The Effect of Adding Different Tea Powders on Bioactive Compounds, Antioxidant and Anti-Inflammatory Activities of Coconut Latte in THP-1 Macrophages via NF-κB Pathway

1. INTRODUCTION

Chronic low-grade inflammation, one of the hallmarks of chronic disease, is the body’s defensive response to stimuli, and it is closely associated with pro-inflammatory mediators, and cytokines. These inflammatory genes are all controlled at the transcriptional level by signalling pathways, such as the Nuclear factor kappa-B (NF-κB) pathway.^[1] NF-κB is a crucial transcription factor, binding to inhibitor kappa B (I-κB) in the cytoplasmic matrix in an inactive form. When the toll-like receptor 4 is stimulated by LPS, inflammatory cascade reactions are triggered, and the NF-κB pathway is activated, leading to phosphorylation, ubiquitination, and degradation of I-κBα. NF-κB is released from I-κB complex, and translocated into the nucleus, therefore regulating the transcription of inflammation-related genes.^[2] Foods such as coffee and tea, rich in bioactive compounds, are regarded as complementary therapies for managing chronic diseases.

Studies have shown that consuming 1 to 6 cups of coffee per day significantly decreases the risk of cardiovascular disease in humans.^[3] The health benefits of coffee are mainly attributed to its richness in polyphenols. Chlorogenic acid has been proven to exhibit anti-inflammatory activity by inhibiting the expression of phosphorylated p38 protein, the transcription and secretion of TNF-α, and upregulating mRNA levels of heme oxygenase-1 (HO-1) in LPS-stimulated RAW 264.7 macrophages,^[4] and reversing TNF-α-induced activation of NF-κB pathway in M03-13 cells.^[5] Ferulic acid reduced oxidative stress and inflammation in diabetic rats by regulating signalling pathways of NF-κB and nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1.^[6] Quercetin has been reported to attenuate LPS-stimulated inflammatory responses by inhibiting mitogen-activated protein kinase (MAPK) and NF-κB pathways’ activation in RAW 264.7 macrophages.^[7]

Black, oolong, and green tea, which are rich in bioactive compounds, have been well reported for their anti-inflammatory activities. Black tea extracts inhibited NF-κB signalling activation and significantly alleviated symptoms stimulated by LPS of inflammatory bowel disease in mice.^[8] Green tea significantly reduced arthritis inflammation in vitro and in vivo by inhibiting MAPK and NF-κB signalling pathways.^[9] Oolong tea extracts significantly reduced LPS-induced TNF-α levels in mouse paw oedema.^[10] It is worth noting that green tea extracts showed a better inhibition of joint swelling and pro-inflammatory cytokines than black tea extracts in adjuvant arthritis rat.^[11] The difference in anti-inflammatory activity of different types of tea can be due to their diversity of polyphenols,^[12] such as epicatechin gallate and epigallocatechin gallate.^[13,14]

However, existing research has focused on the bioactivity of tea or coffee as a single matrix. The potential synergistic effects of combining phytochemicals in tea and coffee have not been systematically investigated. Research on how tea and coffee complexes modulate inflammatory signalling pathways is limited. Therefore, it remains unclear whether adding tea powder to coffee products can provide functional benefits beyond those of a single component. Based on this, this study aimed to evaluate the effects of different types of tea on bioactive compounds, antioxidant, and anti-inflammatory activities of coconut latte. The extracts of tea flavoured coconut lattes (CLs) were measured for physicochemical properties, identified, and quantified for bio-compounds. The THP-1 macrophage model was employed to evaluate the anti-inflammatory activity of CLs by determining the NF-κB pathway proteins, mRNA, and protein levels of inflammatory mediators and cytokines.

2. MATERIALS AND METHODS

3. RESULTS

3.1. Effects of tea on components and antioxidant capacity of NCL

Compared with NCL, the addition of three tea powders significantly (p<0.05) increased both the total contents of phenols and flavonoids, and GCL and BCL showed greater increases than OCL [Figures 1a and b].

