, and to inhibit the activity of iNOS and COX-2 (Paquay et al., 2000, Nagai et al., 2002, Tedeschi et al., 2004). EGCG is found abundantly in green tea as a major polyphenol with a variety of health benefits. However, the molecule is hydrophilic with poor solubility in lipid systems, which, at least partially, accounts for its low absorption by the cells and limited efficacy in the cellular environment. Moreover, cell line studies have suggested that EGCG and other tea catechins are subject to active efflux by multidrug resistance-related proteins, leading to restricted bioefficiency in vivo (Hong, Lambert, Lee, Sinko, & Yang, 2003). In such cases, prodrug strategies may be employed. Ester-based prodrugs are known to provide improved bioavailability and reduced toxicity by occlusion of polar side chains, increasing hydrophobicity and making the hydroxyl groups unavailable for phase II biotransformation or oxidative degradation (Lambert et al., 2006).

In this work, EGCG was structurally modified by esterification with different fatty acids to produce lipophilic ester derivatives with enhanced cellular absorption and bioefficiency in vivo. Our previous study demonstrated that the esters so-prepared exhibited improved antioxidant and antiviral activities (Zhong et al., 2012, Zhong and Shahidi, 2012). In the current study, EGCG was esterified with docosapentaenoic acid (DPA) and the product (EGCG–DPA ester mixture containing mainly tetra- and pentaesters, Fig. 1) was evaluated for its anti-inflammatory activities in terms of inhibition against NO and prostaglandin E2 (PGE2) production as well as iNOS and COX-2 gene expression in murine macrophages. Three isolated EGCG esters (1, tetrastearate; 2, tetraeicosapentaenoate; 3, tetradocosahexaenoate) were also examined for their effect on NO production and iNOS and COX-2 expression, in order to compare the anti-inflammatory efficacy of the esters with that of the original EGCG on a mole basis.

Section snippets

Materials

EGCG (>98%) was supplied by GlaxoSmithKline Consumer Healthcare (Parsippany, NJ). Stearoyl chloride was purchased from Nu-chek Prep Inc. (Elysian, MN). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were prepared as described elsewhere from oils provided by Fuso Pharmaceutical Industries Ltd. (Osaka, Japan) and Martek Bioscience Corporation (Columbia, MD), respectively. DPA was produced via a proprietary procedure from seal blubber oil. RAW 264.7 cells, derived from murine

Inhibition of LPS-induced nitrite production by EGCG–DPA esters

Nitric oxide (NO) is an important oxidative and inflammatory mediator produced by NOS under physiological and pathophysiological conditions. It is a lipid-soluble free radical with a considerably long life and capable of diffusing several cell diameters from its synthesis site (Kruidenier & Verspaget, 2002). NO itself at nanomolar concentrations is not particularly harmful and in some occasions may even exert beneficial effects (Kruidenier & Verspaget, 2002). However, it is the precursor of a

Conclusions

The ester derivatives of EGCG with improved lipophilicity exhibited anti-inflammatory activities in LPS-stimulated murine macrophages, possibly through enhanced cellular absorption and contribution from both EGCG and the fatty acid moieties. The EGCG esters showed inhibition against LPS-induced production of NO and PGE2 in the macrophages. The inhibitory effect is largely related to the ability of the molecules involved to down-regulate the inflammatory mediators (iNOS and COX-2) biosynthesis

Acknowledgements

One of us (F.S.) thanks the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support and AFMNet (Advanced Foods and Materials Network) for partial funding.

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