Winery By-Products In Vitro and In Vivo Effects on Atherothrombotic Markers: Focus on Platelet-Activating Factor

Abstract

Platelet aggregation and inflammation play a crucial role in atherothrombosis. Wine contains micro-constituents of proper quality and quantity that exert cardioprotective actions, partly through inhibiting platelet-activating factor (PAF), a potent inflammatory and thrombotic lipid mediator. However, wine cannot be consumed extensively due to the presence of ethanol. Alternatively, winery by-products are abundant in similar-to-wine micro-constituents that could be used in food fortification and dietary supplements. Also, the vinification process produces millions of tons of by-products worldwide, posing an environmental matter of waste management. Therefore, the purpose of this literature review is to update the existing data concerning the in vitro anti-platelet and anti-inflammatory properties of winery by-product extracts and their possible health effects through controlled clinical trials in humans, specifically focused on their effects on PAF’s actions. Data from in vitro studies report that winery by-product compounds are able to inhibit platelet aggregation against several aggregation factors, as well as to downregulate inflammatory markers. Among their actions, extracts or phenolic compounds present in winery by-products inhibit PAF’s actions, a potent inflammatory and thrombotic mediator. Similar conclusions have been drawn from human supplementation studies, which suggest that winery by-product extracts may have beneficial biological effects on the cardiovascular system. Evidence from long-term studies shows that consumption may lower total and low density lipoprotein (LDL) cholesterol, improve insulin sensitivity, decrease lipid and protein oxidative damage, enhance antioxidant capacity, and have mild anti-inflammatory action toward reducing cytokine expression and levels. Data from the limited postprandial studies report that the acute consumption of winery by-product extracts improves glycemic response and reduces platelet reactivity to aggregatory stimuli. Although wine extracts and phenolic compounds have been reported to inhibit PAF’s actions and reduce the activity of its biosynthetic enzymes, no data exist concerning the influence of winery by-product extracts. In the future, additional long-term randomized controlled trials or postprandial studies are needed to draw definitive conclusions and establish a viable cardioprotective strategy that incorporates the sustainable use of winery by-products.

Graphical abstract

Keywords

PAF / winery by-products / platelet aggregation / inflammation / oxidative stress / cardioprotective effects

1. Introduction

1.1 The Involvement of PAF in Atherothrombosis

Atherosclerosis is a chronic arterial disease with clinical manifestations that include coronary heart disease, stroke and peripheral artery disease. Lipid storage in the arterial wall is gradually developed in atheromatous plaques, the rapture of which causes local thrombosis, leading to partial or total vascular blockage [1].

Oxidative, inflammatory and thrombotic mechanisms are crucial for the initiation and the prolongation of atherosclerosis. The oxidative modification of low-density lipoprotein cholesterol (LDL) is an important step in the initiation of the plaque formation. In the first stages, the peroxidation of polyunsaturated fatty acids (PUFAs) in LDL’s surface occurs and proceeds to the peroxidation of core lipids, resulting in a various range of biologically active molecules capable of activating endothelial cells, such as oxidized sterols, oxidized fatty acids and oxidized phospholipids [2, 3]. Platelet-activating factor (PAF) is a crucial inflammatory and thrombotic mediator, which is thought to play a key role in the initiation and prolongation of atherosclerosis [4] and is a main contributor to the pathogenesis of cardiovascular diseases [5]. Structurally, PAF is a phospholipid and particularly a 1-O-alkyl-2-acetyl-sn-glycero-3-phospho-choline [6]. Several types of cells could synthesize PAF, including platelets and leukocytes upon activation. PAF’s levels in organism are balanced through its biosynthetic and catabolic pathways. PAF can be synthesized by two different enzymatic routes, namely the remodeling and the de novo pathway. The remodeling pathway is believed to produce PAF under inflammatory conditions and involves a structural modification of ether-linked membrane phosphatidylcholines. More specifically, the action of cytoplasmic phospholipase A₂ yields lyso-PAF which is then acetylated by acetyl-CoA: lyso-PAF acetyltransferases (lyso-PAF AT) leading to the formation of PAF. The de novo pathway appears to be responsible for the constitutive production of PAF, which maintains its physiological levels in various tissues and blood. A key step in this pathway is the conversion of 1-O-alkyl-2-acetyl-glycerol to PAF by a specific cytidine diphosphate (CDP)-choline: cholinephosphotransferase (PAF-CPT). As far as PAF’s catabolism is concerned, the most important enzyme involved is a PAF-specific acetylhydrolase (PAF-AH), which cleaves the acetyl-group and forms lyso-PAF. The plasma isoform of PAF-AH is known as lipoprotein-associated phospholipase A₂ (Lp-PLA₂) due to its binding to lipoproteins. PAF is also produced non-enzymatically, during the oxidation of LDL, which results in the inactivation of the associated Lp-PLA₂.

