7  Role of polyphenols in counteracting inflammatory phenomena
Literature data demonstrates that there are approximately 8000 different polyphenolic structures, classified into more than ten classes depending on structural characteristics. Regardless of their differences in chemical behaviour, they are all characterized by having an aromatic ring carrying one or more hydroxyl groups (Bravo, 1998; Del Rio et al., 2013). The polyphenolic molecules originating from diet are involved as key components in counteracting inflammation, mitigating oxidative stress and protecting endogenous compounds from oxidative lesions, regulating the metabolism and promoting a protective phenotype, in improving the endothelial function as well as the platelet function, among several other beneficial actions (Kaliora et al., 2006; Morita et al., 2017; Nitulescu et al., 2017, 2019).
Polyphenols are key dietary components extensively studied for their involvement in preventing CVD, comorbidities of metabolic syndrome, malignancies, etc, all these diseases having an important etiological inflammatory component. For example, a central stage in the expansion of atherosclerosis is dyslipidemia, which together with increased ROS formation leads to LDL oxidation, which was correlated with the activation of pro-inflammatory pathways in the vascular wall. Likewise, it is well established that ROS and pro-inflammatory molecules generate a microenvironment predisposing to endothelial dysfunction. The increased expression of adhesion molecules (VCAM-1, ICAM-1, E-selectin) favours the adherence of monocytes to the endothelium and their infiltration within the vascular wall. As a result, of monocyte diapedesis pro-inflammatory molecules are released (TNF-α, IFNγ, MCP-1), NFκB is up-regulated, interleukins (IL-6 and IL-8) and metalloproteinases (MMPs) are synthesized (Gradinaru et al., 2017, 2018; Hansson and Libby, 2006; Libby, 2012; Libby et al., 2014; Ungurianu et al., 2019).

7.1  Cellular effects
Polyphenols interfere with the inflammatory process in multiple pathways. For example, they alter the enzymatic processes involved in the proliferation and activation of B- and T-cells, as key components of the inflammatory pathway (by inhibiting tyrosine and serine-threonine protein kinases). Likewise, polyphenols blunt the synthesis of pro-inflammatory mediators such as cytokines, chemokines (IL-8, IL-6, TNF-α, VCAM-1 and ICAM-1), and angiogenic factors, NF-кB or iNOS. Further, an inhibitory effect on several pro-inflammatory enzymes was reported, such as COX-2, MAPK or protein kinase-C (PKC) (Alvarez-Suarez et al., 2017; Gasparrini et al., 2017; Hussain et al., 2016). Thus, studies performed on macrophages proved that resveratrol inhibited the IFN-γ-induced NO production and down-regulated the IFN-γ inducible genes. In this respect, resveratrol decreased STAT1 activation (an important transcription factor for IFN-γ-induced genes) and hindered JAK-2 activation (Chung et al., 2011).
Experimental studies arguing the involvement of polyphenols in mitigating inflammation are either performed on cell line models, animal models or have direct clinical implications. In a study exposing human endothelial cells to the effect of Negroamaro and Primitivo extracts or pure compounds - stilbenes (trans-resveratrol, trans-piceid), hydroxycinnamic acids (p-coumaric, caffeic, and caftaric acids) flavonols (kaempferol, quercetin, myricetin) – before stimulation with lipopolysaccharide (LPS), an inhibition in the expression of adhesion molecules was demonstrated. The anti-inflammatory effect was sustained by a reduction of VCAM-1, ICAM-1, E-selectin, MCP-1 and macrophage colony-stimulating factor (M-CSF), ROS intracellular levels with a correlated attenuation of NF-κB and AP-1 activation. Trans-resveratrol also significantly reduced the endothelial expression and release of M-CSF. All tested compounds reduced the adhesion of monocytes to stimulated endothelial cells, this being a key element in the development of atherosclerosis (Calabriso et al., 2016). The treatment of CCD-18Co myofibroblasts cells with red wine extract reduced mRNA levels of ICAM-1, VCAM-1, NF-κB, and PECAM-1, induced by LPS the anti-inflammatory mechanism being dependent on miR-126 (Angel-Morales et al., 2012).
