Grape Infusions: Between Nutraceutical and Green Chemistry

: By tradition, herbal infusions have been mainly consumed for their pleasant taste, but, nowadays, the consumer, along with the pleasantness of drinking a savory beverage, also looks for their health benefits. Grapes and grape/wine by-products are a rich source of health-promoting compounds, presenting great potential for the development of new beverages. Moreover, grape-infusion preparation is no more than a sustainable or green way of extracting polyphenols and other nutraceutical compounds from grapes and grape leaves. In this review, we summarize the benefits of drinking grape infusions and discuss the sustainable processes of extracting potential nutraceutical compounds from grapes and grape by-products, which are often considered fermentation waste and are discarded to the environment without proper treatment. Solvents, Deep Eutectic Solvents (DESs), and Natural Eutectic Solvents (NADESs)


Introduction
Wineries produce a large amount of waste during the grape-harvesting and winemaking periods. It is estimated that 1 billion kg/y of grape stalks are produced worldwide [1] and the total amount of solid waste produced, after winemaking, may well account for over 30% (w/w) of the grapes used [2].
For some time now, there has been a greater demand for sustainable food production. Low use of inputs, zero waste, aggregation of social values, and minimization of environmental impact are essential premises for food production in the world today. To implement sustainable practices in the wine industry, and in all its by-products, aiming to achieve a "Greener Enology/Viticulture", it is necessary to fully understand the constitution of the plants and Enological/Viticultural processes to better profit from their use, in an environmentally friendly way.
Vitis vinifera L. is a species that has always accompanied man over time [3,4]. It is considered one of the most important cultures in the world (77.8 million tons in the world production of grapes in 2018 from 7.4 million hectares [5]), both in terms of economic valuation and occupied area [6]. Wine and spirits are the main products of the vine industry. However, they are not the only products. Thus, fresh grapes, raisins, juices, jellies, molasses, jam, and dishes cooked with vine leaves, are also foodstuffs with nutraceutical characteristics produced from the approximately 5000 cultivars of Vitis vinifera used in the grape industry [4,7,8]. As an example of benefits related to human health, several studies cited by Cosme et al. [9] in grapes and their derivatives, where anti-inflammatory, anticancer, antimicrobial, antiviral, cardioprotective, neuroprotective, and hepatoprotective activities are attributed.
. All parts of this plant have ingredients that are essential for making an infinite variety of value-added products. However, we are going to focus our work on grape berries and leaves once they are the most profitable organs of this species and are described below.

Grape Berry
The vine bear fruit in a cluster (Figure 2A). The fruit is botanically called berry as it reserves the seeds protected by a pericarp inside [11]. Depending on the cultivars, the size and shape of the berries vary greatly. The berries may have a rounded, ovoid, oblate, or elliptical shape [10]. The changes in color, size, aroma, flavor, and texture, observed throughout the development of the berry, are due to changes that occur in the contents of the cell vacuoles [13]. With an integrated structure, three different layers of tissue can be observed in the berries:


Exocarp-includes the cuticle (a waxy layer), epidermis, and hypodermis, consisting of 6 to 8 layers of cells smaller in size than the mesocarp cells [8]. However, between 5 to 18% of the fresh weight of the berry is attributed to the skin [14,15]. The epidermis is formed by tangentially elongated cells, one or two layers. The hypodermis can represent four to five layers of cells, in which the outermost layers of the cells have a rectangular shape, as opposed to the innermost layers in which the cells are polygonal [8,16].  Mesocarp ( Figure 2B)-rich in anthocyanins in red cultivars, occupies between 85-87% of the grape volume. Is made up of rounder and polygonal cells, with thin cell walls, very vacuolized [9,17]. It is exactly in these organelles, the vacuoles, where it is possible during the ripening of the grape to find sugars, organic acids, water, and ions [13].  Endocarp-includes the pericarp in whose locules can be found from two to four seeds ( Figure 2) [18]. Of all parts of the vine, it is in the seeds that the highest concentration of phenolic compounds is found [19].

