Natural Ingredients Common in the Trás-os-Montes Region (Portugal) for Use in the Cosmetic Industry: A Review about Chemical Composition and Antigenotoxic Properties

The natural cosmetics market has grown since consumers became aware of the concept of natural-based ingredients. A significant number of cosmetics have an ecological impact on the environment and carry noxious and chemically potent substances. Thus, the use of natural and organic cosmetics becomes increasingly important since it is clear that topical treatment with cosmeceuticals can help improve skin rejuvenation. A substantial investigation into the benefits that fruits and plants can bring to health is required. Studies have shown that antigenotoxic properties are linked to anti-aging properties. Several studies have shown potential antigenotoxicity in natural ingredients such as Almonds (Prunus dulcis), Elderberry (Sambucus nigra), Olives (Olea europaea), and Grapes (Vitis vinifera). This review presents an overview of research conducted on these natural ingredients, the most common in the Northeast of Portugal. This region of Portugal possesses the most organic farmers, and ingredients are easily obtained. The Northeast of Portugal also has climatic, topographic, and pedological differences that contribute to agricultural diversity.


Introduction
Aging is a physiological process that affects all structures of the organism, with the particularity of each organ and tissue having its own rhythm of aging [1]. It is characterized by the progressive inability to maintain vital functions, it is harmful, and it is considered to be the final stage of human development, ending with death. Oxidative stress is believed to be a significant factor for speeding up the process of aging [2]. Although several theories have been proposed to justify aging, it is a multifactorial process. It involves the cumulative effects of extrinsic influences and an intrinsic molecular program of cellular aging. According to stochastic theories of aging, free radical atoms are unstable due to the loss of electrons and are continuously produced by body metabolism. Damage occurs when they react violently with other molecules in the cell. They can affect cell maintenance and repair if the damage occurs on DNA. So, we grow old because of cumulative damage to our body cells from external and internal sources [1]. The aging of the skin is primarily associated with the intrinsic genome. However, diet, lifestyle, drug and alcohol history, and environmental exposures are other factors that influence skin aging by affecting DNA [3]. On the other hand, deterministic theories maintain that aging is a direct consequence of a genetic program; the genome and molecular structure are a type of molecular clock [1].
There are many ways in which we are exposed to toxic substances: through the air we breathe, the food we eat, the water we drink, the clothes we wear, cosmetics, radiation exposure, which also has harmful effects. Toxic substance exposure is much more problematic today than it would have been in the past. The environmental repercussions include DNA damage, and this genome instability leads to diseases such as cancer, degenerative Table 1. Major chemicals found, their uses, and problematic properties.

Chemical Uses Problematic Properties Comments Reference
Iodopropynyl butylcarbamate Preservative Allergenic The Cosmetic Ingredient Review concluded that IPBC was safe as a cosmetic ingredient at concentrations less than or equal to 0.1%, but IPBC should not be used in products intended to be aerosolized because of the negative effect on the lungs. [19] Butylparaben A preservative used in personal care products Endocrine disruptor The available data for butylparaben show strong evidence that this compound has estrogenic effects in vivo in Uterotrophic assays performed in immature females. One in vivo study has shown adverse effects on sperm counts following perinatal exposure, while there are conflicting results on the influence of butylparaben on sperm count/quality following exposure of young male rats. Butylparaben causes vitellogenin induction in fish. [20] Resorcinol Numerous uses, including rubber and resins, in cosmetics, pharmaceuticals and hair dye

Endocrine disruptor
Resorcinol has been shown to affect thyroid function as well as estrogen and glucose metabolism. According to human case reports, resorcinol exerts anti-thyroid functions. Resorcinol's long-term administration to permeable (damaged) skin can cause myxoedema (reduced thyroid function). Cessation of exposure causes the myxoedema to disappear. In the human study investigating dermal uptake in healthy individuals, the dermal barrier avoids uptake of resorcinol. [20] Ethylhexyl Methoxycinnamate (OMC)

