*5.4. Phenolic Acids*

Caffeic acid, identified in Maqui, has also been noted for its antiplatelet properties. Studies have shown that this compound possesses antithrombotic activity on mouse brain arterioles in vivo, and inhibits platelet aggregation in vitro stimulated by various agonists (ADP and thrombin) [126,127]. It has been described that this polyphenol is a potent compound that increases the level of cAMP-dependent protein phosphorylation in collagen– platelet interactions [128]. Studies report its antithrombotic action at doses of 1.25–5 mg/kg, an effect related to its capacity to suppress phosphorylation of ERK, p38, and JNK, which leads to cAMP elevation, and it negatively regulates P-selectin expression and activation of αIIbβ3 [126–128]. Meanwhile, Nam et al. showed that this polyphenol decreases the production and release of thrombogenic molecules in human platelets. This effect was not only mediated by TxA2 but also by the decrease in serotonin released by collagen by inhibiting the phosphorylation of JNK1 [129]. This phenolic compound at 25–100 μM further inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca2+ mobilization, and αIIbβ3 integrin activation [127].

The potential of ferulic acid has been related to the activation of cAMP and cGMP signaling [113,114]. This compound inhibits dose-dependent (50–200 μM) platelet aggregation induced by platelet agonists (ADP, thrombin, collagen, AA, and U46619). Additionally, it attenuates intracellular Ca2+ mobilization and TxA2 production. It also increases cAMP, cGMP, and vasodilator-stimulated phosphoprotein (VASP) levels while decreasing phospho-MAPK and phosphodiesterase (PDE) in washed rat platelets [113]. Studies report that ferulic acid has an antithrombotic effect in the in vivo model of acute thromboembolism and decreases the expression of αIIbβ3/FIB and AKT phosphorylation in thrombin-stimulated platelet activation [114].

Both ellagic acid and ferulic acid inhibit platelet activation in vitro, induced by ADP and collagen [130]. Phenolic acid derivatives have also been shown to be novel antiplatelet targets, even more potent than their phenolic precursors. Dihydrocaffeic acid and dihydroferulic acid at doses of 0.01–100 μg/mL 1 μM decreased ADP-stimulated platelet activation, measured as P-selectin expression and fibrinogen binding [131].

AA, arachidonic acid; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Akt, protein kinase B; CD63, a membrane protein associated with lysosome 3; CD40L, a ligand of the membrane protein; COX-1, cyclooxygenase; CRP, collagen-related peptide; ERK: extracellular signal-regulated kinase; FIB, plasma fibrinogen; FXa, factor Xa; cGMP, cyclic guanosine monophosphate; GP, glycoprotein; 5-HT, serotonin; αllbβ3, GPVI, glycoprotein VI; GPIIb/IIIa, glycoprotein IIb–IIIa; JNK: c-Jun N-terminal kinase, MAPK, protein kinase activated by mitogens; MW, molecular weight; OH\*, hydroxyl radical; PAF, platelet activating factor; PDE: phosphodiesterase; PG, glycoprotein; PF4, platelet factor 4; PI3, phosphoinositol 3 kinase; PKB, protein kinase B; PKC, protein kinase C; PLC γ2, phospholipase C; Rantes, regulatory chemokine beta; ROS, reactive oxygen species; Ser, serotonin; Syk, tyrosine protein kinase; β-TG, beta thromboglobulin; TGF-β1, transforming growth factor-beta 1; TRAP-6, thrombin receptor activator for peptide 6; TxA2, thromboxane; VASP, phosphoprotein stimulated by vasodilator.

The secretion of α and dense granules, the release of adenosine triphosphate (ATP), and the mobilization of Ca2+ are the main markers of activation reported to date by mechanisms involving the AA-derived pathway and GPVI receptor, among others. Some receptors for collagen (GPVI), thrombin (protein activated receptors, PARs), and TxA2 activate phospholipase C (PLC). The aforementioned receptors generate diacylglycerol (DAG) and inositol triphosphate (IP3), activating the protein kinase C (PKC) and intracellular Ca2+ release, leading to granule secretion [98,101,102]. On the other hand, adenylyl cyclase (AC) favors the conversion of ATP into cyclic adenosine monophosphate (cAMP). The activation of phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) could mediate the phosphorylation of endothelial nitric oxide synthase (eNOS), increasing the production of platelet nitric oxide (NO). This leads to the production of cyclic guanosine monophosphate (cGMP), and stimulates the activation of mitogen-activated protein kinases (MAPK), thus promoting granule secretion and activation of platelets [31,118].