Many experimental data revealed that the atherogenic actions of oxidized LDLs (ox-LDL) could be attributed to PAF and PAF-like lipids (oxidized phospholipids), which render these molecules as the initiators of atherosclerosis [4, 7]. In early stages of atherogenesis, oxidized phospholipids participate in the activation of the immune system, while in late stages, they contribute to platelet aggregation and plaque disruption [8]. Activated endothelial cells express several types of adhesion molecules that are recognized by monocytes, which are recruited and infiltrated into the subendothelial area. PAF triggers inflammatory response in endothelial cells and at the same time activates blood cells through its specific receptor (PAFR) which is a member of the G-protein-coupled seven-transmembrane receptor superfamily. The interaction of PAF with its receptor leads to increased endothelium permeability which is a crucial event in the initiation of atherosclerosis [9]. Activated endothelial cells rapidly produce and display PAF as well as P-selectin on their cell surface, which act as juxtacrine signals for activation and adhesion of neutrophils and monocytes. Cytokines, such as tumor necrosis factor-\alpha (TNF-\alpha) and interleukin-1 (IL-1), promote the synthesis of E-selectin and PAF [4]. This is a two way interaction since PAF promotes the production of cytokines [10, 11], such as IL-6 and IL-8, as well as chemokines such as monocyte chemoattractant protein-1 (MCP-1). Monocytes differentiate into macrophages that uptake ox-LDL, leading to their differentiation into foam cells [12]. Data support that ox-LDL interacts with PAFR in macrophages to increase CD36 expression and ox-LDL uptake, leading to enhanced foam cells formation [13]. Platelets are also important cells in atherothrombosis. Several agonists interact with platelet membrane receptors and facilitate aggregation, such as thrombin, thromboxanes, arachidonic acid (AA), PAF, adenosine diphosphate (ADP), serotonin, fibrinogen, collagen, von Willebrand factor (vWF). Platelet aggregation is mediated through integrin \alpha2b\beta3, that has the ability to bind extracellular agonists such as fibrinogen and vWF and is the combined result of reduced cyclic AMP (cAMP) levels and increased intracellular Ca²⁺ levels [14]. PAF’s and PAF-like molecules’ interaction with its membrane receptor (PAFR) results in AA and metabolites release, thromboxane A₂ (TXA₂) biosynthesis, intracellular Ca²⁺ levels increase, substrate phosphorylation, degranulation and platelet aggregation. PAF is synthesized by activated platelets and remains cell associated leading to a tethering and juxtacrine signaling system at the platelet surface which mediates rolling and tight adhesion of neutrophils, through interaction of Mac-1 (CD11b/CD18) with fibrinogen bound to integrin \alpha2b\beta3 [15]. As the atheromatous plaque evolves, a fibrous membrane is being developed, in which, dead cells are accumulated. Previous data suggest that local PAF production within the atherosclerotic plaques may contribute to intra-plaque neoangiogenesis [16, 17].