UV-filter Endocrine disruptor
There is strong evidence that OMC can affect the endocrine system in vivo. Slight but significant increases in uterine weights have been seen in both intact immature and adult ovariectomized rats. In a 2-generation study, a significant decrease in sperm cell number was seen. In contrast, another reproductive study has shown developmental OMC exposure to cause several adverse reproductive effects in offspring, including reduced reproductive organ weights, reduced reproductive hormone levels, reduced sperm counts, and neurobehavioural effects. OMC can also interfere with the hypothalamo-pituitary-thyroid axis in vivo, as many studies have shown reduced levels of thyroxine in the blood. OMC affects the transcription of genes involved in hormonal pathways, including vitellogenin, in most fish studies. [20] Glyoxal Preservative Antimicrobial Mutagenic As for the in vitro studies, Glyoxal is reported to be a mutagen in renaturation assays, unscheduled DNA synthesis (UDS) assays, the Ames assay, the Escherichia coli SOS chromotest, the Bacillus subtilis liquid rec-assay, the rat hepatocyte primary DNA repair test (single strand breaks found, but no DNA cross-linking), sister chromatid exchange assays, Chinese hamster ovary (CHO) and Chinese hamster V79 chromosome aberration assays, the CHO/HGPRT gene mutation assay (only with metabolic activation), the mouse lymphoma L5178y/TK+/− system, and in vivo in the rat, where UDS and increased alkaline elution of DNA were seen in glandular stomach tissue and single strand breaks in liver tissue DNA (not seen in kidney, spleen, pancreas, and lung). It was negative in the C3H/I0T1/2 cell transformation assay and the in vivo mouse micronucleus assay. For the in vivo studies, Glyoxal was mutagenic in most assays. Glyoxal inhibited the effect of DMN in a short-term oral study in rats. [21]  Propylparaben is associated with estrogenic and antiandrogen activity, affecting sperm function and prenatal development, among others. The substance has been detected in biomonitoring studies and human urine and milk. [22,23] Zinc Pyrithione Preservative Antimicrobial CRM Category 1B Zinc pyrithione, which has been classified as a category 1B carcinogen, is now prohibited for use in cosmetic products. [24] Consumers exposed themself to these chemicals, perhaps daily. Since the genotoxic agents are present in many cosmetics, a substantial investigation into the benefits that plants and fruits can bring to health is required since it is clear that topical treatment with cosmeceuticals can help improve skin rejuvenation [25]. Identifying products with antigenotoxic effects is among the most promising research areas in recent years since they might protect against DNA damage and its consequences. Studies have shown that antigenotoxic properties are linked to anti-aging properties [4,26], and these properties are essential to revert genotoxic effects. Therefore, this review presents an overview of research conducted on natural ingredients common in the Northeast of Portugal with an antigenotoxic effect.

Characterization of Trás-os-Montes Region
Trás-os-Montes is limited to the west by the province of Minho, to the south by the Douro, to the east by the Douro River, and to the north by Spain. The climate is sub-Atlantic/continental and Mediterranean, represented by "Terra Fria de Planalto", "Terra Fria de Montanha", "Terra Fria de Alta Montanha", "Terra de Transição" and "Terra Quente". The climate is influenced in the west by the humidity from the Atlantic, in the east by the cold and dryness from the continent, and in the south by the heat [27]. The ecological zoning of Trás-os-Montes highlights the domains: Atlantic (50%), Iberic (26%) and Mediterranean (24%) and four agrotypes, "Granito e Xisto," "Meia Encosta Nordestina," "Terra Fria Transmontana" and "Terra Quente Transmontana" [28]. Physiography is dominated by mountain and sub-mountain hypsometry, from 450-700 m (natural limit of vine culture) to 1500 m [29]. The dominant soils are of granitic origin and the like, associated with schist [30]. In the Valleys of Vila Pouca de Aguiar, Chaves, Vila Real, and Boticas (<700 m), there is a greater variety of crops, such as vines, olive trees, fruit trees, wheat, potatoes, rye, maize, and permanent pastures [27]. This review will focus especially on Elderberry, Almonds, Olives, and Grapes.
Elderberry is commonly found in the north, especially in the Varosa Valley, which because of the surrounding mountains, produces a microclimate favorable for the development of this species [31,32]. The almond tree is one the most widely planted tree crops in the Trás-os-Montes area, occupying an area of 19,206 hectares [33]. The most common varieties are "Parada", "Casanova", "Verdeal" and "Pegarinhos" [34]. Trás-os-Montes is the second Portuguese olive growing region, currently representing between 12 and 15% of the national production of olive oil [35]. The more important varieties are "Cobrançosa", "Madural" and "Verdeal" [36]. Portugal has a wine-growing area of 1/4 to 1/5 of the surface of the significant wine-growing countries in Europe. Of the 343 grape varieties listed, about 230 varieties are considered indigenous to Portugal or the Iberian Peninsula, reflecting the vast and unique Portuguese viticultural genetics. Trás-os-Montes area has 40 indigenous varieties in cultivation [37]. As such, natural ingredients are easily obtained in this area [38]. It is also a region with the most organic farmers, and the climatic, topographic, and pedological differences predispose this region for agricultural diversity [16].