Another study tested the effects induced by a mixture of four main catechins found in green tea (epicatechin – EC, epigallocatechin – EGC, epigallocatechin gallate – EGCG, epicatechin gallate – GCG), as well as each one alone, on neutrophils isolated from healthy subjects. The catechin compounds induced anti-inflammatory effect (reduction of IL-1β and IL-6, TNF-α, HOCl synthesis and mieloperoxisase), together with the stimulation of antioxidant enzyme activities and Nrf2, along calcium release as well as increased phagocytic capacity, thus proving anti-inflammatory and immunomodulatory actions (Marinovic et al., 2015). In a separate study, EGCG reduced ICAM-1, NF-ĸB and IĸB expressions and reduced ROS levels upon TNF-α-induced inflammation in human retinal pigment epithelial cells. The results suggest the possible therapeutic involvement of EGCG in blocking TNF-α-mediated eye inflammation (Thichanpiang and Wongprasert, 2015). Moreover, in a study evaluating the effect of epicatechin and catechin on arachidonic acid-activated platelets and, respectively, on platelet-HUVEC interaction, the tested compounds induced a reduction of sICAM1, sVCAM1, and sE-selectin levels, as well as an increase of NO bioavailability, proving an anti-inflammatory effect and the ability to counteract endothelial dysfunction, but only when platelets were harvested from peripheral artery disease patients and not from healthy subjects (Carnevale et al., 2014).
Ellagic acid (EA) was tested for its ability to inhibit MIF-induced chemotactic migration of PBMCs, showing a failure to inhibit this response when PBMCs were stimulated with MIP-1α, but high specificity for MIF-induced effect (Sarkar et al., 2015). Chlorogenic acid exerted a dose depended anti-inflammatory effect showed by the reduction of IL-1β, TNF-α and IL-6 levels as well as the inhibition of NO synthesis and expression of COX-1 and iNOS in LPS-stimulated murine macrophages and microglial cells (Hwang et al., 2014).
An in vitro experiment evaluated the results induced by various flavonoids on the arachidonic acid release from rat neutrophils; kaempferol, luteolin, quercetin and amentoflavone reduced the inflammatory response and inhibited the activity of β-glucuronidase and lysozyme (Tordera et al., 1994). Furthermore, the positive outcome of Brassica oleracea extracts (water and methanol respectively) for CVD risk were examined; for this purpose, HUVECs were exposed to the extracts (24 h) and then to TNF-α stimulation. Exposure to the extracts from Brassica oleracea significantly reduced the TNF-α induced expression of E-selectin, ICAM-1 and VCAM-1 (Kuntz and Kunz, 2014). Another study demonstrated the capacity of extracts from Mango (Mangifera indica L.) to counteract TNF-α effects on HUVECs, through the inhibition of IL-6, IL-8, COX-2 and ICAM-1, while restoring the expression of eNOS usually down-regulated by TNF-α (Mura et al., 2015). Interestingly, in an animal model, for example, supplementation of standard chow with cacao phenolic compounds in apolipoprotein E-deficient mice, lead to an anti-inflammatory effect justified by a reduction of VCAM-1 and ICAM-1 expressions, a decrease of oxidative stress markers and a reduction of cholesterol accumulation in plaque compared to control (Natsume and Baba, 2014). Likewise, propolis (green, red or brown) treatment induced a reduction of MCP-1, VCAM, PECAM, FGF, PDGF, VEGF, MMP-9 and upregulated tissue inhibitor of metalloproteinases 1 (TIMP-1) in initial atherosclerotic lesions in LDL receptor gene knockout mice fed a diet rich in cholesterol for inducing atherosclerotic lesions; only red propolis upregulated heme oxygenase 1 (HO-1) and TIMP-1 in advanced atherosclerotic lesions (Daleprane et al., 2012).

7.2  Anti-inflammatory potential of polyphenols in humans
The anti-inflammatory effects induced by polyphenols in human studies with dietary interventions and weight related outcomes are heterogeneous, as summarized from recent literature (Table 7 ). Thus, in a clinical study with double-blind, randomized, cross-over design, the effect of a commercially available polyphenols rich extract was tested, the study including patients having more than two metabolic risk factors. Results showed a reduction of MCP-1 and MIF, but not of other markers of CVD risk (CRP, IL-6, HDL, adiponectin and oxidized LDL) (Broekhuizen et al., 2011). In a separate clinical study with cross-over design, overweight patients consumed a high-carbohydrate, moderate-fat meal supplemented with strawberry/placebo juice. The anthocyanin supplementation blunted the postprandial inflammatory response (reduced CRP level) induced by the high fat diet and reduced the postprandial insulin response (Edirisinghe et al., 2011). Furthermore, a double-blind study focused on hypercholesterolemia subjects, receiving a purified anthocyanin mixture, resulted in a reduction of their CRP, sVCAM-1 and IL-1β levels. Similar results were obtained for HepG2 and porcine iliac artery endothelial cells exposed to the same purified anthocyanin mixture (reduction of LPS-induced VCAM-1 secretion) (Zhu et al., 2013). Patients with early atherosclerosis, received simple or EGCG-enriched olive oil. Independent of EGCG addition, olive oil reduced inflammatory markers and endothelial dysfunction (sICAM, white blood cells, monocytes, lymphocytes and platelets) (Widmer et al., 2013).
