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By Paula Silva, María Rodríguez-Pérez and Emma Burgos-Ramos

In this review, we explored the potential of a zebrafish model to investigate the antioxidant effects of key components of the Mediterranean diet, namely, olive oil and wine, in the context of preventing age-related diseases, particularly cardiovascular conditions. This paper explores the spectrum of observational studies to preclinical investigations and ultimately converges toward potential translational insights derived from animal experimentation. This review highlights the potential and underutilization of zebrafish as an experimental model in this domain. We highlighted the genetic proximity of zebrafish to humans, offering a unique opportunity for translational insights into the health benefits of olive oil and wine. Indeed, we wanted to focus on the potential of zebrafish to elucidate the health benefits of olive oil and wine while calling for continued exploration to unlock its full potential to advance our knowledge of age-related disease prevention within the Mediterranean diet framework.

The Mediterranean Diet

The MD is a dietary plan based on Crete’s traditional eating habits. The term “Mediterranean Diet” was coined by Ancel Keys, an American physiologist, in 1960, during the publication of his book How to Eat Well and Stay Well [1]. The scientific evidence for its existence before 1960 is controversial, but it is reasonable to consider a broader temporal context, reaching back to biblical times or even earlier [2]. Interestingly, the strength of the MD is closely linked to ancient biblical culture. In fact, when we look for the seven key components of the traditional biblical diet, namely, wheat, barley, grapes, figs, pomegranates, olives, and honey, we recognize some MD components [2]. Olive oil and wine, the foods discussed in this review, were also included in biblical diets. The evolution of the MD is intertwined with the development of Western civilization. Initially, people in the region adapted their food consumption to the seasons, which was determined by climate and agriculture [3].
The origins of the MD reflect the cultural, societal, and economic growth of the region. The Latin term “Mediterranean” means “the sea in the middle of the earth”, highlighting its historical role as a meeting point between southern Europe, northern Africa, and western Asia. The word “diet” itself is derived from the Greek word “diaeta”, which encompasses not only food but also one’s overall lifestyle. The Mediterranean region spans numerous countries, including Italy, Greece, Spain, southern France, Turkey, parts of North Africa, and the Middle East, resulting in a rich diversity of ingredients and culinary traditions within the MD.
Although the MD has often been associated with the “eternal trinity” of wheat, olive oil, and wine, it also embodies the essence of traditional agricultural practices and dietary habits, which are marked by a culture of sharing and reciprocity [4]. For instance, wealthy urban Greeks favored a diet rich in vegetables, grains, legumes, olive oil, and wine. Barley and wheat were used for oatmeal and bread, whereas various legumes such as fava beans, chickpeas, lentils, lupines, and peas were either prepared in specific dishes or incorporated into flour for bread and oatmeal [5]. A typical MD is characterized by several key features: substantial consumption of vegetables, fruits, legumes, and grains (including complex carbohydrates and dietary fiber), limited total fat intake (<30%), low saturated fat intake (<10%), emphasis on monounsaturated fats, and moderate alcohol consumption (primarily wine) [6]. Over time, the introduction of new ingredients and influences from regions such as Asia and the Americas, including tomatoes, potatoes, maize, beans, and cane sugar, have resulted in changes in Mediterranean cuisine, expanding its culinary horizons beyond indigenous roots. Throughout different historical eras, cultures, religions, agricultural practices, and economic circumstances, the emphasis on food elements within the MD has varied. Additionally, factors such as climate, economic challenges, and scarcity have played a significant role in shaping this diet rather than relying on intellectual foresight or deliberate dietary planning, which is often the approach adopted in modern times to create popular diets. This might explain why it has been challenging to precisely characterize this dietary regimen, as it exhibits unexpected complexity and variations across different countries and historical periods. In 2010, the MD gained recognition as an Intangible Cultural Heritage by the United Nations Educational, Scientific, and Cultural Organization (UNESCO). The official submission made to UNESCO characterizes the MD as “a social tradition rooted in a collection of competencies, wisdom, customs, and traditions that encompass everything from the natural environment to culinary practices. These encompass activities such as cultivation, harvesting, fishing, preservation, processing, cooking, and, most notably, consumption, particularly within the Mediterranean region” [7]. In 2011, the Mediterranean Diet Foundation in Spain, in collaboration with experts in the field, revised the “classic” MD pyramid to accommodate changes brought about by modernization and to integrate cultural and lifestyle aspects [8]. A literature review by Davis et al. (2015) attempted to establish an integrated definition of the MD. In conducting their analysis, the authors considered a variety of criteria, including general descriptive terms, recommended serving sizes of key food groups, and nutrient content [9]. Based on this comprehensive review, the authors defined daily dietary intake as follows: vegetables, 3–9 servings; fruits, 0.5–2 servings; cereals, 1–13 servings; and olive oil, up to 8 servings. In terms of energy content and macronutrient composition, the MD typically consists of approximately 2220 kcal/day, with fat accounting for 37% of the total calories [9]. Regardless of the definition adopted, there is a consensus regarding the health benefits of the MD. Since Ancel Keys’ pioneering research revealed that the dietary practices of Mediterranean countries are associated with longer lifespans and reduced incidences of coronary heart disease (CHD), many more studies have been published [1]. In the following section, we present a summary of the effects of the MD on longevity and cardiovascular diseases (CVDs) derived from observational studies. In our opinion, reliable information regarding dietary effects in humans, especially in those suffering from a specific illness or disorder, requires clinical investigation based on well-constructed preclinical data relevant to the intended human population. Moreover, modern research requires animal studies, such as the analysis of signaling pathways under various conditions or examination of novel dietary approaches. Basic research can benefit from combining animal and human data. Therefore, it is necessary to work with animal models that share genetic similarities. This helps confirm the changes observed in animals after specific treatments in humans. Thus, translational research using observational studies allows for the identification of associations between basic research and humans, even before new experimental strategies are approved and tested in randomized controlled trials (RCTs).