Elderberry (Sambucus nigra L.)
Overview: The Elderberry is a species of the Caprifoliaceae family. It grows in most parts of Europe, North Africa, West Asia, and the USA [39]. The flower blossoms generally in May or June [40]. The fruit matures over six to eight weeks from July to September [41].
Flowers and berries have a large spectrum of applications such as cosmetics, for skin and eye lotions; in the kitchen, for liquors, wine, jelly, and chutneys; in the food industry, as a natural dye; and in medicine, for cold, flu and phlegm [42]. Various studies suggest that Elderberry helps to reduce enhanced production of inflammatory mediators [43], has beneficial effects on blood pressure [44], diabetes, and obesity [45,46], has antiviral, antibacterial, and antifungal activity [47], protects against UV radiation [48] and has laxative and diuretic activity [49]. Tree leaves and barks present diuretic and healing properties. They should only be used for external use since they cause poisoning due to cyanogenic glycosides, the most common being sambunigrin and prunasin. They also contains m-hydroxysubstituted glycosides, such as zierin and holocalin. These compounds are toxic and life-threatening because they can be hydrolyzed, resulting in the release of cyanide [46,50].
Elderberry fruit also has high antioxidant activity. According to Duymuş et al. [55], elderberry extract shows antiradical activity (towards DPPH), having an IC 50 value (concentration needed to scavenge 50% of free radicals) of 123 µg/mL. Espín et al. [56] demonstrated lower radical scavenger capacity towards DPPH than other studied sources of anthocyanins and natural and synthetic antioxidants. Obied et al. [57] also investigated the antioxidant potential of Elderberry, with results indicating that elderberry fruit can protect colon cells against harmful effects of oxidative stress. Elderberry berries have a vibrant chemical composition. According to Imenšek et al. [58], the consumption of 100 g of berries could cover 13% of the recommended daily intake of calcium for women and men. The cellulose content is vital for lowering the risk of developing type II diabetes and heart diseases. Elderberry has antioxidant and anticancer activity thanks to the high content of vitamin C and anthocyanins. It has a protective influence in many chronic degenerative diseases due to the high protein level and seven essential amino acids [52]. Therefore, using fruits, flowers and leaves may constitute a potential protective agent against growth and unfavorable effects of oxidative stress in the human body [54,59].
Elderberry flowers contain much higher amounts of phenolic compounds than the fruits and leaves [54]. The main phenolic compounds found are chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 3-and 5-feruloyl quinic acid, and dicaffeoylquinic acid. As for flavonols, glycosides of quercetin, kaempferol, and isorhamnetin are present in elderflowers [60]. Another group present in elderflowers is flavanols, including catechin, epicatechin and procyanidin trimer, and flavanones [61]. They also possess higher antioxidant activity than berries and leaves. A study performed by Kołodziej and Drożdżal showed that the time to reduce DPPH concentration by 50% was 23-75 s for flowers and fruit 91-133 s. A study performed by Imenšek et al. [58] analyzed the macro and micronutrients in various parts of the Elderberry plant. Elderberry leaves have the highest calcium content (1.38 ± 0.04%) and significantly higher magnesium content (0.73 ± 0.05%) than other plant parts. Phosphorus was also found but in significantly lower proportions (0.30 ± 0.02%). As for micronutrients, iron (115 ± 4 mg/kg DW), manganese (67.0 ± 4.6 mg/kg DW), zinc (39.7 ± 2.0 mg/kg DW), and strontium (35.0 ± 2.2 mg/kg DW) were detected.