Considering the studies about the antiplatelet activity of previous tested chemical compounds that have also been identified in Maqui, and the reviewed inhibition mechanisms of platelet activation, we propose a scheme of how the bioactive compounds present in Maqui extracts exert their effects (Figure 5).

**Figure 5.** Antiplatelet mechanistic proposal for Maqui. AC, adenylate cyclase; AKT, protein kinase B; cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; PI3, phosphoinositol 3 kinase; PIP2, phosphatidylinositol bisphosphate; GP, glycoprotein; MAP, mitogen-activated protein kinase; P2Y12/P2P1, ADP receptor; TxA2R, thromboxane receptor.

#### **6. Limitations and Future Perspective**

Inhibition of platelet function has long been used to prevent and treat CVD [63]. Although antiplatelet drugs currently exist, this therapy has been accompanied by side effects, such as bleeding. Recent efforts focus on the search for and development of new therapeutic agents, together with the healthy habits that we must promote to contribute to adequate cardiovascular health.

In general, healthy eating is promoted, which includes minimally processed foods and foods rich in bioactive products such as fruits, nuts, seeds, beans, vegetables, whole grains, vegetable oils, yogurt, and fish [132]. Epidemiological studies have provided evidence for the protective role of healthy diets in the prevention of CVD. For example, eating two or three kiwis a day for 28 days reduces platelet aggregation induced by collagen and ADP. On the other hand, raw garlic and some of its preparations are recognized as antiplatelet agents [63]. As the above, Maqui plays a fundamental role among healthy fruits due to its wide biological potential, as highlighted in this work.

Maqui is known to be rich in phenolic compounds, and phenolic compounds have a well-recognized role in reducing the risk of chronic diseases, increasing healthy life years, and promoting healthy aging [133]. The protective mechanisms of foods rich in polyphenols may not only depend on their content of nutrients and bioactive compounds but also include their food matrix properties that affect glycemic load and energy density, among others [132].

The extraction procedure affects the chemical composition of the Maqui extracts; variables such as temperature, sonication, extraction time, and solvent are some of the parameters that would influence the chemical profile identified for this species [119]. Concerning the solvent used for extractions in Maqui matrices, it has been shown that hydroalcoholic mixtures are the most used solvents for the extraction of bioactive components from berries [134]. The biological potential of aqueous and ethanolic extracts of Maqui, which have a high content of polyphenols, has been reported [28,46]. Hydroalcoholic extract presented higher polyphenolic content, with antioxidant and antimicrobial properties meaning it is the most promising extract for pharmaceutical purposes [51]. As previously described, Maqui has the highest phenol content compared to other berries, and this depends substantially on the solvent used for extraction [16,19].

Sonication is an ideal alternative to obtain extracts from Maqui berries with high bioactivity [49] while the extraction time can also modify the antioxidant capacity of Chilean Maqui berries [48]. Three operating conditions of the extraction process were compared by modifying the type of solvent (methanol, ethanol, and acetone), solvent concentration (20, 60, and 100%), and extraction time (15, 127.5, and 240 min). Results show that after a certain extraction time there is a final equilibrium between the solutes of the matrix and the extraction solvent [28].

Metabolites such as polyphenols, flavonoids, and anthocyanins are the main ones that have been identified in Maqui extracts. Polyphenols may possess antiplatelet properties, but their coadministration may not be safe [135]. It is important to make the correct and precise determination of both the composition and the amounts of the phenolic compounds that we consume to avoid toxicity or unwanted side effects. Of the total number of trials with polyphenols in the last 20 years, 20% analyzed vascular and endothelial responses, and trials on platelet function and thrombosis are lacking [135,136]. In vivo and trial studies evaluating potential polyphenol–drug interactions are needed to address this limitation [123].

On the other hand, studies refer to the protective effects of flavonoids against druginduced toxicity. It was concluded that flavonoids, both dietary and derived from plant medicines, can exert protective effects against drug-induced toxicity [135,137,138]. These compounds are generally recognized as safe, due to the long history of use and consumption of foods rich in flavonoids. The total daily intake of flavonoids in Europe is estimated to be around 428 ± 49 mg, with 136 ± 14 mg being monomeric [135,139], while flavonoid-rich beverages and vegetables can reach 1000 mg/day of flavonoids [135,140].