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Figure 1:

The total phenolic, flavonoid contents, and antioxidant activity of CLs. (a): Total phenol contents. (b): Total flavonoid contents. DPPH• (c) and ABTS+ (e) Radical scavenging ability, ferric reducing ability of plasma (g). IC₅₀ values for DPPH• radical scavenging activity (d), ABTS+ radical scavenging activity (f), and ferric reducing ability (h). NCL: Coconut latte, BCL: Coconut latte with black tea, GCL: Coconut latte with green tea, OCL: Coconut latte with oolong tea. The results were expressed as means ± SD (n = 3). Different letters represent a significance (p < 0.05).

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DPPH, ABTS radical scavenging abilities, and FRAP of all groups significantly increased with increasing concentration from 0 to 1mg/mL. Although the DPPH radical scavenging ability of the GCL did not change from 0.6 to 1 mg/mL, GCL still exhibited the strongest DPPH radical scavenging ability within this concentration range. At concentrations of 0-1 mg/mL, the antioxidant activities (DPPH, ABTS radical scavenging capacity, and FRAP) of each group were GCL > BCL > OCL > NCL, respectively. The IC₅₀ values for DPPH radical scavenging ability were 224.48, 365.65, 531.60, and 636.09 μg/mL, respectively [Figures 1c and d, p < 0.05]. The IC₅₀ values for ABTS⁺ free radical scavenging ability were 327.54, 421.25, 620.14, and 743.05 μg/mL, respectively [Figures 1e and f, p < 0.05]. The IC₅₀ values for FRAP were 27.62, 34.59, 51.71, and 58.45 μg/mL, respectively [Figures 1g and h, p < 0.05]. These results indicated that tea powders, especially green tea, significantly increase NCL’s antioxidant activity. Besides, the antioxidant activity trend of CLs were generally consistent with their total phenolic and flavonoid contents.

3.2. The cytotoxicity of CLs on THP-1 macrophages

The cell viability of THP-1 macrophages treated with four CLs are shown in Figures 2(a-d). 0.2 mg/mL of four CLs was non-toxic and used for further cell study.

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Figure 2:

Effects of tea types on cytotoxicity and inflammatory gene expression in NCL. Cellular viability of NCL (a), BCL (b), GCL (c), OCL (d). The relative mRNA expression of COX2 (e), IL1B (f), IL13(g), IL8 (h), IL10 (i), IL6 (j), HO1 (k), NRF2 (l). NCL: Coconut latte, BCL: Coconut latte with black tea, GCL: Coconut latte with green tea, OCL: Coconut latte with oolong tea. The results were expressed as means ± SD (n = 3). Different letters represent a significance (p < 0.05).

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3.4. The IL-1β secretion and NF-κB signalling activation

All four CLs significantly reduced IL-1β secretion, without effects by tea or tea type [Figure 3a], which were consistent with those results from mRNA expression of IL1B.

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Figure 3:

Effects of tea types on the expression of the NF-κB pathway and TNF-αin coconut latte. (a): IL-1β levels at CLs. Protein expression levels from CLs (b), I-κBα (c) TNF-α (d), pNF-κB (e), NF-κB (f). NCL: coconut latte, BCL: coconut latte with black tea extract, GCL: coconut latte with green tea extract, OCL: coconut latte with oolong tea extract. The results were expressed as means ± SD (n = 3). Different letters represent a significance (p < 0.05).

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LPS stimulation significantly (p < 0.05) enhanced the phosphorylation of NF-κB, TNF-α, and IκBα [Figures 3c-f] as compared to control. Importantly, all CLs (except NCL) markedly (p<0.05) suppressed NF-κB phosphorylation and TNF-α expression. Moreover, CLs significantly upregulated I-κBα expression, with a more pronounced effect observed in tea-supplemented samples, and GCL showed the highest increase in I-κBα expression.