Potential Application in the Cosmetic Industry
Elderberry possesses ingredients favorable for cosmetic formulation, such as anthocyanins, which that can reduce oxidative stress by scavenging free radicals, making them a potential anti-aging agent [62]. Elderberry flowers are potential sources of active substances, which in contrast to the antioxidants commonly added to cosmetics, are not subjected to degradation under the influence of ultraviolet radiation and exhibit a high biological activity [48,63]. Limonene and linalool, monoterpenic compounds, are often employed in perfumes, creams, and soaps [64]. Cellulose enhances hydration's physical and structural properties and the skins' oil holding capacity [65].
Relevant studies of Elderberry: Only two studies have been developed since 2012, showing that Elderberry has no mutagenic effect and a high in vitro activity ( Table 2). 3.2. Olives (Olea europaea L.) Overview: Olives are the fruit of the Olive tree, a species of the Oleaceae family. The olive tree is one of the most ancient cultivated fruit trees, and the use of Olives has been ascertained in the late Stone Age at the Kfar Samirin site in Israel [68]. In the Graeco-Roman civilization, olive oil and wine were closely associated due to the similarities in their transformation process and economic importance. They were used not only in daily life but also in trade, religious rites, and art. Since prehistoric times, the olive tree has been of significant cultural importance in that region and still has symbolic and religious significance today [69]. Olive trees are usually distributed in the coastal areas of the eastern Mediterranean basin, the contiguous coastal areas of south-eastern Europe, northern Iran at the south end of the Caspian Sea, western Asia, and northern Africa [70]. The best olive oil should be acidic [71], from the first cold pressure, preferably from of organic farming [36].
Olive oil is rich in molecules with antioxidant and anti-inflammatory functions, such as ω-3 polyunsaturated fatty acids, ω-9 monounsaturated fatty acids, and phenolic compounds [89]. Olive oil is recommended in the diet of pregnant women as it favors the healthy development of the brain and nervous system of the baby before and after birth [90,91]. It also allows better bone mineralization [92]. Olive oil prevents the accumulation of fats in the liver, lowers blood pressure, prevents atherosclerosis, and prevents thrombosis [93][94][95][96]. Olive oil can benefit another group of dementia-related pathologies called tauopathies [97]. It could also prevent diseases related to oxidative damage, such as coronary heart disease, stroke, and several types of cancer [98]. Olive oil could also have an anti-aging effect. Olive oil is widely used in soaps and massage oils [99]. It is an excellent vehicle for macerating aromatic plants and flowers for therapeutic and culinary uses.

Potential Application in the Cosmetic Industry
Virgin olive oil provides a safe and stable emulsion delivery system [100]. The antioxidant activity of olives makes them a candidate for moderating the effects of the aging process on the skin by limiting biochemical consequences of oxidation [101] due to its high squalene content and β-sitosterol, and richness in oleic acid (a skin softener). As such, virgin olive oil is ideal for directly protecting the skin [102]. Oleuropein is used in cosmetics due to its antioxidant, antiviral, antimicrobial, anti-inflammatory, skin protecting, and anti-aging properties [65]. Fatty acids increase hydration, softness and elasticity and act as a protective barrier [65,103].
Relevant studies of Olives: Several studies have been developed since 2005, showing that Olives have an antigenotoxic effect, antioxidant potential, protect cells from induced DNA damage, and minimize cytotoxicity (Table 3).