Although the benefits of phenols have been demonstrated in different in vitro and in vivo models, there are few reports evaluating the possible effects of Maqui extracts on platelet aggregation [93]. Although there are clinical studies that show that Maqui extract or capsules are safe for human consumption [74,75], studies that evaluate their administration over longer periods are required.

Although in vitro studies help to understand the possible health contributions of Maqui berries, these may not be fully applicable to humans, as in the case of anthocyanins, such as delphinidin and anthocyanin, which have been shown to have limited bioavailability. In animal and human studies, anthocyanins are poorly absorbed and thus show low bioavailability [73]. Furthermore, several groups have suggested that dietary cyanidins and delphinidins may be subject to extensive biotransformation in humans, most likely involving the colonic microbiota [73,141].

Despite the above, it appears that anthocyanins are more bioavailable than is perceived, and their metabolites are present in the circulation for ≤48 h after ingestion. A study carried out on eight male volunteers showed that cyanidin-3-*O*-glucoside, a main component of the Maqui berry, has a non-negligible bioavailability of 12.38 ± 1.38%. Blood concentrations of cyanidin-3-*O*-glucoside and cyanidin-3-*O*-glucoside conjugates were observed to appear between 1 and 2 h after the intake of 500 mg of cyanidin-3-*O*-glucoside [142].

There are few in vivo and in vitro studies comparing Maqui with other fruits and berries to determine the benefits and weaknesses of Maqui compared to other foods with similar bioactive compound composition. There is a lack of studies to establish how much Maqui should be consumed daily to see this effect at physiologically relevant concentrations.

The natural bioactive ingredients of Maqui have antiplatelet effects with multiple targets on the platelet, while the synergistic effects of the phenolic compounds could enhance the antiplatelet activity. Due to the above, there exists an interest in the cardiovascular benefits of Maqui berries, so daily consumption of this berry can prevent the development of CVD, although detailed studies are needed to reinforce its clinical utility.

#### **7. Conclusions**

Due to the prevalence of CVD, new antiplatelet drugs are needed to prevent and treat arterial thrombosis as well as other CVDs. Some bioactive compounds, such as polyphenols and anthocyanins in fruits and vegetables, have been reported to inhibit platelet activation and so reduce the risk of CVD.

The broad chemical profile of Maqui (flavonoids, anthocyanins, and phenolic acids) is directly related to its high biological potential. The current knowledge about Maqui's antioxidant, anti-inflammatory, and hypoglycemic effects suggests that a diet including Maqui could aid in the prevention of CVD, with more studies being required to prove this hypothesis. The fundamental mechanisms which this species influences have been mainly related to the inhibition of lipid peroxidation, decrease in cholesterol and blood glucose levels, as well as a decrease in oxidative stress. Studies are needed to establish how much Maqui should be consumed daily to see this effect at physiologically relevant concentrations.

Additionally, this species can be studied in detail for antiplatelet purposes, since to date, there have been few authors who have highlighted this potential in the fruits of Maqui. Our findings highlighted that the main mechanism by which the compounds identified in this species act is related to the metabolic pathways of the AA and GPVI receptors. Although anthocyanins are the main phenolic compounds in this berry, antiplatelet activity may be directly related to the presence of a specific compound or enhanced by the synergy of several phenolic compounds.

This review allowed us to investigate the antiplatelet and cardioprotective activity of the bioactive compounds present in Maqui and highlight the areas in which much remains to be investigated. Without a doubt, we have shown that Maqui is an interesting target in the search for new antiplatelet therapies.

**Author Contributions:** L.R. and A.T.: conceptualization, conducting the research, writing the original draft. H.V. and I.W.: conceptualization. I.P.: conceptualization and writing the original draft. E.F. and S.W.: conceptualization and conducting the research. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been funded by ANID/REDES190112 "INTERNATIONAL NETWORK ON THE STUDY OF ENDOPLASMIC RETICULUM STRESS IN PLATELET FOR PREVENT CARDIO-VASCULAR DISEASE IN GLUCOLIPOTOXIC MILIEU".

**Acknowledgments:** We would like to thank ANID-FONDECYT N◦1211136, ANID-FONDECYT N◦1220339, ANID-FONDEQUIP EQM200049, ANID/FONDECYT N◦3220099 and Interuniversity Center for Healthy Aging, Code RED211993. Andres Trostchansky thanks Comisión Sectorial de investigación Científica (CSIC), Uruguay Grupos N◦536 and Proyecto Donaciones Especiales (Medical Plus), Fundacion Manuel Perez, Uruguay.

**Conflicts of Interest:** The authors declare no conflict of interest.