3.5. The metabolite profiles in CLs

Non-targeted metabolomics revealed distinct metabolite profiles among the CLs [Figure 4a]. A clear separation was revealed among four groups by principal component analysis (PCA) [Figure 4b], and a total of 3150 metabolites of 20 categories were detected [Figure 4c]. Phytochemicals have attracted substantial attention due to their established roles in antioxidant and anti-inflammatory activities.^[22] Compounds 44, 53, and 64 differential metabolites were identified in the comparisons between NCL and BCL, OCL, and GCL, respectively [Figure 4d-f]. A total of 31 metabolites were shared across all three comparisons. A total of 14 metabolites were notably enriched in GCL. Four metabolites were enriched in OCL, while ten metabolites were enriched in NCL [Figure 4h].

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Figure 4:

Overview of metabolite distribution and differential analysis in CLs. Heatmap of all detected metabolites (a), PCA of sample distribution (b), Pie chart of metabolite classification (c), Volcano plots of differential metabolites of NCL vs BCL (d), NCL vs OCL (e) and NCL vs OCL (f), Venn diagram (g) and heatmap (h) of 31 shared differential metabolites. NCL: coconut latte, BCL: Coconut latte with black tea, GCL: Coconut latte with green tea, OCL: Coconut latte with oolong tea. The results were expressed as means ± SD (n = 3). Different letters represent a significance (p < 0.05).

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3.7. Correlation analysis of polyphenols and inflammation

The polyphenol profiles of four CLs were subjected to a PCA plot [Figures 5a and b]. The total contribution rate in the PCA plot was 84.9%, in which PC1 and PC2 explained the variance of 64.4% and 20.5%, respectively. BCL and GCL were positioned on the right side, characterized by main polyphenols, antioxidant mediators (NRF2, HO1, and I-κBα). BCL and GCL exhibited negative correlations with antioxidant IC₅₀ values, pro-inflammatory cytokines, and mediators, reflecting large negative loadings on PC1. In contrast, NCL and OCL are located on the chart’s left side, representing samples with minor polyphenols and showing strong negative loadings on PC1. NCL and OCL were correlated positively with antioxidant IC₅₀ values, pro-inflammatory mediators, and cytokines.

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Figure 5:

Principal component analysis (a, b) and Spearman correlation analysis (c) of CLs and indicators. NCL: Coconut latte, BCL: Coconut latte with black tea, GCL: Coconut latte with green tea, OCL: Coconut latte with oolong tea.

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Spearman correlation analysis was performed for polyphenols and inflammatory mediators. Most polyphenols exhibited significant negative correlations with pro-inflammatory mediators (IL6, IL8, IL10, IL13, IL1B, and COX2), antioxidant IC₅₀ values, pNF-κB and TNF-α, while most polyphenols positively correlated with NRF2, HO1, and I-κBα (p<0.01, |r|>0.5) [Figure 5c]. These results indicated that most polyphenols exhibited significant anti-inflammatory effects, consistent with enhanced antioxidant capacities of BCL and GCL rather than NCL and OCL.

4. DISCUSSION

In this study, the total phenol and flavonoid contents in CLs showed similar trends, with the highest contents in GCL and BCL, followed by OCL and NCL [Figures 1a and b]. The antioxidant capacity results [Figures 1c-f] showed that teas significantly enhanced the free radical scavenging ability of NCL, which results from the polyphenols in tea with strong free radical scavenging ability.^[23] Moreover, the total phenol and flavonoid contents were significantly negatively correlated with their antioxidant capacity. It has been demonstrated that green tea was generally better than oolong and black tea in scavenging free radicals and enhancing antioxidant enzyme activity, which is closely related to green tea’s lower degree of fermentation and retention of more free phenolic structures.^[24]