Almonds (Prunus dulcis)
Overview: The Almond tree is a species of the Rosaceae family. It is the oldest nut crop of southwest Asia, and from that region it has diffused to other regions and continents [106]. Hippocrates was the first to discuss the use of almonds for cold and other phlegmatic disorders [107]. Due to the successive Greek, Roman, and Arab invasions, almond cultivation spread in a narrow horizontal band westward through the Mediterranean to Spain [106]. Almonds can be eaten as dried fruit and are also used for pastry and liquors. The almond shell is used for biofuel [108].

Potential Application in the Cosmetic Industry
Sweet almond oil is used in the cosmetic industry, especially in dry skin creams, anti-wrinkle and anti-aging products. It enhances the glow and fairness of the skin. It is used in over 280 cosmetic formulations in concentrations up to 50% [119]. When mixed with white wine and honey, it can be applied to urticaria and wound healing [120]. Almond oil is one of the most popular oils used in aromatherapy and massage therapy since it is suitable for any skin type. Since it contains large amounts of vitamins E and K, it helps skin regeneration and elasticity [121].
Relevant studies of Almonds: Table 4 shows the results of antigenotoxic studies of Almonds. Fermented nuts are considered to be not genotoxic in HT29 cells [124]

Grapes (Vitis vinifera)
Overview: Grapes are the fruit of the Winegrape, a species of the Vitaceae family. It is one of the most consumed fruits globally, mostly in juice and wine, but some are destined for fresh consumption or dried into raisins [127,128]. The archaeological record suggests that cultivation of the domesticated grape, Vitis vinifera subsp. vinifera began 6000-8000 years ago in the Near East from its wild progenitor, Vitis vinifera subsp. sylvestris [128]. After pruning in January, clusters are formed in the spring, and it is during the summer that the Grapes gain color, aroma, and taste. Between September and October, when the Grapes are already ripe, that is when their weight, color, and acidity present the ideal conditions for wine production, the harvests take place [129].
Grapeseeds contain 8-20% of oil and are a by-product of the winemaking process, and their oil content is traditionally extracted using either mechanical techniques or an organic solvent. Cold-pressing is a method of oil extraction involving no heat or chemical treatment and may retain more health-beneficial components [132][133][134]. The total amount of polyphenols extracted from grapeseed oil by the cold-pressing method is about 2.9 mg/kg. They are a rich source of catechins and procyanidins [135]. Grapeseed oil contains many phenolic compounds, including flavonoids, carotenoids, phenolic acids, tannins, and stilbenes. However, the polyphenols content in grape seed oil is low (0.013-0.019% of total phenolic compounds) [136]. They also contain palmitic, 11-eicosenoic, oleic, linoleic, α-linolenic, and arachidic acids [137]. Flavan-3-ols are also present in the seed, namely catechin (67.71 ± 11.37 mg/100 g) and epicatechin (57.93 ± 22.12 mg/100 g) [130]. Transresveratrol is also present in grapes (0.62 ± 0.24 mg/kg). Benzene compounds can also be found in grapes, the most abundant being benzyl alcohol (540-1140 µg/kg of grape) and homovanillic alcohol (380-960 µg/kg of grape). Grapeseed oil has a great vitamin E content, ranging from 1 to 53 mg per 100 g of oil [138]. Vitamin E contributes to has a high antioxidant activity and neuroprotective and antitumoral properties [139,140]. For this reason, grape seed oils have been suggested to delay the aging process and prevent some chronic diseases. Grapeseed oil has antioxidant, anti-inflammatory, antimicrobial, and antitumoral properties [132,136,139,141]. It has also been used as edible oil.
In winemaking, a large amount of grape pomace is produced that is rich in polyphenolics and highly beneficial for human health, as phenols are helpful for skin ultraviolet (UV) protection. Incomplete extraction of compounds such as polyphenols during winemaking leads to about 70% of the initial active substances remaining in grape pomace waste, 20-30% in peels, and 60-70% in seeds [142][143][144]. Some groups of polyphenols present in V. vinifera L. grape pomace are flavanols (flavan-3-ols), flavonols, anthocyanins, condensed tannins, and proanthocyanidins [145].