Other pathophysiological conditions such as insulin resistance (in the presence or absence of hyperglycemia) promote the risk for cardiovascular disease. Atherosclerosis and plaque progression can be facilitated in an insulin resistant state along with the presence of hyperglycemia, dyslipidemia, hypertension and pro-inflammatory conditions. Impaired insulin signaling may also take place in endothelial cells, macrophages and vascular smooth muscle cells, as these types of cells express the insulin receptor [18]. In diabetic patients, PAFR is up-regulated and hyperglycemia is associated with the downregulation of Sirtuin 1 (SIRT1) that can be abolished by the PAFR antagonist CV3988 [19]. PAF has also been reported to contribute to the progression of non-alcoholic fatty liver disease (NAFLD) through its ability to activate several signaling pathways (5-hydroxytryptamine, inositol triphosphate, AA, diacylglycerol) that eventually promote insulin resistance, inhibition of lipid \beta-oxidation, accumulation of triglycerides (TG), oxidative stress and inflammation in the hepatocytes as well as its ability to induce platelet activation that is, also, involved in NAFLD progression [20].

1.2 Biological Actions of Wine’s Micro-Constituents

The beneficial properties of wine consumption were firstly established in 1979 when an inverse association between red wine consumption and cardiovascular disease mortality was reported [21]. The term “French Paradox” was initially established in 1992, leading to the escalation of scientific research concerning wine consumption. The term describes the epidemiological observation that the French had relatively low incidence of coronary heart disease despite their rich-in saturated fatty acids diet and despite the presence of similar risk factors with other populations [22]. The J-shaped association between moderate wine consumption and stroke incidence as well as cardiovascular mortality has been stated in a meta-analysis. In contrast, the above study found a weaker association concerning beer consumption and cardiovascular disease and no association for other alcoholic beverages [23]. More recently, a meta-analysis has confirmed the protective effects of wine consumption regarding all-cause mortality as opposed to alcohol consumption from non-wine sources [24].

According to scientific literature, the beneficial effects of wine consumption are mainly attributed to its bioactive compounds. In this area, our previous studies revealed that wine contains potent micro-constituents that exert anti-platelet effects and among other actions, reduce PAF-induced platelet aggregation and PAF biosynthetic enzymes activities in leukocytes both in vitro and in vivo [25, 26, 27, 28]. Nevertheless, the fact that wine contains ethanol should be taken into consideration since excess ethanol consumption is detrimental for health through various mechanisms. A recent meta-analysis reported that all-cause mortality is highly associated with ethanol consumption and concludes that concerning alcohol consumption, “the risk of all-cause mortality, and of cancers specifically, rises with increasing levels of consumption, and the level of consumption that minimizes health loss is zero” [29]. However, the same group 4 years later, based on new statistical models modulated their statement to “there is strong evidence to support recommendations on alcohol consumption varying by age and location” [30]. Also, it should be mentioned that they did not take under consideration the type of alcohol consumed or the frequency of drinking during the week. There are data supporting that regular moderate wine consumption is not associated with negative effects and in some cases, especially cardiovascular diseases, it is associated with positive outcomes [31, 32].

Considering the above data and the existence of phenolic compounds in wine, several researchers have focused on the health effects of individual phenolic compounds, such as resveratrol, in the form of dietary supplements [33, 34]. However, wine is a complex mixture of compounds and its health benefits may be attributed to the co-existence of other micro-constituents apart from phenolic compounds. Due to existing evidence, it can be hypothesized that wine contains a micro-constituent’s mixture in proper quality and quantity that provide cardiovascular protection through synergistic action. This hypothesis is supported by the fact that individual phenolic compounds do not or exert weaker biological actions compared to their mixtures or wine extracts [35].

On the other hand, it should be noted that the isolation process of these beneficial wine micro-constituents is non-profitable from an economic aspect. Alternatively, winery by-products are cheap and rich-in similar-to wine constituents sources. Especially, during the vinification process of red grapes, the grape skins and seeds are present in the must, which is fermented for a significant amount of time. Many beneficial micro-constituents are being transferred to wine and yet, the by-products are still an abundant-in bioactive compounds source. The vinification process produces millions of tons of by-products which also poses an environmental matter of waste management. In this direction, their sustainable use is highly considered [36].

1.3 Composition of Winery By-Products

Winery by-products consist of grape pomace (GP) (seeds and skins), stems, leaves and lees. Several environmental factors (such as grape variety, cultivation) as well as grape processing procedures can affect the chemical composition of winery by-products. Grapes are subjected to various invasive processing stages, yet, their chemical composition concerning bioactive compounds is not significantly altered [37].