Table 7 Clinical data regarding the efficacy of polyphenols on inflammatory phenomena.
Design Population Type of intervention Inflammatory outcome Reference
Randomised, double-blind, cross-over study 34 subjects with two or more metabolic risk factors 4-week intervention: 500 mg polyphenols/day vs. placebo ↓MCP-1 and MIF, unchanged levels of HDL, adiponectin, CRP Broekhuizen et al. (2011)
Cross-over study 24 overweight adults high-carbohydrate, moderate-fat meal together with a strawberry beverage (SB) or placebo SB:↓ CRP and postprandial insulin Edirisinghe et al. (2011)
Randomised, double-blind 150 hypercholesterolemic subjects 24-week intervention: 640 mg purified Anthocyanin/day ↓ CRP, sVCAM-1 and IL-1β Zhu et al. (2013)
Randomised, double-blind 52 patients with early atherosclerosis, 4-month intervention: 30 mL olive oil or 30 ml of EGCG-supplemented olive oil ↓ sICAM, white blood cells, monocytes, lymphocytes and platelets Widmer et al. (2013)
Single-centre, randomised, two-arm, double-blinded, placebo-controlled study 36 pre-hypertension patients, randomized to control or treatment groups 6-week intervention: grape seed extract, 150 mgx2/day ↓BP (SBP, DBP)↑ fasting insulin and insulin sensitivityNo change of sICAM-1 Park et al. (2016)
Single blinded crossover trial 38 patients with untreated mild hypertension 8-week intervention: 300 mL/day cold-pressed 100% chokeberry juice and 3 g/day chokeberry powder ↓IL-10 and TNF-α Loo et al. (2016)
Randomized, double-blind, placebo-controlled clinical trial 50 hyperlipidemic patients 4-week intervention: V. arctostaphylos fruit extract x2/day no effects on HDL and CRP↓ total cholesterol, LDL-C, TG, MDA Soltani et al. (2014)
Placebo controlled study 20 study subjects and 19 placebo 4-week intervention: 361 mg polyphenols and 120 mg vitamin C x2/day No significant effects on apolipoproteins, adiponectin, CRP, ICAM-1, E-Selectin or t-PA Mullan et al. (2016)
Placebo controlled study 16 trained cyclists 7-day intervention: 30 mL of Montmorency tart cherry concentrate x 2/day ↓lipid hydroperoxides, IL-6 and hsCRP Bell et al. (2014)
Double-blind crossover study 49 healthy male subjects with APOE genotype 8-week intervention: 150 mg/day quercetin or placebo (3 phases, three-week washout periods) ↓ waist circumference and postprandial SBP, moderately increased levels of TNFα Pfeuffer et al. (2013)
Randomised, double-blind, placebo-controlled, cross-over trial 18 healthy volunteers One-tine administration of 51 mg of oleuropeine ↓of IL8 production Lockyer et al. (2015)
Cross-sectional study 1997 females Frequency questionnaires assessment of total intake of flavonoids Higher anthocyanin and flavone intake were associated with significantly lower peripheral insulin resistanceHigher anthocyanin intake was also associated with lower CRP concentrationsHigher anthocyanin-rich intake was associated with lower insulin and inflammation Jennings et al. (2014)
Randomized, controlled dietary study 27 subjects with metabolic syndrome 400 g fresh bilberries/days vs. control diet ↓ CRP, IL-6, IL-12, Kolehmainen et al. (2012)
The effects of grape seed extract on pre-hypertension patients were tested. Results of the study in question, showed a reduction of blood pressure (both systolic and diastolic), with improved fasting insulin and insulin sensitivity after 6 weeks of supplementation, but no significant change in sICAM-1 plasma concentration (Park et al., 2016). Furthermore, in a single blinded crossover trial including patients with untreated mild hypertension, cold-pressed chokeberry juice and dried chokeberry powder induced a reduction of IL-10 and TNF-α levels (Loo et al., 2016).