Grape Leaves
Although the grapevine is a particularly important crop in the world economy, and the use of grape leaves in gastronomy is already very ingrained in some cultures, namely in Europe, North Africa, and the Middle East [4,8,20], it would be important to foster new ways of valuing this nutritionally rich by-product [21][22][23]. Thus, knowledge of the ultrastructure of the leaves of the different cultivars is important to assess the viability of their use in human food. According to Vilela and Pinto [24], some varieties of Vitis vinifera have crystals of calcium oxalate in the leaves, making its use unfeasible for human consumption, due to the potential risk of causing kidney stones [25].
Typically, the vine leaf has a hand-like shape, with three lobes ( Figure 3A) [26][27][28], although its shape may vary with the cultivar. With a heterogeneous mesophyll, it is easily recognized that the upper surface of the leaf is structurally different from the lower surface. Thus, associated with the upper surface the palisade chlorophyll parenchyma is identified, where the cells are rod-shaped, close together and with few spaces between them. The lower surface also allocates chlorophyll parenchyma, but with round cells with an irregular contour and many intercellular air spaces, named spongy parenchyma (Figure 3B,C). This difference in the structure of the chlorophyll parenchyma associated with the two pages of the leaf implies an uneven distribution of chlorophyll on both sides of the leaf. Thus, the leaf shows different coloring on the two pages, darker green on the upper surface and light green on the lower surface [29]. The entire leaf is traversed by a vascular system organized in bundles composed of xylem and phloem ( Figure 3B,C), with greater representation in the main vein. Associated with these, it is still possible to identify support tissue, mostly fibers [8] The entire leaf is covered externally by a protective tissue, the epidermis, where stomata are mostly distributed on the lower surface. .