IL1B, IL13, IL8, IL10, and IL6 are important inflammatory cytokines produced by activated macrophages and play a key role in chronic inflammatory diseases.^[25] CLs (except NCL) significantly inhibited LPS-induced IL1B, IL13, IL8, IL10, and IL6 gene expression [Figures 2f-j]. These results were consistent with a previous study that extracts of green, black, and oolong tea showed significant anti-inflammatory activity in vitro by effectively inhibiting the production of inflammatory cytokines production.^[26] IL13, IL8, and IL10 were inhibited to a similar extent by BCL, OCL, and GCL, indicating consistent responses to tea polyphenols. The correlation results showed that IL13, IL8, and IL10 were negatively correlated with a variety of polyphenols [Figure 5c]. The inhibition is more dependent on the synergistic effect of polyphenols rather than the difference of a single component, which can explain why BCL, OCL, and GCL showed similar inhibitory effects on these three cytokines. A study showed that different polyphenols synergistically produce pro-inflammatory cytokines in LPS-induced RAW264.7 cells better than a single compound.^[27] The inhibitory effects on IL1B and IL6 were obviously dependent on tea type, with the greatest inhibition by GCL. The quantitative results of polyphenols showed that the content of catechins (epigallocatechin, catechin, epicatechin gallate, epigallocatechin gallate) in GCL was the highest (1821.6 ± 21.5μM, Tables 1 and 2), which has been shown to have been showed anti-inflammatory activity. Epigallocatechin gallate and epicatechin gallate significantly reduced the expression of IL-8 and IL-6 in LPS-induced dental pulp cells.^[28] Catechin inhibited significantly TNF-α, IL-6, and IL-1β levels in streptozotocin-induced diabetic retinopathy rats.^[29] This further confirmed higher content of catechins for higher anti-inflammatory activity of GCL.

COX-2 was a key enzyme in the inflammatory response that was induced by LPS and other stimuli. CLs significantly inhibited LPS-induced COX2 transcription [Figure 2e]. Besides, COX2 mRNA levels were negatively correlated to major polyphenols [10 out of 16, Figure 5c], including phenolic acids, flavonoids and catechins, which had been shown to inhibit COX-2 expression by inhibiting NF-κB and MAPK signalling pathways’ activation.^[30] Chlorogenic acid inhibited the expression of COX-2 and NF-κB phosphorylation in RAW264.7 cells treated with LPS.^[31] The addition of tea (except OCL) had a better inhibitory effect on COX2 gene expression, due to a significant increase of total phenols and flavonoids in NCL [Tables 1 and 2, Figure 1], especially catechins. It has been reported that catechins not only downregulated COX2 mRNA expression in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner,^[32] but also downregulated COX-2 expression in human dental pulp cells.^[33] GCL and BCL inhibited COX2 transcription better than OCL due to higher content of 8 major polyphenols (> 50 μM in GCL) in BCL and GCL [Tables 1 and 2].

Nrf2 is a key transcription factor regulating oxidative stress and inflammatory responses. Under oxidative stress, Nrf2 will translocate into the nucleus and activate HO-1 expression, which is a rate-limiting enzyme of heme degradation to alleviate oxidation damage,^[34] thereby protecting from oxidative stress [Figure 6]. CLs significantly upregulated the expression of NRF2 (except NCL) and HO1 [Figures 2l and k]. Ferulic acid in coffee has been shown to provoke Nrf2 nuclear translocation and upregulate mRNA and protein expressions of HO-1 in human lens epithelial cells.^[35] In this study, the addition of tea further upregulated NRF2 (except in BCL) and HO1 mRNA levels. This difference may be due to the higher contents of p-coumaric acid (3.814±0.394, 10.186±0.108) and rutin (33.76±0.095, 79.451±0.487) in OCL and GCL (compared to NCL and BCL). It was supported by a previous study that p-coumaric acid significantly increased HO-1 and Nrf2 expressions in the colonic tissue of mice with colitis. Rutin significantly upregulated the expression of Nrf2 and HO-1 and inhibited activation of the NF-κB pathway in liver injury,^[36], showing its great anti-inflammatory activity.