Olive Oil and Wine

In the MD, there are two outstanding liquid foods: olive oil and wine. Ancient civilizations were great consumers of olive oil not only in their daily diets but also as a medicine and source of light with which to illuminate their homes during the Middle Ages. All observational data indicated that our ancestors observed some beneficial effects of olive oil on health. However, the chemical substances or constituents responsible for these effects were not investigated. Wine has been a popular beverage for millennia, with historical evidence dating back to around ten thousand years. Wine has played a significant role in numerous medicinal applications throughout the history of humankind. Approximately five decades ago, scientific research was initiated to investigate the beneficial effects of moderate alcohol consumption on cardiovascular mortality. With the discovery of the French paradox, there has been a substantial boost in biological studies related to wine [60].

Olive oil is a crucial liquid food for the MD as it is the primary source of fat and offers numerous health benefits. EVOO is especially beneficial as it retains its organoleptic and nutritional properties after extraction and contains high levels of MUFA, tocopherols, and polyphenols. From a chemical point of view, 98–99% of the total weight of EVOO is represented by fatty acids, especially MUFA such as oleic acid. Tocopherols, polyphenols, and other minor constituents represent the remaining 1–2% which have antioxidant properties that contribute to the health effects of EVOO. EVOO has been linked to various preventive and therapeutic health benefits for different pathologies, including cardiovascular and inflammatory diseases, obesity, cancer, and neurodegenerative alterations associated with aging [61,62].
Several observational studies have shown that the minor constituents of olive oil mediate its health benefits, attenuate CVD prevalence, reduce the risk of aging-associated diseases, decrease weight in obese patients, and increase longevity [62,63,64]. Over the last decade, positive feedback from the scientific community has motivated researchers to further investigate the role of the minor constituents of olive oil in the treatment of various global pathologies. Olive oil has been attributed to its antioxidant, anti-inflammatory, antiatherogenic, antithrombotic, antiaging, neuroprotective, and antimutagenic properties, with many yet to be discovered [63,65,66]. Several in vitro studies have demonstrated the beneficial effects of olive oil. However, simple and suitable animal models are required in order to determine their nutraceutical advantages.