Nutraceutical Compounds of Grape Berries and Grape-Leaves
The economic importance of grape and wine production in many countries is widely recognized. In the 21st century, world wine production has remained relatively stable. In 2016, it reached 267 million hectoliters, with geography very dispersed throughout the world, with Europe holding a dominant position with 60% of production, Figure 4 [30].  [30].
The relevance of grape production and its by-products is not limited to economic factors, but also its nutritional and functional properties. This material is considered a rich source of bioactive compounds, thus being of interest to the cosmetics, pharmaceutical, and food industries.
The phenolic compound's potential protective role has been widely studied. Its antioxidant properties are attributed to its ability to remove free radicals and inhibit the formation of reactive species during metabolic processes, to prevent damage to biomolecules such as lipids, proteins, and nucleic acids, contributing to the prevention of cell damage [31,32]. Even more, it may involve block cell propagation and apoptosis as well [33].
Polyphenols are a major class of bioactive phytochemicals [33]. Cassidy and co-workers [34] estimated the existence of more than 8000 known phenolic compounds, which include several subclasses such as phenolic acids, flavonoids, stilbenes, lignans, and tannins.
Structurally based on a 15-carbon skeleton arranged in three rings (C6-C3-C6) (see Figure 5) [31], flavonoids comprise a large group of phenolic compounds, including flavan-3-ols, flavonols, flavones, isoflavones, flavanones, and anthocyanins [35]. Most flavonoids are transformed in the small intestine before being absorbed. Although most ingested flavonoids are absorbed in the small intestine, there is also significant absorption in the large intestine [36,37]. Readers should note the example of green tea in which, according to Selma et al. [38], a part of its flavonoids is metabolized in the small intestine before being absorbed, and another part is metabolized by the large intestine microbiota forming phenolic acids. Crozier et al. [36] also mention that the colon bacteria influence the absorption of several flavonoids, which may determine their bioavailability in the systemic circulation.
Among the various foods rich in flavonoids are tea, grapes, and red wine [32]. The grape seed extract was recently shown to improve the disturbance and metabolic function of intestinal flora in rats [39]. Several studies showed that ingestion of flavan-3-ol from various dietary sources has positive effects on cardiovascular disease, reducing the risk of diabetes, cholesterol levels, blood pressure [40,41]. What is more, Arts et al. [42] found an inverse relationship between coronary heart disease and consumption of flavanols, flavonols, and flavones. Peter et al. [43] report an 11% reduction in the risk of cardiovascular disease demonstrated by the ingestion of three cups of tea a day. Age-related vascular problems can also be avoided by consuming flavanols and flavonols [44]. Anthocyanins, as with other polyphenols, also have anti-inflammatory properties and positive vascular effects [34]. Indeed, ingestion of flavonoids and anthocyanins has led to reduced mortality related to cardiovascular disease [45].
Over the past two years, many meta-analyses have shown a positive relationship between cardiovascular disease and COVID-19 disease severity [46][47][48][49][50]. On the other hand, resveratrol is known for its antiviral effects against several respiratory tract viruses [51] including MERS-CoV [52]. So, several studies have already anticipated the potential implementation of resveratrol in the treatment of COVID-19. Yang et al. [53] demonstrated that resveratrol inhibits SARS-CoV-2 replication in cultured Vero cells and, as a result, its potential utility as a new therapy has been proposed. Wahedi et al. [54] recognized that resveratrol is a promising candidate for drug development against COVID-19. Furthermore, with its proven antithrombotic effects, resveratrol has been proposed as an adjunct treatment to delay and improve vascular thrombosis in the course of COVID-19 [55]; it has a beneficial effect on the modulation of inflammation without compromising the adaptive immune response [56]. Given its antioxidant effect, it may also contribute to the prevention of viral protein binding in host cells [57]. The association of resveratrol with essential minerals is beneficial in reducing the mortality of patients with COVID-19 [58]. Resveratrol is also recognized as a cardioprotective supplement for mitigating cardiotoxicity associated with chloroquine/hydroxychloroquine treatment in SARS-CoV-2 patients, enhancing its antiviral potency [59].
Many studies have confirmed the epidemiological associations between regular consumption of fruits and vegetables and an inverse relationship with the progression of several types of cancer. It has been shown that polyphenols found, for example, in red wine, grapes and fruit juices, influence carcinogenesis, and cancer development at the cellular level [33]. Phenolic compounds are also involved in the prevention of diabetes disease, due to their proven remedial benefits in reversing the metabolic processes of type 2 diabetes mellitus [60,61].
According to Montalbano et al. [62], several authors have focused their studies on white grape juice extract (WGJe), claiming it is rich in flavonoids. The study on mitochondrial activity, in an ex vivo experimental model of activated lymphocytes from human subjects, stands out, and the results highlight the potential use of WGJe in improving mitochondrial functioning, leading to aging delay [63]. Moreover, Montalbano and collaborators [62,64], in two studies, evaluated the anti-obesity effect in vivo WGJe in the induced diet in obese zebrafish and found that the flavonoid-rich white grape juice extract leads to a reduction in adipose tissue and an anti-effect-obesity, which may represent a new approach to solving the problem of excess weight.
White grape juice extract (WGJe) also possesses neuroprotective and anti-microbial activities, and those have been tested, both in vivo and in vitro models. Filocamo et al. [65] found that WGJe can exert both bacteriostatic and bactericidal activity, inhibiting, in vitro, Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli. Moreover, WGJe repressed the biofilms formation of E. coli and Pseudomonas aeruginosa, with a dose-dependent effect. Giacoppo et al. [66] investigated, in vivo, the possible neuroprotective role of a polyphenolic white grape juice extract (WGJe) in an experimental mice model of autoimmune encephalomyelitis, the most used model for multiple sclerosis. The mentioned authors found that the oral administration of WGJe (20-40 mg/kg/day) may exert neuroprotective effects against multiple sclerosis, diminishing both clinical signs and histological score typical of disease (lymphocytic infiltration and demyelination).
Even after the fermentation/maceration processes of the grapes, more than 70% of polyphenols remain in the pomace, which makes it an interesting source of nutraceuticals in food supplements, to enrich beverages, or even as a substitute for synthetic antioxidants [80]. Grape seeds can also be used to produce grape seed oil, rich in unsaturated fatty acids and phenolic compounds [81,82] or as individual food supplements in the form of grape seed powder or grape seed extract, becoming a valuable source of health-promoting nutraceuticals [83][84][85][86]. The cosmetic industry also invests in facial creams, facial serums, anti-aging creams, and anti-wrinkle creams, among others, which include grape polyphenols [87].
According to Pastrana-Bonilla et al. [19] and Rockenbach et al. [88] grapes and grape leaves are a rich source of phenolic compounds, although their concentration will vary according to the structure evaluated: seeds (2178.8 mg/g gallic acid equivalents), skin (374.6 mg/g) or leaf (351.6 mg/g).
In different concentrations, grape berry tissues are still rich in other types of compounds [99]:


Sugars such as glucose, fructose, and sucrose.  Organic acids such as malic, tartaric, and citric acid.  Aroma precursors, which could be volatile and non-volatile.
In addition to its wide use in the gastronomy of some cultures, the leaves of the vine have also been used for medical purposes, such as to stop bleeding, inflammation, pain, and diarrhea [89]. Leaves, usually discarded by grape farmers, are a rich source of vitamins, minerals, crude fiber, and phenolic compounds [4,100].
Resveratrol and some of its derivatives are present in various extracts of grape leaves, regardless of variety. Trans-and cis-resveratrol and astringin (a stilbenoid), are the stilbenes that can be mentioned because they are present in some red grape varieties such as Syrah, Merlot, Vranac, and white varieties such as Marastina and Posip [104]. The phase of the plant's vegetative cycle is also a determining factor in the concentration of phenolic compounds, increasing with vegetative development: May (2911 mgL −1 gallic acid equivalents), September (3339 mgL −1 gallic acid equivalents) [104].

Ways of Extracting Nutraceutical Compounds from Grapes and Grapes by-Products
Grape marc, fermented or semi-fermented, is constituted by high contents of biodegradable compounds and solid by-products, consisting mainly of grape stalks, seeds, skins, and wine lees. Grape marc is either sent to distilleries for obtaining ethanol or grape-spirit, also called "Aguardente Velha" or Cognac when submitted to double distillation, rectification, and wood-aging 105, or is discarded as natural waste. Recently, the productive use of such by-products can offer economic advantages once they constitute an abundant feedstock for biodiesel production in wine-producing countries 106, or they can be used-due to a high level of polyphenols that possess antioxidant and radical scavenging activities 78,107]-to obtain nutraceutical compounds, such as polyphenols, used in food supplementation 108, in new nutraceutical beverages formulations 109, in pharmaceutical formulations 110 and even in cosmetic products 111.
To avoid spoilage and time-degradation of grape marc, drying is used as a preservation method, inhibiting the growth of microorganisms, and delaying chemical reactions 112,113. Natural solar drying, or, when the sun is not available, hot-air drying is the most used method to produce dried enological and agricultural products due to the smaller operating cost. However, phenolic, and other nutraceutical compounds, that can be extracted from these biological wastes are heat-sensitive substances 114, therefore temperatures lower than 60-70 °C are considered suitable for the pre-treatment of grape marc and other agricultural by-products to retain its bioactive compounds 24,115.
In the case of grape marc, when the focus compounds are soluble or weakly bound, such as polyphenols, the most common technique of extraction is solid-liquid extraction, mainly based on organic solvents (SLE) 87. The most used procedures, in the laboratory, are mechanical agitation and Soxhlet extraction (SOX) ( Figure 6). In the 1st step ( Figure  6A) the solid matrix is placed in SOX thimble and the solvent is heated under reflux. Then, condensation and extraction with "fresh" solvent is carried out. Solutes are transferred from the extraction chamber into the reservoir ( Figure 6B). Continuous repetition of the extraction occurs ( Figure 6C). Finally, exhaustive extraction is complete ( Figure 6D) 116. The total extraction time ranges between 12-24 h, making this a time-consuming technique. Additionally, the most common extractors use large amounts of purified solvent (several hundred milliliters) causing potential environmental pollution 116,117.
Solid-liquid extraction at a semi-industrial scale can be performed using ethanol/water solutions. Escobar-Avello et al. [118] worked with grape canes (vine shoots), a viticultural by-product containing high levels of phenolic compounds. They used grape canes of Vitis vinifera cv. Pinot noir, and a 750 L pilot-plant reactor under the following conditions: T = 80 °C, time 100 min, solid/liquid ratio 1:10. The extraction solvent was constituted by ethanol: water solution (80:20, v/v).
Other solid-liquid extraction solvents, including green solvents and methods, will be referred to further in this manuscript. Due to the disadvantages presented (loss of compounds due to hydrolysis, oxidation of the target compounds during extraction, long extraction time, and environmental pollution 119 this technique, and other similar ones, tend to be replaced by novel extraction methodologies including mechanical extraction by ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) 1, and greener technologies such as supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) 120. The latter can achieve high yields of bioactive compounds with the use of food-grade and non-toxic solvents [121,122]. Degrading enzyme mixtures of the grapes cell wall polysaccharide can also be used, leading to the recovery of compounds with bioactive properties [123,124].