Phenolic acids contain benzoic and cinnamic acid derivatives, with hydroxycinnamic acids being the largest subcategory in winery by-products. These molecules are usually bound to glycosylated derivatives, shikimic, quinic or tartrate acid esters. Gallic acid is the most frequent hydroxybenzoic acid derivative in stems, skin and seeds, syringic acid in stems and procatechuic acid in grape pomace. Hydroxycinnamic acids are present in grapes and especially in skins. Mainly, caftaric and coutaric acid are found in white grape skins, whereas chlorogenic acid is found in red grape skins [38]. The relative amount of phenolic acids is also dependent on the extraction solvent. In our previous study, the aqueous ethanolic GP extract contained mainly vanillic acid, gallic acid, syringic acid, salicylic acid and p-coumaric acid [39].

Flavonoids refer to a group of compounds composed by low molecular weight molecules, with a structure of 15 carbon atoms. Winery by-products’ flavonoids are integrated by flavonols, flavanols, anthocyanins and proanthocyanidins [40]. Flavonols are characterized by the existence of a double bond between C2 and C3 carbons and a hydroxyl group at C3 carbon. Different sugars can be connected to flavonols resulting in glucosides, galactosides and diglucosides. In red variety stems, quercetin is abundant in the form of quercetin-3-glucoronide, -3-glucoside, -3-galactoside and -3-ramnoside. Red and white varieties express a similar compounds profile and the red varieties have higher concentrations of these molecules [38]. Flavanols have a hydroxyl group in the C3 carbon and no carbonyl group in the C4 carbon and they are the main substances responsible for the astringency of wine. Catechin is the main molecule in both red and white varieties and its concentration appears to be 4-fold higher than epicatechin [40]. Anthocyanins are responsible for the color of red grapes and wine and they possess an aromatic ring (A) bound to a heterocyclic ring (C) that contains oxygen, which is bound to a third aromatic ring (B). The aromatic ring B is bound to sugars or organic acids and is responsible for the different colors of anthocyanins. Malvidin-3-glucoside and peonidin-3-glucoside are the most abundant anthocyanins found in grape skins [37, 38]. Condensed tannins or proanthocyanidins consist of flavanol monomer subunits and their structures vary depending on the subunits compounds, the polymerization degree and the coupling sites. Regarding red varieties the most frequent proanthocyanidin is procyanidin B3, whereas in white varieties is procyanidin B1. Procyanidins are mostly present in white varieties compared to the red ones and especially in stems compared to skin and seeds [38]. In accordance with the aforementioned, we have shown that an aqueous ethanolic GP extract contained mainly catechin, epicatechin, quercetin and malvidin-3-glucoside [39].

Stilbenes are phenolic compounds with two aromatic rings attached to an ethylene bridge. They are mostly found in grape skins and also in stems and seeds. Grapes produce stilbenes for protection from ultraviolet (UV) radiation [38]. Resveratrol is the most common stilbene and in our previous study we confirmed its existence in an aqueous ethanolic GP extract from red varieties. Tyrosol and hydroxytyrosol that belong to the simple phenolic compounds category were also detected in the above aqueous ethanolic extract [39].

The aim of this review is to update the existing data concerning the in vitro anti-platelet and anti-inflammatory properties of winery by-product extracts as well as their possible health effects through controlled clinical trials. Especially, focus is given on the potent inflammatory and thrombotic lipid mediator PAF.

2. Methods

The PubMed and Scopus databases were reviewed from 2000 up to December 2023, using the following keywords: “grape pomace”, “grape seed”, “grape skin”, “grape marc”, “wine by products”, “winery by products”, “winemaking by products”. The search yielded 1948 publications from the PubMed database and 4873 items from the Scopus database. The titles of the studies were screened and the appropriate ones were reviewed and included in the manuscript. Additional searches were also carried out using keywords suitable for each individual table of studies. The flowchart of the literature search is presented in Fig. 1.