Wine is a notable source of polyphenols, some of which have antibacterial, antifungal, antiviral, antineoplastic, and anti-inflammatory properties. Furthermore, it has been reported that their therapeutic use is beneficial in several diseases, including CVDs and other diseases associated with aging. These pharmacological effects are primarily associated with their antioxidant capacity [67]. Owing to its greater content of antioxidant substances released from the grape skin and seeds, red wine is considered to have a more protective effect. In a bottle of red wine, the total polyphenol content is around 1.8 g/L, whereas in a bottle of white wine, it ranges from only 0.2 to 0.3 g/L of polyphenols. During the process of producing white wine, the skin and seeds are promptly removed from the must, which is then left for fermentation. Since in vitro antioxidant capacity is closely linked to total polyphenol content, white wines exhibit approximately five to ten times lower antioxidant activity than red wines. White wine also contains significant amounts of hydroxycinnamic acids, tyrosol, and hydroxytyrosol, which are known for their antioxidant properties [65,68]. One noteworthy aspect of this diversity is the variation in antioxidant content among the different types of wine. The specific antioxidants and their levels can vary depending on the grape variety, terroir, and winemaking technique. In recent years, there has been a growing interest in organic winemaking, including its subsets, such as biodynamic, natural, and clean wine. Organic winemaking emphasizes environmentally friendly practices and avoids the use of synthetic chemicals. This approach benefits not only the environment, but also the antioxidants present in wine [69]. Understanding the intricacies of winemaking methods is crucial. For example, biodynamic winemaking incorporates holistic farming principles, enhancing the overall vitality of the vineyard and potentially leading to unique antioxidants in grapes. Natural wines are crafted with minimal intervention, allowing grapes to express themselves fully and potentially preserving a broader range of antioxidants [69]. Additionally, the rise of clean wine, which focuses on transparency regarding what goes into the bottle, ensures that consumers receive wines free from additives and unnecessary processing, retaining the natural antioxidants present in grapes. In this evolving landscape, wine enthusiasts seek not only exquisite tastes but also health-conscious choices, exploring the diverse world of antioxidants in different types of wine, especially those emerging from organic winemaking and its subsets [69].
As previously mentioned, numerous observational studies have been conducted over the past few decades to investigate the health effects of the MD, including moderate alcohol consumption (mainly wine). Moderate alcohol intake is associated with reduced CVDs risk and overall mortality in both healthy and unhealthy individuals. Observational studies examining the correlation between alcohol consumption and mortality have demonstrated that individuals who refrain from consuming alcohol have a greater likelihood of death and cardiovascular incidents [70,71]. In the ATTICA study, an interesting pattern emerged regarding the relationship between low wine or beer consumption and the risk of developing CVD. When individuals consumed less than one glass per week, a distinct and striking correlation was observed, indicating a decreased risk of CVD for wine (hazard ratio: 0.40, 95% CI: 0.17–0.98) and beer (hazard ratio: 0.43, 95% CI: 0.20–0.93) compared to individuals who did not consume alcohol. However, no significant relationship was observed between abstainers and individuals who consumed more than one glass per week. Nonetheless, compared with subjects who consumed less than two grams of ethanol per day, those who consumed between 2 and 10 g, 10 and 20 g, and more than 20 g of ethanol per day had a higher hazard ratio [72]. The Moli-sani study, a large-scale cohort study, examined the association between alcohol consumption and the onset of HF and ventricular fibrillation. The study revealed that moderate alcohol consumption, ranging from 10 to 20 g/day, was associated with a reduced risk of HF; however, it did not exhibit a comparable effect on AF when compared to individuals who abstained from alcohol. In addition, this study examined certain biochemical parameters, including HDL and total cholesterol, in relation to alcohol consumption. Those who consumed more than 48 g of alcohol per day had the highest HDL levels (63 mg/dL). Nonetheless, their total cholesterol levels were the highest in the entire study cohort, averaging 226 mg/dL. In contrast, non-drinkers had HDL levels of 54 mg/dL, and occasional drinkers had total cholesterol levels of 207 mg/dL. The authors found that the mean reduction in the risk of total mortality associated with alcohol consumption was 11%. Additionally, alcohol consumption of >20 g/day was linked to a 13% reduction in the risk of total mortality. Considering the deaths attributed to cardiovascular causes alone, these trends were consistent. Notably, the correlation between alcohol consumption and mortality was similar between men and women. Furthermore, the observed benefits were more pronounced in individuals who favored wine, suggesting that the observed benefits might be attributed to components other than ethanol [73]. In summary, observational studies have demonstrated a protective effect of moderate alcohol consumption on the occurrence of cardiovascular events; however, the precise mechanisms involved are still not sufficiently understood.
Animal trials to study the antioxidant effects of specific foods such as olive oil and wine offer numerous advantages. It helps to identify active components, provides experimental control, establishes dose–response relationships, offers mechanistic insights, has practical applications, and can be cost-efficient, reducing the expense of sociosanitary treatment of determinate pathologies. These insights can contribute to a deeper understanding of the health benefits associated with the components of the MD and can inform dietary recommendations. Wine and olive oils were selected for studies on the antioxidant effects of the MD because of their prominence in the diet, distinct antioxidant profiles, existing scientific interest, ease of isolation and control, consumer relevance, and documented positive health outcomes. Studying these specific components allows researchers to explore the mechanisms responsible for the potential health benefits of the diet.