Ultrasound (UAE) and Microwave (MAE)-Assisted Extraction
The winemaking processes of grape crushing and maceration, especially in red winemaking, are the primary ways of extracting nutraceutical compounds from grapes, such as polyphenols 125 and, of course, they are extracted into the wine, making it a beverage rich in this type of nutrients. However, even during red winemaking, only a minor part of grape phytochemicals is extracted into the wine, leaving the pomace or grape marc, still rich in phenolic compounds (mainly flavonoids, phenolic acids, stilbenes, and anthocyanins), dietary fibers, proteins, lipids, and minerals. White grape marc is not subject to maceration, keeping almost all phytochemicals of grapes, so they represent a hugely important source of bioactive compounds 126.
Accentuated Cut Edges (ACE) is a newly developed grape-must extraction technique. It can be applied to mechanically break grape skins into small fragments while maintaining seed integrity 127. This technique enhances the extraction of phenolic components while avoiding the extraction of bitter and astringent compounds from seeds 128, thus it does not affect the textural characteristics of wines.
In an article published by Grillo et al. 1 two techniques are mentioned that prevent the usage of organic solvents. Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE).
Ultrasound is a key technology in achieving the objective of sustainable "green" chemistry and extraction. By using ultrasound, several benefits can be achieved: (i) full extractions are completed in a short time with high reproducibility results, (ii) reduction in the use of solvents, (iii) simple equipment manipulation and work-up, (iv) final products with higher purity, (v) post-treatment of wastewater elimination, and (vi) consumption of only a fraction of energy needed for a conventional extraction (Soxhlet extraction, maceration, or Clevenger distillation) 129. When ultrasound waves cross a medium at a frequency of 20-100 kHz 130 several physical phenomena develop, and macro-turbulence and micro-mixing are generated in the case of liquid-solid systems. The combined effect of several independent factors (fragmentation, erosion, capillarity, destructuring, and sonoporation) leads to the disruption of, for instance, vegetal matrices, and a higher solvent-penetration coefficient, thus significantly improving mass transfer kinetics during an extraction procedure [129,130]. Labanca et al. 131 investigated the effect of different extraction processes on the polyphenolic profiles of V. vinifera L. cv. Aglianico leaf extracts and the antioxidant, anticholinesterase, and antityrosinase activities. Ultrasound-assisted extraction combined with a solvent made by ultrapure water and ethanol (50:50) led to an extraction yield of 13.81%. So, this methodology allows the use of green solvents, such as ethanol (EtOH) and water (H2O), leading to the production of edible extracts and products. Gerardi et al. 132 exploiting strategies for reuse of skins separated from grape pomace as an ingredient of functional beverages using ultrasound-and microwave-assisted extraction (MAE) of and acidified water as a solvent, were able to obtain a high value of Trolox equivalents antioxidant capacity (high value of antioxidant capacity assay based on scavenging of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical anions (ABTS •-)). MAE, when employed for the extraction of bioactive compounds, presents the advantage of significantly reducing the extraction time 133. This technique has been employed for polyphenols extraction from wine waste improving the efficiency of polyphenol extraction from grape marc by 57% 80,134.

