Coffee can be described as the life-saving artifact of contemporary workers, and the "coffee culture" that comes with it is full of every moment of life - whether at home, in the office, or in various social occasions, we can see coffee. Coffee, gradually associated with fashion, modern life, work and leisure.
At the same time, there is a growing number of scientific research related to coffee. Previous studies have shown that drinking coffee not only does not damage the heart, but also greatly reduces the incidence of cardiovascular disease and prolongs life.
Recently, a study from the University of Copenhagen in Denmark showed that adding milk to coffee can enhance the anti-inflammatory effect of coffee and double the anti-inflammatory effect of immune cells. The study, titled "Phenolic Acid-Amino Acid Adducts ExertDistinct Immunomodulatory Effects in Macrophages Compared to Parent PhenolicAcids," was published in the Journal of Agricultural and Food Chemistry.
1. Research background
When bacteria, viruses, or other foreign substances enter the body, our immune system protects us by deploying white blood cells and chemicals, a response commonly known as inflammation.
Caffeic acid (CA) and chlorogenic acid (CGA), phenolic acids commonly found in plant-derived foods and beverages, and their adducts corresponding to cysteine (Cys) have been detected in coffee-containing beverages. Although CA and CGA have antioxidant and anti-inflammatory activities, the immunomodulatory activity of Cys adducts (CA-Cys and CGA-Cys) is unknown. Therefore, the scientists synthesized adducts, studied their immunomodulatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 cells, and compared them with the activity of parent phenolic acids.
The RAW 264.7 cell line was used because numerous studies have shown that the biological activity of phenolic compounds is similar in RAW 264.7 cells and primary human immune cells. First, the researchers investigated the effects of CA-Cys and CGA-Cys on cell transcriptome responses to explore whether CA-Cys and CGA-Cys can regulate cell signaling at the transcriptional level. Next, the anti-inflammatory activity of CA-Cys, CGA-Cys with CA and CGA was compared by studying tumor necrosis factor-α (TNF-α) in PGE2 production, interleukin-6 (IL-6), and LPS-activated cells.
2. Research methods
1. Synthesis and purification of CA-Cys and CGA-Cys adducts
Quinones for CA and CGA are produced using periodate resins. Stir the reaction mixture with a molar ratio of 1:1 between CA (or CGA) and Cys at room temperature and stir in phosphate buffer (pH 6.5) for 24 h. Thereafter, purification was performed using a semi-preparative high performance liquid chromatography (HPLC) system with the Eurospher 100-5 C18 Column (300 × 8 mm diameter, 5 μm particles). Dry and dissolve the mixed fraction in water and store at -20 °C. CA–Cys and CGA–Cys stock solutions were available at concentrations of 204 μg/mL (681.6 μM) and 210 μg/mL (443.6 μM), respectively. The purity of CA-Cys and CGA-Cys (verified based on HPLC chromatograms) was 99.8% and 99.7%, respectively.
2. Cell culture
RAW 264.7 macrophages (ATCC-TIB-71) were cultured in DMEM and contained 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Keep the cell culture at 37 °C in an environment containing 5% CO2.
3. Cell stimulation
264.7 cells (1× 105 cells per ml) divided into 24-well plates adhering to the wall for 2 h; Then dissolve 10, 50, or 100 μM of CA, CA–CYS, CCA, or CGA-Cys in Milli-Q water and add serum-free DMEM for 30 min pre-inhibition; Then add 500 ng/mL LPS to stimulate the cells for 6 and 24 h. After 264 h of stimulation with the corresponding phenolic acid or adduct, the mRNA of RAW 7.6 cells is extracted according to the RNeasy kit. After 24 h of culture, collect the supernatant from the cells and store at -20 °C until further analysis. The samples are named CA, CA-CYS, CCA, or CGA-Cys, representing LPS-activated cells exposed to CA, CA-CYS, CCA, or CGA-Cys, respectively.
4. Cell viability test
Assess cell viability using the PrestoBlue assay.
5. The secretion levels of PEG2, IL-6 and TNF-α were determined by ELISA.
6. RNA sequencing
RNA extracted from cultured cells is used for RNA sequencing, and sequence data is available at GEO.
7. PCR to check the level of RNA transcription.
8. Vacuum measurement
Cells are collected after 24 h of stimulation and washed with warm (37 °C) serum-free DMEM. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) is used to stain cells at 30 °C for 37 min. DCFH-DA is then removed by washing three times with warm (37 °C) serum-free DMEM before measuring fluorescence intensity using flow cytometry.
3. Research results
In the study, the researchers applied artificial inflammation to immune cells in order to explore the anti-inflammatory effect of polyphenols binding to proteins. Among them, some cells received different doses of polyphenols that reacted with amino acids, while others received only the same dose of polyphenols; The control group received nothing. The researchers observed that immune cells treated with a combination of polyphenols and amino acids were twice as effective at fighting inflammation as cells that added polyphenols alone.
Intracellular ROS in RAW 7.100 macrophages stimulated with LPS cultured with 264 μM MCA, CA-CYS, CCA, CCA–CYS.
In fact, coffee beans are rich in polyphenols, while milk is rich in protein. Therefore, in this study, the researchers found that in coffee with milk, there is also a reaction between polyphenols and proteins.
CA, CA-Cys, CGA, and CGA-Cys inhibit pro-inflammatory cytokine secretion in RAW 264.7 cells.
III. Summary
The reason why the addition of Cys to phenolic acids caused such a drastic change in macrophage activity requires further study. It has been shown that polyphenols are internalized by innate immune cells, which are then transported to lysosomes. It can therefore be speculated that the addition of Cys regulates this internalization and/or trafficking process, but further experiments are needed to verify this. In addition, some of the inflammatory molecules observed by the researchers from transcriptomic analysis were inhibited, while others were enhanced, suggesting a complex regulation of the anti-inflammatory effects of CA and CGA. The mechanisms by which these phenolic acids regulate the inflammatory response of macrophages may be multifaceted, involving multiple pathways, such as inhibition of NF-κB signaling and autophagy.
At present, the results of this study provide the first set of unique data on the immunomodulatory activity of phenolic acid-amino acid adducts, indicating that the biological activity of these well-studied phenolic acids is substantially altered by the formation of Cys adducts; This is important considering that such adducts can be present in food products. The results of this study can be used as an important reference for the application of phenolic compounds and amino acid-forming adducts in future functional foods or pharmaceuticals to regulate metabolic, neurological, or immune-related diseases.
It can be seen that coffee and milk have anti-inflammatory miracles! But drink coffee in moderation, don't be greedy for cups~
Source: https://pubs.acs.org/doi/full/10.1021/acs.jafc.2c06658
Written by Catherine