Zebrafish as a Potential Model to Illustrate Olive Oil and Wine Antioxidant Effects

Traditionally, mammalian models, especially rats and mice, have been the primary models used for research in food science, although their complexity and relatively slow rate of development often compromise the rapid progress in resolving fundamental nutritional questions. However, new animal models such as zebrafish and frogs, whose genomes are fully sequenced and very similar to humans, have emerged [98]; 70% of human genes have at least one zebrafish ortholog. Zebrafish have several advantages as model organisms because of their short life cycle, strong reproductive ability, easy rearing, and low cost (Figure 1). Therefore, the use of zebrafish in nutritional studies provides a valuable reference for researchers in the field of food science [99].
Zebrafish are emerging as very high-throughput and genetically tractable vertebrate models for replicating the developmental environment of disease progression in CVD pathophysiology. A diverse range of gene editing tools have been developed to propagate the modified contractile apparatus or CVD pathophysiology in multiple zebrafish generations, thereby enabling the production of multiple, stable zebrafish generations for the purpose of studying mutagenesis [100]. Zebrafish have a great potential for cardiac function disorders. Their heart rate is similar to that of humans compared to other model organisms. Electrocardiogram (ECG) analysis of adult zebrafish underscores the high similarity in electrophysiology between the adult zebrafish heart and that of humans. Although zebrafish hearts are small, techniques have been developed to measure ECG profiles in embryos. These methods have identified similar ECG features in embryos as early as three days post-fertilization (dpf), which mirrors those observed in adult zebrafish (Figure 1) [101]. Atherosclerosis (AS) is a multifaceted chronic disease that poses a significant threat to human health, and it is a fundamental factor underlying numerous CVDs. In clinical practice, one of the most prevalent diagnostic indicators of AS is the presence of abnormal blood lipid levels, which are frequently observed in patients with AS due to disturbances in lipid levels. Typically, cholesterol is transported to peripheral tissues via LDL and subsequently returned to the liver via HDL through its cholesterol reverse-transport function, ultimately leading to its elimination. However, when exposed to oxidative stress, LDL is frequently oxidized into ox-LDL, which is avidly engulfed by macrophages, giving rise to foam cells. This disrupts normal cholesterol metabolism. These foam cells actively contribute to the formation of atherosclerotic plaques and rupture can precipitate ischemic heart disease or stroke. Ox-LDL also has detrimental effects on AS by damaging the vascular endothelium, stimulating the migration and proliferation of smooth muscle cells, and activating platelets. Consequently, inhibition of LDL oxidation has emerged as a promising avenue for enhancing lipid metabolism, preventing the onset of AS (Figure 1) [102]. Hyperlipidemia can be effectively induced in zebrafish through both dietary and genetic strategies, thus providing valuable models for screening novel lipid-lowering compounds. Currently, there are two established zebrafish genetic models of hyperlipidemia: apolipoprotein C-II (apoc2) and LDL receptor (ldlr) mutants [103,104]. In accordance with the clinical phenotype observed in human patients with APOC2 deficiency, zebrafish mutants deficient in Apoc2 exhibit hypertriglyceridemia when fed a standard diet. Apoc2-deficient zebrafish larvae exhibit lipid accumulation and lipid-laden macrophages within their vasculature, similar to the early atherosclerotic lesions observed in humans and mice [103]. In contrast, ldlr mutants develop moderate hypercholesterolemia when fed a standard diet. This hypercholesterolemic state is further aggravated following a short-term, 5-day high cholesterol diet (HCD) regimen initiated 4.5 days post-fertilization (dpf) (Figure 1) [104]. Feeding zebrafish a high-cholesterol diet (HCD) closely mimics certain processes observed in the early AS stages, including hypercholesterolemia, lipoprotein oxidation, vascular lipid accumulation, and recruitment of myeloid cells to the vasculature [105]. It was observed that feeding zebrafish an HCD for up to 10 days, beginning at 5 dpf, resulted in increased expression of inflammatory markers, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). Additionally, there is a reduction in the expression of the gene encoding