Supercritical Fluid Extraction (SFE) and Pressurized Liquid Extraction (PLE)
Supercritical Fluid Extraction (SFE) is a technique that can be applied on a laboratory and industrial scale and is characterized by being considered a green procedure. CO2 is the most commonly used solvent, and it does not contaminate the final extract [135]. The supercritical form of CO2 (SC-CO2) increases its diffusion properties and allows its penetration into the material resulting in a high mass transfer in a low extraction time, and its critical point is quite easily reachable, even at an industrial scale, compared to other molecules [136]. Another advantage of SFE is that CO2, being a non-polar solvent, can be combined with other more polar green solvents, such as EtOH [135]. The first scientific work on SFE from grape by-products used CO2 modified with 5% methanol at 350 bar and 50 °C for the extraction of phenolic compounds [137]. Another similar work involved the parameters being changed to 60 °C, 250 bar, and 20% ethanol as a CO2 modifier, which was performed by Chafer et al. [138], aiming to extract polyphenols from five grape skin varieties. Pressurized liquid extraction (PLE) relies on the use of "green" solvents at controlled temperature and pressure to extract target components from various matrices 139. This technique also named Accelerated Solvent Extraction (ASE) is carried out statically by applying heat and pressure to extraction solvents and samples 140. PLE has been applied to extract anthocyanins and total phenolics from dried red grape skin 141; trans-resveratrol from grapes 142 and several antioxidants' compounds from different varieties of red grape pomace 121 and from Vitis vinifera leaves 143.

Ionic Liquid (ILs) Solvents, Deep Eutectic Solvents (DESs), and Natural Eutectic Solvents (NADESs)
Greener technologies are being studied for nutraceutical compounds extraction, considering the need to increase efficiency, selectivity, and low energy consumption. One already available solution is the use of alternative solvents, generally reported as green solvents: ionic liquid (ILs) solvents, deep eutectic solvents (DESs), and natural eutectic solvents (NADESs) 144. Eutectic solvents are homogenous liquids whose melting points are lower than the individual melting points. The name comes from the Greek "εύ" (eu = well) and "τήξις" (tēxis = melting) 144.
Ionic liquids (ILs) are melted salts that remain liquid at room temperature, and their most important characteristic is having lower melting points than their elements. They are the result of the combination of organic cations with organic or inorganic anions [145]. DESs are ILs' equivalents and include salts, carbohydrates, amino acids and polyols, and others. They are less toxic and more environmentally friendly than ILs. They are, also, characterized as being more viscous than water and conventional organic solvents [146]. These types of solvents can be formed of natural eutectic compounds from plant metabolites and their derivatives. In this case, they are called natural eutectic solvents (NADESs). The most common is choline chloride (ChCl), organic acids such as citric, malic, maleic, and acetic; sugars (glucose, fructose, sucrose, trehalose), and terpene alcohols [145].
The application of DESs or NADESs as pioneering green solvents has increased in recent years applied in the extraction of bioactive compounds from grape/wine by-products.