For the in vitro anti-platelet actions, the search focused on human platelet aggregation studies using winery by-product extracts. For the in vitro anti-inflammatory actions, studies where human cells (mostly cell lines) were incubated with winery by-product extracts and inflammatory proteins or signal transduction molecules were evaluated, were included. Cell proliferation, cytotoxicity and cell migration assays were out of the scope of this review. Also, controlled clinical studies that administered winery by-product extracts in the form of dietary supplements in human subjects were included in this review. Outcomes including effects in weight management, satiety and hematological markers were not included.

3. In Vitro Anti-Platelet Actions of Winery By-Products

The studies examining the in vitro anti-platelet effects of winery by-products (Table 1, Ref. [39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51]) have used human washed platelets, whole blood or platelet-rich plasma (PRP) against several aggregation factors. The winery by-product extracts examined, were mostly dietary supplements from grape skins (GSK) or/and seeds (GS). In this context in our previous study GP extracts from four red grape varieties (Sour Black, Syrah, Cabernet Franc, Cabernet Sauvignon) were prepared using different solvents and were tested for their ability to inhibit platelet aggregation (in PRP) against several agonists. The results indicated that the extracts’ anti-platelet effect was not grape-variety dependent, whereas the solvent used for the extraction that determines the extracted micro-constituents was the crucial factor. In specific, the aqueous ethanolic GP extracts exhibited more potent anti-platelet action compared to the lipoid extracts using the Bligh-Dyer method, while the aqueous and the hexanoic extracts exhibited no anti-platelet effect [39]. In our early studies [25, 26], potent anti-platelet actions against PAF were reported in the wine lipid extracts using the Bligh and Dyer method. However, this method utilizes chloroform and methanol, which are not suitable solvents for products consumed by humans. Therefore, the discovery of potent inhibitors of platelet aggregation in aqueous ethanolic extracts is promising for the development of dietary supplements and food enrichment.

ADP as an aggregation factor was used in four studies. Luzak et al. [41] reported that 1 µg/mL of extract exhibited weak anti-platelet action as it failed to inhibit platelet aggregation above 15%. Bijak et al. [42] examined an extract at 15 µg gallic acid equivalents (GAE)/mL which reduced the area under the curve (AUC) by 38.9%. Out of the nine grape varieties examined by another group, only the Petit Verdot GP extract significantly inhibited platelet aggregation against ADP by 67.1% [43]. In our previous study, the aqueous ethanolic GP extracts exhibited more potent action against ADP compared to the lipoid extracts (Bligh-Dyer method), with half maximal inhibitory concentration (IC₅₀) values being 181.2 \pm 82.3 µg and 293.2 \pm 102.7 µg extract respectively [39].

Thrombin was used in four studies and the GS extracts’ IC₅₀ values ranged between 5–25 µg/mL [44, 45, 46, 47]. Malinowska et al. [47] examined a hyperomocysteine state in healthy subjects’ washed platelets and reported inhibition of platelet aggregation by the extracts’ compounds. Thrombin receptor activating peptide (TRAP) was also used as an aggregatory factor with IC₅₀values ranging between 10–1000 µg of GS/ GP extract/mL. Also, the aqueous ethanolic GP extracts inhibited platelet aggregation more effectively against TRAP compared to the lipoid extracts (Bligh-Dyer method) as the IC₅₀ values revealed: 156.3 \pm 97.5 µg and 284.8 \pm 131.8 µg of extract respectively [39]. Another study that examined GS and GSK extracts concluded that each extract could inhibit platelet aggregation against TRAP separately as well as that their combination led to additive inhibition of platelet aggregation, enhancement of NO release, and prevention of superoxide production [48].

The synergistic action between the GS and GSK constituents is also observed in the study of Shanmuganayagam et al. [49] where collagen, as agonist, was used. Specifically, the combination of the two extracts, at concentrations where each one separately did not cause inhibition of platelet aggregation, led to a 40.5% inhibition of platelet aggregation [49].