On the other hand, there are studies that do not confirm the positive effects of natural compounds on inflammatory markers regarding cardiovascular risk, especially on CRP, even if they prove to yield other beneficial outcomes (for example, on LDL). Thus, Soltani et al. examined the effects of a Vaccinium arctostaphylosfruit extract on hyperlipidemic adult patients and even if no significant effects were observed regarding HDL and CRP, a significant reduction of total cholesterol, LDL, triglycerides and malondialdehyde (MDA) levels was obtained (Soltani et al., 2014). Furthermore, Mullan et al. showed that administering a vitamin C and polyphenol-rich beverage didn't induce a reduction of metabolic or inflammatory markers (apolipoproteins, CRP, ICAM-1, E-selectin) (Mullan et al., 2016).
Results regarding the effect of polyphenols on inflammation were obtained not only in metabolically impaired patients, but also in healthy subjects. Thus, in a placebo-controlled study, the effect induced by Montmorency tart cherry concentrate on the inflammatory and redox status markers in subjects simulating road cycle racing was examined. Trained cyclists received the concentrate or placebo for 7 days, after which their oxidative stress and inflammatory markers (IL-6 and CRP) were reduced, highlighting the ability of the tested extract to improve the stress response in physical exercise conditions (Bell et al., 2014).
Furthermore, in a study with crossover, double-blind design, 49 healthy males with APOE genotype received 150 mg/day quercetin or placebo for 8 weeks, in three phases, separated by three-week washout periods. Quercetin administration led to a decrease of waist circumference and postprandial systolic blood pressure, as well as a moderately increased levels of TNFα (Pfeuffer et al., 2013). In a separate study with cross-over, double-blind, randomized, placebo-controlled design including 18 healthy subjects, the effect of oleuropeine (51 mg) on a single occasion was tested. Blood of the subjects was collected and cells were stimulated with LPS, showing a significant decrease of IL-8 production (Lockyer et al., 2015).
Flavonoid-rich products have also been discussed for their beneficial effect on insulin resistance-related metabolic complications possibly via the upregulation of genes involved in insulin sensitivity (Engin et al., 2018). In a recent review, the beneficial effect of flavonoid-rich products in a cross-sectional study including 1997 females aged 18–76 years, the correlation between the total flavonoid intake as well as their subclasses (assessed from food questionnaires) and insulin resistance/inflammation was evaluated. Results showed higher anthocyanin and flavone intake were associated with significantly lower peripheral insulin resistance and CRP concentrations (Jennings et al., 2014). In a randomized controlled dietary study, results showed that consuming a high anthocyanins diet is associated with the reduction of CRP, IL-6, IL-12, and LPS concentrations indicating a positive effect on the long term cardiometabolic risk (Kolehmainen et al., 2012).
According to literature data, polyphenols blunt the inflammatory response in clinical setting, thus reducing the cardio-vascular risk factors for hypertensive, obese or diabetes mellitus patients. Positive effects on inflammation and redox stress markers were also obtained in healthy subjects and, moreover in trained athletes, who seem to benefit from the metabolic point of view from receiving polyphenols supplements.
Just a small number of published studies investigated if n-3 PUFAs may interact with the polyphenols and if a combined diet may have an enhanced beneficial impact. Until now only the beneficial effects on HDL-levels (as a “surrogate” for the anti-inflammatory effect) were identified (Ahn et al., 2020; Annuzzi et al., 2014; Bub et al., 2019).
In an 8-week randomized controlled dietary study on overweight patients, a combined diet of high polyphenols and marine PUFAs- diet showed beneficial, although non-additive effects on plasma triglycerides. Also, the reduction in oxidative stress (measured by urinary 8-isoprostane) was observed, probably as an effect of polyphenols. A negative interaction between PUFAs and polyphenols was observed for some variables (chylomicron cholesterol and apo B-48 in large VLDLs), which suggest a possible kinetic interaction between PUFAs and polyphenols (Annuzzi et al., 2014). This kinetic finding is also in line with some preclinical data that suggested that dietary polyphenols interact with the metabolism of n-3 PUFAs and increase blood EPA and DHA level (Toufektsian et al., 2011).
Recently, in a three monocentric, parallel-arm, double-blind, randomised, dietary intervention trial, 250 mg DHA and 320 mg grape anthocyanins administered as bioactive-enriched foods, for 4 weeks, had positive effects on serum triglyceride or HDL levels (Bub et al., 2019). These results are partially in line with a later cross-sectional analysis in the Korean population that suggested beneficial effects exhibited only in men by high amounts of proanthocyanidins or n-3 PUFAs on HDL levels. In addition, n-3 PUFAs intake increased polyphenols uptake (Ahn et al., 2020).
In conclusion, large-scale cohort studies with a combined diet PUFAs + polyphenols are needed to confirm the limited available data and to investigate possible additive effects.