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Figure 6:

Molecular mechanisms of CLs in attenuating inflammatory responses in THP-1 macrophages. ROS: Reactive oxygen species, ETGE: Glutamate–threonine–glycine–glutamate motif, DLG: Aspartate–leucine–glycine motif, ARE: Antioxidant response element, STATs: Signal transducers and activators of transcription, JAK1: Janus kinase 1, TYK2: Tyrosine kinase 2, TRIF: TIR-domain-containing adapter-inducing interferon-β, TLR4: Toll-like receptor 4.

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CLs significantly inhibited the protein expression of TNF-α (except NCL) and NF-κB phosphorylation induced by LPS [Figure 3]. It was found that gallic acid in coffee increased the level of cytosolic I-κBα and suppressed NF-κB activation induced by LPS in A549 lung cancer cells.^[37] The inhibitory effect was greater after adding tea powder, which can be related to the catechins rich in tea. A previous study showed that epigallocatechin gallate significantly decreased the phosphorylation of NF-κB in nude mice.^[38] Epicatechin gallate prevented inflammatory response in hypoxia-activated microglia and cerebral oedema by inhibiting NF-κB signalling pathway activation.^[39] GCL had the best effect on inhibition of NF-κB phosphorylation, followed by OCL and BCL [Figure 3]. This can be related to the significantly increased contents of rutin and quercetin, which showed a significant negative correlation with pNF-κB. Rutin has been shown to have a significant effect on inhibiting Nrf2/HO-1 and NF-κB signalling pathways in the arthritic rats.^[40] Quercetin has been displayed to inhibit the nuclear translocation of NF-κB in HepG2, peripheral blood mononuclear cells (PBMCs), RAW264.7, and other cells.^[41] Moreover, quercetin has also been found to inhibit oxidative stress, NF-κB activation, and iNOS overexpression in the liver of streptozotocin-diabetic rats.^[42]

The PCA plot showed that NCL was negatively correlated with most polyphenols, while BCL, OCL, and GCL had more polyphenols with potential anti-inflammatory and antioxidant properties, and their distribution was negatively correlated with pro-inflammatory cytokines [Figure 5]. This was consistent with the PCA distribution of all substances in the metabolomics results that BCL, OCL, and GCL were on the positive half axis of the PCA plot, while NCL was on the negative half axis [Figure 4b]. These results were consistent with our previous studies, which showed that the content of polyphenols in camellia and papaya oils is negatively correlated with pro-inflammatory cytokines.^[18,43] It can be supported by another study that extracts from different parts of echinacea purpurea with high polyphenol contents (rich in flavonoids and phenolic acids) significantly negatively correlated to pro-inflammatory cytokines (such as IL-6 and IL-8), indicating that polyphenols played a significant role in inhibiting inflammation.^[44] BCL and GCL were more related to more polyphenols [Figure 5] than OCL, resulting in their higher anti-inflammatory activities. These results were in agreement with a previous study that polyphenols in green or black tea significantly inhibited the expression of monocyte chemoattractant protein-1 in micethan oolong tea.^[45]

5. CONCLUSION

The total contents of phenols and flavonoids of NCL were increased by tea in the order of GCL=BCL>OCL, and antioxidant activities of NCL were significantly increased in the order of GCL>BCL>OCL. NCL showed few anti-inflammatory activities by decreasing (increasing) the transcription of COX2, IL13, IL8, and IL10 (HO1) and IL-1β secretion, which were significantly increased by tea, especially GCL via greater inhibition of all above genes (except IL13), and IL1B, IL6, NRF2 and I-κBα expression, the inhibition of pNF-κB and TNF-α expression. Sixteen polyphenols were identified and quantified in four CLs, and tea increased significantly (greatest in GCL) in their contents. Spearman correlation analysis suggested that polyphenols in CLs played an important role in both antioxidant and anti-inflammatory effects. These results indicated that the tea, especially GCL, increases the functionality of the coconut latte, which is helpful for developing functional coffee.