PAF, apart from its pro-inflammatory actions, is considered a potent platelet agonist. High PAF levels, acting through its specific plasma membrane receptor (PAFR), enhance both inflammation and thrombosis. Indeed, the ex vivo platelets response to PAF or the in vitro inhibition of PAF-induced platelet aggregation, aside from estimation of the anti-platelet ability, is considered as an indirect method to estimate PAF’s actions in organism and bioactive compounds’ anti-inflammatory action [4]. We have shown that GP extracts inhibited PAF-induced platelet aggregation, with the aqueous ethanolic extracts being more potent inhibitors compared to the lipoid ones (Bligh-Dyer method) [39]. In another research study, GSK extracts from different grape varieties also inhibited PAF-induced platelet aggregation with IC₅₀ values ranging between 19.7–26.6 µM GAE [50].

The fact that the winery by-product extracts are able to inhibit different aggregation factors (PAF, ADP, thrombin/TRAP, collagen), reveals that their anti-platelet action is achieved through different signaling pathways and is probably attributed to the synergistic action of all the extracts’ components [49]. The anti-aggregatory actions could be achieved through various mechanisms such as inhibition of agonist binding to its receptor, inhibition of signal transduction enzymes namely phospholipase C (PLC), protein kinase C (PKC) and cyclooxygenase (COX), inhibition of phospholipid oxidation, or through free radicals scavenging. Catechin, the most abundant flavanol detected in GP, has been shown to modulate signal transduction mediated by the PAR (thrombin/TRAP receptor), GPVI (collagen receptor), and P2Y12 (ADP receptor) receptors, integrin \alpha2b\beta3 and PLC [52]. Epicatechin is reported to be able to reduce the expression of integrin \alpha2b\beta3 induced by ADP and epinephrine [53]. Quercetin has been reported to inhibit cyclooxygenase (COX), thereby reducing TXA₂ formation as well as attenuating signal transduction through the TP receptor (TXA₂ receptor) [52]. It has also been shown that quercetin inhibits signal transduction from other receptors such as P2Y12 [52, 54], PAR, GPVI, integrin \alpha2b\beta3 as well as pathways mediated by PLC and protein kinase B (PKB)/Akt [52]. Tyrosol and resveratrol have been shown to inhibit PAF-induced platelet aggregation [55]. Resveratrol’s anti-platelet actions have also been reported against the PAR [52], P2Y12 and GPVI receptors, PLC action [52, 56] as well as the COX- and TXA₂-mediated pathway [52]. Taking into consideration the data from the studies that examined winery by-products and individual compounds, the potential mechanisms of anti-platelet action are presented as a whole and summarized in Fig. 2.

4. In Vitro Anti-Inflammatory Actions of Winery By-Products

In studies examining the anti-inflammatory actions of winery by-products in cell lines, the winery by-products constituents were mostly received by dietary supplements produced by GS, GSK or GP (Table 2, Ref. [57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69]). In the majority of these studies, cells were stimulated by a pro-inflammatory mediator. Lipopolysaccharide (LPS) was mostly used [57, 58, 59, 60, 61, 62], as well as ox-LDL [59], hydrogen peroxide (H₂O₂) [63], tumor necrosis factor-\alpha (TNF-\alpha) [61, 62, 64], Campylobacter jejuni [65], advanced glycation end products (AGEs) [66] and high glucose concentration [67].

The cells used in these studies were frequently macrophages, endothelial cells or epithelial cells and several biomarkers of inflammation were measured. In 6 studies, cells incubated with winery by-product extracts were able to modulate the function of transcription factors, mainly nuclear factor kappa B (NF-\kappaB) [61, 63, 64, 67, 68, 69], in mRNA and protein levels. The primary mechanism of NF-\kappaB activation is the inducible degradation of I\kappaB\alpha triggered through its site-specific phosphorylation by a multi-subunit I\kappaB kinase (IKK) complex. Winery by-product extracts were capable of inhibiting the phosphorylation and the nuclear NF-\kappaB translocation, resulting in reduced pro-inflammatory genes expression and therefore reduced inflammation. Indeed, several cytokines, chemokines and adhesion molecules have been found to be reduced in mRNA expression and protein levels by winery by-product extracts (Table 2). Also, several enzymes involved in the inflammatory process such as COX have been found to be downregulated in mRNA level [60, 68, 69].