the anti-inflammatory protein peroxisome proliferator-activated receptor-γ in the endothelium before myeloid cell accumulation and lipid deposition occur [106]. Numerous studies have demonstrated that telomeres are shorter in human arterial tissue located in vascular beds that are susceptible to AS than in those located in sites that are resistant to AS. Furthermore, the telomere length of white blood cells is shorter in individuals with conditions such as hypertension, diabetes, and coronary artery disease than in unaffected controls (Figure 1) [107]. The regulation of telomere length in zebrafish resembles that in mammalian organisms, making it an ideal model for investigating the link between telomeres and vascular aging (Figure 1) [108]. In conclusion, the zebrafish model has emerged as a valuable tool for advancing our understanding of CVDs, particularly AS and hyperlipidemia. This versatile model combines the practical advantages of in vitro systems and the physiological relevance of mammalian models. Zebrafish share a substantial degree of genetic and functional similarity with humans, making them an ideal platform for investigating the intricate mechanisms underlying these diseases.
Figure 1. Advantages of zebrafish model and how it can be used to study cardiovascular diseases. Simple representation of zebrafish cardiovascular system. From the heart, O2-poor blood goes to the ventral aorta, which branches out, molding the afferent branchial arteries and leading the blood to the gills. Exchanges occur, and the blood becomes rich in oxygen, draining into the efferent branchial arteries and the dorsal aorta. From these vessels, blood travels to the anterior and posterior capillary beds, in which it becomes oxygen-poor again; then, it re-enters the heart through the caudal and cardinal veins. Figure also includes a characterization of the small fish heart, highlighting its four chambers: Sinus venosus, atrium, ventricle, and bulbus arteriosus.
Zebrafish present an exceptionally promising model organism, exhibiting significant potential for unraveling the mechanisms associated with ROS in crucial human diseases. Studies have demonstrated their ability to evaluate antioxidant responses to oxidative stress in vivo, thus making them a rapid and straightforward experimental model. Researchers have explored the effects of oxidative stress on disease progression in both transgenic and wild-type zebrafish. Remarkably, zebrafish embryos are invaluable for conducting in vivo experiments and devising protocols for measuring the oxidative stress in living organisms. Recent advancements in zebrafish research include the generation of homozygous null mutants using a gene-targeted approach. This innovation allows for precise study of oxidative stress-mediated toxicity [107]. Zebrafish are also used as a model with which to evaluate the antioxidant efficacy of polyphenols, including those found in olive oil and wine. Resveratrol (5 µM), an important wine polyphenol, was shown to prevent oxidative stress and mitochondrial damage in zebrafish embryos (Figure 2) [109]. Administration of quercetin (1 µg/L) significantly enhanced the activities of SOD, GPX, and CAT, as well as the expression of the respective genes. Moreover, the expression of inflammatory genes in the liver and intestine of zebrafish is reduced (Figure 2) [110]. In addition, a recent study showed that zebrafish fed a diet supplemented with 200 mg/kg of hydroxytyrosol, the main polyphenol present in EVOO, alleviates fat accumulation, oxidative stress, and mitochondrial dysfunction (Figure 2) [111]. In a previous study, quercetin (50 and 100 mg/kg; administered intravenously) was found to inhibit lipopolysaccharide-induced behavioral changes, neuronal disruption, and levels of TNF-α, IL-1β, lipid peroxidation, and acetylcholinesterase in adult zebrafish (Figure 2) [112]. Administration of gallic acid resulted in a reduction in the neutrophil inflammation index in a zebrafish inflammatory model induced by a high-cholesterol diet (Figure 2) [113]. Arteaga et al. (2021) evaluated the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, β-carotene, and astaxanthin) that are naturally present in foods using a zebrafish embryo model. They found that each compound, except β-carotene, had a protective effect against oxidative stress-induced lethality (Figure 2) [114].