Potential Application in the Cosmetic Industry
Grapes can be used in cosmetics as follows: bud extract, flower extract, fruit extract, fruit powder, fruit water, juice, juice extract, leaf extract, leaf oil, leaf/seed skin extract, leaf water, leaf water, leaf wax, root extract, seed, seed extract, seed powder, shoot extract, skin extract, skin powder, vine extract, and vine sap. The seed extract is used in 463 cosmetic formulations, fruit extract in 219 cosmetic formulations, and leaf extract is used in 78 cosmetic formulations [146]. Grape seeds contain fiber (40%), oil (16%), protein (11%), sugars, and minerals being a rich source of proanthocyanidins. Proanthocyanidins are potent antioxidants and have free radical scavenger activities [147]. The various components of grapes make them an excellent ingredient to be added to cosmetic formulations. Resveratrol's proven ability to penetrate the skin barrier and anti-aging activity makes it an excellent complement for cosmetic formulation. It can also stimulate fibroblasts' proliferation and increase the concentration of collagen III [148]. Phenolic acids and flavonoids, such as ferulic acid, caffeic acid, gallic acid, and proanthocyanidins, are efficient protectors by reducing oxidative stress and may be essential in cosmetic surgery formulation for post-sun skin care [149]. Grapes also provide phenolic components, like anthocyanins, gallic acid, catechin, epicatechin, conjugated flavonoids, oleic, linoleic, and linolenic acids, counteracting symptoms of epidermal aging and delaying the process of photoaging [150,151]. Currently, to optimize sun protection and photostability, sunscreens use natural antioxidant composition. Scientific evidence has shown the benefits of polyphenols' topical and oral use from some plant species against UV radiation, including Vitis vinifera [120,121,146,152].Relevant studies of Grapes: Table 5 shows relevant studies developed on Grapes since 1999.  Peel, seed, leaf, and stalk of strawberry grape Seed and stalk extracts together with the leaves of the grape showed high antigenotoxic activity [159]