It is worthy to mention that although wine extracts have been reported to inhibit PAF’s action in platelets and also to reduce the activity of its biosynthetic enzymes [35], no published data exist concerning the influence of winery by-product extracts. However, apigenin and keampferol, quercetin, naringin hesperidin as well as epicatechin-3-O-gallate and epigallocatechin-3-O-gallate were found to be inhibitors of the lyso-PAF AT and the PAF biosynthesis [70].

5. In Vivo Biological Actions of Winery By-Products Extracts in Humans

Several studies examined the health effects of winery by-products, mostly as dietary supplements, in humans (Table 3, Ref. [71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110]). Concerning the effects of winery by-products on lipid biomarkers, in most studies total cholesterol was reduced in the group receiving treatment compared to control group [71, 72, 73, 74, 75, 76, 77] as well as LDL cholesterol [72, 73, 74, 77]. Winery by-products consumption had mild effect on triglycerides (TG) levels as they were reduced in two studies from which, in one, hyperlipidemic subjects participated in an 8-week intervention [72] and in the second study TG reduced after the consumption of a high-fat lunch meal compared to the placebo treatment [78].

Research has also focused on the effect on glycemic biomarkers, such as insulin levels or sensitivity and glucose levels, although few significant changes regarding the treatment group compared to control were reported. The intake of 600 mg GS extract for 8 weeks managed to improve insulin sensitivity and reduce fasting insulin levels in 40 volleyball athletes [79]. The daily consumption of 20 g bread or cookies made from flour enriched with GP for 16 weeks reduced postprandial insulin levels during an oral glucose tolerance test (OGTT) in subjects with at least one metabolic syndrome (MS) criteria [80]. The acute consumption of a GP extract under postprandial conditions also reduced the insulin and improved insulin sensitivity in 12 healthy men [81]. The intake of GS extract significantly reduced the postprandial glucose 15 and 30 minutes after the meal consumption as well as the glucose response AUC in healthy volunteers [82]. No effect was reported when hemoglobin A1c (HbA1c) levels were measured, possibly due to the long time needed for alterations to be detected in this particular biomarker [76, 83, 84].

Many studies assessed the extracts’ effects on cardiovascular indexes. Flow mediated dilation (FMD) was increased after the 12-week consumption of a yogurt enriched with GS extract, but not after a yogurt enriched with both GS and quercetin, in subjects with cardiovascular risk factors [85]. FMD was also increased after a single dose of GS extract, when hypertensive subjects performed exercise or during resting conditions compared to pre-supplementation [86]. The same study reported reduction in their mean systolic (SBP) and diastolic blood pressure (DBP). Blood pressure was also reduced in subjects with MS receiving either 150 or 300 mg of GS extract compared to the control group [87]. SBP alone was significantly reduced in hypertensive subjects receiving 300 mg of GS extract [83], whereas DBP alone, in women with menopausal symptoms after consumption of 100 or 200 mg of extract for 8 weeks [88].

