Investigating the anti-inflammatory properties of epigallocatechin gallate from green tea Camellia sinensis (L) O.Kuntze in mammalian innate immunity

Abstract

Humans are hypersensitive to lipopolysaccharide, a gram-negative bacterial endotoxin that stimulates an inflammatory response by binding to the extracellular domain of TLR4 of innate immune cells. Through the MyD88-dependent pathway, NO (a signalling molecule) mediates the inflammatory response to clear pathogenic infection and promote wound healing. This pathway involves the activation of transcription factors, AP-1 and NF-κB, which promote the production of pro-inflammatory cytokines such as TNFα. However, dysregulated inflammation leads to chronic inflammation, causing autoimmune diseases including rheumatoid arthritis and asthma, among others. Considering the shortfalls of currently available treatments such as NSAIDs, there is a growing need for novel treatment strategies. Studies suggest that EGCG, a phenolic compound found abundantly in green tea (C. sinensis), possesses anti-inflammatory activity. This study sought to test this hypothesis. Firstly, 70% methanol was used to extract phenolic compounds and caffeine from three commercial green tea brands (Lipton, Tetley and BST) available on the South African market. Then the EGCG and caffeine contents of these green tea products were measured using UPLC analysis. Additionally, the total phenolic contents (TPCs) were measured for comparison using the Folin-Ciocalteu method. Secondly, the anti-inflammatory properties of EGCG were investigated in an acellular system and in RAW 264.7 cells using the Griess assay to test for NO production. Thereafter, MTT assays were conducted on the macrophages to determine cell viability after exposure to EGCG. Lastly, the role of EGCG on TLR4 signalling during inflammation was tested in the context of the MyD88-dependent pathway. To do this, NF-κB expression was measured using luciferase activity assays in LPS-stimulated HEK 293 cells treated with EGCG. Additionally, ELISAs were performed on stimulated wild-type immortalised bone marrow-derived macrophages (iBMDMs) to monitor the expression of TNFα in response to treatment with EGCG. This study determined the average total phenolic content of Lipton, Tetley and BST to be 18%, which falls within the range of 15 – 20% reported in previous literature. Lipton contained a significantly (P < 0.05) higher %TPC than each of the other brands. Furthermore, it was established that these brands contain between 0.8 – 0.9% caffeine and approximately 1% EGCG by dry weight. Although the literature indicated that the caffeine content was only slightly lower that the reported range of 1.1 – 2.0%, it was noted that the observed EGCG content was much lower than the expected value of 3%. Furthermore, EGCG exhibited the highest NO-scavenging activity at concentrations of 100 µM in an acellular system, and 200 µM in RAW 264.7 cells. Minimal cytotoxicity towards the cells was observed at a concentration of 200 µM, although previous literature showed that such a high concentration of EGCG was toxic to human astrocytoma U373MG cells. Moreover, TLR4 signalling assays revealed that 10 µM EGCG significantly down-regulated NF-κB expression in LPS-stimulated HEK 293 cells, however, EGCG had no significant effect on the expression levels of TNFα in LPS-stimulated iBMDMs. Hence, it was concluded that further study was required before a sound conclusion can be drawn regarding the role of EGCG in inflammation since the evidence was inconclusive.