Discussion
Micronucleus, Comet Assay, and SMART (in Drosophila melanogaster) are widely used tests for genotoxicity of potential substances and detect DNA damage evidenced by strand breaks in various organisms and tissues. Excessive generation of free radicals may result in DNA damage. At least two major human problems, aging, and carcinogenesis, involve DNA damage. The possible protective effect of these natural ingredients against oxidative DNA damage should be investigated. We are exposed to noxious substances daily. Table 6 presents different assays used in genotoxicological studies, as well as the advantages and disadvantages. In vivo genotoxicity assays are performed on Drosophila melanogaster. It has been used extensively to analyze many chemicals with different action mechanisms, showing a vast ability to detect genotoxic effects. [160,166,167].
Easy to perform Inexpensive Shows high sensitivity, specificity, and accuracy Detects loss of heterozygosity by deletions, point mutations, mitotic recombination, and nondisjunction [166,168] Time-consuming Delay in the development of the larvae [168,169] Micronucleus Test A small, chromatin-containing round-shaped body that is visible in the cytoplasm of cells. Can originate from acentric fragments, segregation of chromosomes, dicentric chromosome breakage, chromosome instability, or aggregation of double minutes. A rise in the frequency of micronucleated cells is a biomarker of genotoxic effect that can reflect exposure to agents with clastogenic (chromosome breaking; DNA as target) or aneugenic (aneuploidogenic; effect on chromosome number; mostly non-DNA target) modes of action. [160,[170][171][172][173][174].
Reliable identification of cells that have completed only one nuclear division Easy to perform Less time required Inexpensive Allows the detection of chromosome and genome mutations and co-detection of apoptosis and necrosis, measuring the extent and progression of nuclear division in a dividing cell population Detects dicentric bridges, chromosome loss, nondisjunction, excision repair events, and Hypoxanthine guanine phosphoribosyl transferase variants Allows automatic scoring [175,176] Low sensitivity Detects only acentric fragments Cell division is needed for effective use [176] Chromosomal Aberration Test It can be performed in primary peripheral blood lymphocytes or established cell lines, such as Chinese hamster ovary cells. Recognizes agents that cause structural chromosome aberrations (clastogenesis) produced by various mechanisms [177,178].
Allows identification of all chromosome mutation types and co-detection of mitotic indices [178] Need for cell cultivation and highly skilled and experienced personnel Time-consuming Expensive Automatic scoring is not possible [176,179] Bacterial Reverse Mutation Test Uses amino acid-requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substituting, adding, or deleting one or a few DNA bases pairs. Detects chemicals that induce mutations that revert mutations present in the tester strains and rehabilitate the functional capability of the bacteria to synthesize an essential amino acid. [180][181][182][183].
Allows replicates to be made and results to be obtained relatively quickly Allows inexpensive study of a large number of test materials inexpensively Enables identification of the molecular mechanism effect of test material Very versatile test [183,184] May not be appropriate for the evaluation of certain classes of pharmaceutical, for example, highly bactericidal compounds such as antibiotics, and those which are thought (or known) to interfere specifically with the mammalian cell replication system Can produce false positives Possible interference from biological samples, e.g., plant extracts that contain amino acids (histidine) with the test system [180,184] These natural ingredients have been shown to possess a variety of biological activities and to hold therapeutic promise. Elderberry presents fewer studies in comparison with the other natural ingredients. The difficulty of accessing such natural ingredients in some countries and the fact that it is a wild tree can be some of the reasons for the lack of studies on this tree. On the other hand, grapes and olives have various in vivo and in vitro studies. Also, some of their studies have been performed in D. melanogaster and human lymphocytes. D. melanogaster is one of the preferred organisms for toxicological research. In recent decades, it has been used as a model to elucidate human diseases and also for toxicological studies [185][186][187]. The use of D. melanogaster in experimental studies met the standard of the European Centre for the Validation of Alternative Methods (ECVAM): reduction, refinement, and replacement (3Rs) of laboratory animal usage. D. melanogaster as a model raises few ethical concerns [188]. The short life cycle, the distinct developmental stages, the availability of various tools and reagents, known genome sequence, and the physiological similarity of D. melanogaster with humans (namely on dietary input, xenobiotic metabolizing system, antioxidant enzymes, and DNA repair systems) make them an excellent in vivo model organism to rapidly test toxicity in the whole organism and elucidate the molecular mechanisms underlying the toxicity [160]. In biomedical sciences, experimental evidence has implicated oxidative stress in the pathophysiology of several disease conditions [189]. However, the precise role of oxidative stress in the pathology of diseases is far from being known. Therefore, the study of oxidative stress in animal models is of particular importance at present. D. melanogaster has been used to assess oxidative stress and antioxidant markers [190,191]. They have been used in genotoxicity and antigenotoxicity studies. In vivo tests for detecting somatic or, germinative mutations are especially valuable.
Human lymphocytes are used as surrogate tissue, as they are easily obtained, are available in large numbers, do not require cell culture, are diploids, and are almost all in the same phase of the cell cycle. Although the Comet Assay is well accepted among the scientific community, there are issues regarding standardization among laboratories [192]. Therefore, new methods for DNA damage assessment would be beneficial to improve research on DNA damage repair and antigenotoxicity.

Conclusions
The present review synthesizes the most accurate evidence of the antigenotoxic capacity of some natural ingredients common in the Northeast of Portugal. Almonds, Grapes, Olives, and Elderberry proved to have an antigenotoxic effect. Natural occurring antigenotoxicity in natural ingredients could strongly counteract genome instability.
Even though these ingredients are already being used in cosmetics, the lack of antigenotoxicological studies makes it crucial to investigate further how to incorporate them in cosmetics to benefit human health. Studies have shown that plants, fruits, and vegetables with antigenotoxic properties show promising results for the cosmetic industry [193][194][195]. Additional investigation can be carried out, namely, evaluating the cosmetic properties of the natural ingredients towards promoting DNA integrity. Using Comet Assay and SMART, evaluating genoprotection, longevity, and prolificacy of the natural ingredients in D. melanogaster could reveal exciting results.

Acknowledgments:
The authors would also like to thank Malcolm Purves (professional translator) for the manuscript proofreading and editing.

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