Regarding the extracts’ antioxidant properties, studies examined the oxidative damage on lipids, proteins and DNA as well as their effect on antioxidant enzymes or molecules. The majority of the studies that measured lipid peroxidation products after the consumption of the extract capsules observed reduction in malondialdehyde (MDA) levels [71, 73, 76, 79, 89, 90], in lipid peroxides [91] as well as in thiobarbituric acid reactive substances (TBARS) levels [92]. Two studies assessed the extracts’ effects on protein carbonyls (PC), in which their levels where reduced, compared to the control group [89] and only compared to baseline values [80]. In our study, the acute consumption of a GP extract reduced postprandial PC levels compared to the control group only in overweight/obese women [92]. Few data exist concerning the effects on DNA oxidation, as the two research groups that measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels did not observe any changes [93, 94]. The antioxidant molecule glutathione in its reduced form (GSH) was increased in volleyball players that received 600 mg of GS extract for 8 weeks [79] compared to baseline values and to the control group, whereas in other studies GSH [95] and GSH/glutathione oxidized (GSSG) ratio [96] were increased compared to baseline values but not to the control group. The effect on antioxidant enzymes can be assessed by the change in their levels or their activity. Superoxide dismutase (SOD) and catalase (CAT) levels were significantly elevated in chronic kidney disease (CKD) patients [89] that received 2 g of GS extract for 6 months. For SOD, the same effect was found in type II diabetic patients receiving 200 mg daily for 8 weeks, whereas, SOD levels were reduced in chronic obstructive pulmonary disease (COPD) patients receiving 150 mg daily for the same period [71]. In our study, postprandial SOD activity was reduced only in normal-weight women compared to the control group after the consumption of a GP extract along with a high-fat meal [92]. SOD activity was increased in one study in overweight or obese subjects [73] but only compared to baseline values. In the same study, glutathione peroxidase (GPx) activity was enhanced only compared to the control group. GPx levels were elevated after the daily administration of 400 mg GS extract for 2 months in obese subjects compared to the group receiving resveratrol and compared to the control group [94]. Paraoxonase (PON) was examined in one study where hyperlipidemic subjects received 200 mg of GS extract for 8 weeks, leading to its activity enhancement compared to baseline values and to the control group [72]. Assays developed for the measurement of the antioxidant capacity in serum and plasma such as total antioxidant capacity (TAC), trolox equivalent antioxidant capacity (TEAC), total radical trapping antioxidant parameter (TRAP), oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP), were performed in several studies examining GS extracts and favorable results were reported [74, 84, 90, 91, 94, 97].

Concerning the extracts’ actions on inflammatory markers, research focused on cytokines, acute phase proteins (C-reactive protein, CRP in particular), adhesion molecules and inflammatory genes expression. Significantly reduced levels of high sensitive CRP (hs-CRP) were reported after 8 weeks of GS oil consumption for 39 overweight/obese subjects, compared to baseline levels and to the control group [98]. Other studies reported reduced CRP levels [95, 99] compared to baseline values. The majority of cytokines examined were TNF-\alpha and interleukin-1\beta (IL-1\beta). In 28 smokers receiving 200 mg GS extract for 8 weeks TNF-\alpha levels were reduced compared to baseline and to the control group [96]. In the rest of the studies TNF-\alpha [73, 98] and IL-1\beta [73] were reduced only compared to baseline levels. Intercellular adhesion molecule-1 (ICAM-1) protein levels did not change in hyper-cholesterolemic and/or hypertensive subjects [83, 85], whereas, downregulation of the ICAM-1 gene expression was reported after the 8-week consumption of 400 mg GS extract in obese subjects compared to the control group, as well as compared to the group receiving resveratrol [94].

The anti-platelet effects of extracts were examined only by three of the studies listed in Table 3. Two of them reported favorable results in the postprandial state by reducing platelet sensitivity to ADP [100, 101] and epinephrine [100]. No data exist, yet, concerning PAF as agonist for platelet aggregation.

6. Conclusions

In this review, we provide a brief overview of the atherothrombotic process in order to further investigate the effects of winery by-product extracts. Data from in vitro studies report that winery by-product compounds are able to modulate platelet function, as well as to downregulate inflammatory markers. Among their actions, extracts or phenolic compounds present in winery by-products inhibit PAF’s actions, a potent inflammatory and thrombotic mediator. In addition, they modulate its levels either through direct inhibition of its metabolic enzymes or through indirect reduction of oxidative stress leading to lower LDL oxidation and PAF or PAF-like molecules production. Similar conclusions have been drawn from the human supplementation studies stating that winery by-product extracts may exert favorable biological actions towards the cardiovascular system. Evidence from long-term studies show that consumption may lower total and LDL cholesterol, improve insulin sensitivity, decrease lipid and protein oxidative damage, enhance antioxidant capacity and a have mild anti-inflammatory action towards reducing cytokine expression and levels. Although data from postprandial studies are limited, the acute consumption of winery by-product extracts seems to improve glycemic response as well as reducing platelet reactivity to aggregatory stimuli. Nutritional supplements or foods fortified with winery by-products could provide a safe and cost-effective complementary treatment towards atherothrombotic complications. Nevertheless, further randomized controlled long-term or postprandial trials need to be conducted in order to draw definitive conclusions for providing a useful cardioprotective approach that includes the sustainable use of winery by-products.

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