A polyphenolic extract derived from wine lees has a noteworthy impact on the regulation of lipids in zebrafish metabolism. The impact of this extract on lipid metabolism in zebrafish embryos resulted in a decrease in fat reserves and modifications in the expression of crucial genes associated with lipid transport, lipogenesis, and oxidation. Alterations in the concentrations of stearic and oleic acids, as well as polyunsaturated fatty acids and total fatty acids, within the phospholipid and triglyceride fractions of zebrafish embryos, provided evidence of the remodeling and antioxidant capabilities of this polyphenolic extract. This observed effect cannot be attributed to a single active compound but rather arises from the presence of multiple active compounds that exert additive or synergistic pharmacological effects (Figure 2) [115]. An eight-week feeding experiment was conducted to evaluate the impact of grapevine leaf extract on growth, oxidative enzyme activities, immune response, and the expression of antioxidant genes in zebrafish. Three hundred and sixty zebrafish were provided and fed varying levels of leaf extract (0, 0.5, 1, and 2 g kg−1). The research revealed that the leaf extract resulted in an enhancement in the serum and mucus activity of crucial enzymes, such as CAT, SOD, and GPx, which are crucial in eradicating excessive free radicals. Along with an increase in antioxidant enzyme activity, a noticeable reduction in the levels of malondialdehyde was observed. In conclusion, the addition of 0.5−1 g kg of Vitis vinifera leaf extract polyphenols to the fish feed serves to enhance the antioxidant defense system (Figure 2) [116].
Figure 2. Zebrafish model to assess the olive oil and wine antioxidant capacity.
Achievements already made using zebrafish as a research model are of paramount importance for advancing our understanding in various fields, including CVDs, AS, hyperlipidemia, and antioxidant studies. Traditionally, mammalian models, such as rats and mice, have been predominant in food science research, but the complexity of these models and their relatively slow rate of development have often hindered rapid progress in addressing fundamental nutritional questions. However, the emergence of zebrafish as a highly versatile and genetically tractable model has revolutionized our ability to investigate these complex issues. Zebrafish have been used to evaluate the antioxidant effects of various polyphenols found in food. However, it is noteworthy that relatively few studies have used this model specifically to investigate the polyphenols present in two key dietary components, olive oil and wine. Studies conducted with individual polyphenols or in combination can play a crucial role in exploring potential antioxidant mechanisms. Furthermore, the zebrafish model has significant potential for evaluating the benefits of byproducts generated during olive oil and wine production. These by-products, which often contain bioactive compounds, have the potential to be used as dietary supplements. The antioxidant potential of these byproducts could help us to understand their health-promoting properties and develop novel dietary supplements that could enhance overall well-being.


Zebrafish have emerged as a valuable but underutilized model for exploring the antioxidant effects of olive oil and wine within the context of the MD. This review highlights the potential benefits of these dietary components in mitigating age-related diseases, particularly cardiovascular conditions. Specifically, the genetic resemblance between zebrafish and humans presents a promising avenue for converting research findings into insights into the health benefits of olive oil and wine. The unique advantages of zebrafish, such as their small size, rapid development, and transparent embryos, make them an economical and efficient model for conducting experiments involving liquid foods, such as olive oil and wine. These features provide a unique opportunity to investigate the effects of these compounds in a controlled and tractable manner. However, it is essential to acknowledge that zebrafish may not fully recapitulate the intricacies of human physiology and metabolism in an experimental model. This limitation underscores the need for caution when extrapolating the zebrafish findings to human health outcomes. In the future, it will be crucial to encourage further exploration of zebrafish models in this context. Researchers should extend the utilization of zebrafish to the investigation of the constituents of the MD and their impact on age-related ailments. In conclusion, it is imperative to conduct more comprehensive studies that delve deeper into the molecular and physiological mechanisms at work and to compare the findings of zebrafish with those of other animal models and clinical trials.

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