Enzyme-Assisted Extraction (EAE)
EAE is based on the rupture of cell wall components, making the linked phenolic compounds leak into the media (wine, during the vinification process), or facilitating the recovery of those present in the cell vacuoles. This technique presents many advantages, as it reduces the use of toxic solvents and extraction time, is environmentally friendly, and is extremely specific (acting directly on specific linkages or compounds) 147,148.
Averilla et al. 149 combined the use of heat treatment and enzymes to extract resveratrol and its glycosides piceid (a stilbenoid glucoside and a major resveratrol derivative) from the grape skin ( Figure 7A,B). The grape peel was subjected to a heat treatment which altered cell wall characteristics and increased its susceptibility to the enzymes polygalacturonase (PG; poly(1,4-α-d-galacturonide) glycan hydrolase) and β-glucanase. Polygalacturonase is responsible for fruit cell wall degradation 150 once it hydrolyzes (1,4) -α-d-galactosiduronic linkages ( Figure 7C); β-glucanase hydrolyzes a non-reducing end of the polysaccharide (1,3) -β-d-glucan 151. Other important enzymes that can be used are: (i) Cellulases, composed of enzymes endoglucanases, exoglucanases, and beta-glucosidases, acting in synergy. The endoglucanases act on the inner region of the cellulose microfibrils ( Figure 7B) by releasing oligosaccharides, the exoglucanases act on the cellulose fiber ends releasing units of glucose or cellobiose (formed by two units of glucose), and, finally, beta-glucosidases act on cellobiose releasing glucose units 152; (ii) Endoproteases (trypsin and chymotrypsin) that degrade proteins that bind phenolics 151; (iii) Tannase, which catalyzes the hydrolysis of tannins in gallic acid and glucose 152; and (iv) Glucoamylases that take action under non-reducing ends of starch molecules, generating units of β-D-glucose 153,154. Other enzymatic preparations, that were studied by some authors in grape/wine by-products, are referred to in Table 1 [155][156][157][158]. In Table 1 we also summarize some works using supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), natural eutectic solvents (NADESs), and enzyme-assisted extraction (EAE) techniques in grape and grape/wine by-products aiming at extraction compounds from grape/wine by-products, that can be used as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries. Table 1. Examples of extraction methods, grape/wine residue used, main compounds extracted, and extraction conditions/solvents used.

Extraction Conditions/Products Used and Quantity of Compounds Recovered
Ref.

MAE and/or UAE (Only at lab scale)
Grape canes Trans-resveratrol and trans-ε-viniferin.
Compounds were analyzed qualitatively by comparing their retention times and UV spectra with authentic standards. Extract yield-12.32% (2.45 mg GAE/mL total phenols and 7.08 mg AAE/mL antioxidants).
The fatty acid composition of the extracts was determined by GC. Before chromatographic analysis, the fatty samples were prepared in the form of fatty acid methyl esters (FAME).
Grape marc
HPLC analysis of polyphenolic compounds.
HPLC-DAD-ESI-MS analysis. The optimal conditions to maximize the extraction were ChClU at 30% and extraction temperature of 100 °C.

Anthocyanins
Extracts qualitative analysis by UHPLC-Q-TOF-MS. Total anthocyanin contents (TACs) were measured using the pH differential spectrophotometric method.

Wine lees
Phenolic compounds, anthocyanins, and flavanols Separation, identification, and quantification of anthocyanin and non-anthocyanin phenolic compounds were performed by UHPLC-ESI-Q-TOF-MS.
For the quantitative/qualitative analysis of the extracts, studies have indicated that it is important to accurately identify and quantify the levels of phenolics, especially given the complex chemistry of the grape/wine by-products. The determination of phenolic compounds remains difficult due to several limitations: (i) numerous types of phenolic compounds with a wide range of polarities, (ii) different degrees of stability, (iii) low concentrations of the compounds, and (iv) the matrix effect with interferences by impurities and compounds. A widely used approach for Total Phenolic Compounds (TPC) quantification is the Folin-Ciocalteu method [163,170,176] where the absorbance of the resulting complex of phenolic and Folin-Ciocalteu reagents, measured at 510 nm-700 nm by a spectrophotometer, indicates the level of TPC. The final calculation for the TPC is provided in units of mg gallic acid equivalents (GAE) per g of fresh or dry material. Liquid chromatography, namely HPLC, has also been used to measure phenolic compounds, aiming to identify or quantify specific molecules. This separation technique has evolved aiming at achieving better resolution, speed, and sensitivity. Thus, several high performance liquid chromatographic techniques have being applied in phenolics quantification of grape/wine by-products such as: HPLC-DAD (high-performance liquid chromatography (HPLC) with a diode-array detector (DAD)) [131,157], UPLC-ESI-MS (ultra-performance liquid chromatography (UPLC)-electrospray mass spectrometry (ESI-MS)) [161], TLC (thin-layer chromatography) [165], HPLC-ESI-MS/MS (ultra-performance liquid chromatographyelectrospray tandem mass spectrometry) [168], UHPLC-Q-TOF-MS (ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry) [178] and UHPLC-ESI-Q-TOF-MS (ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometric method) [158], and HILIC-FLD (hydrophilic interaction liquid chromatography) [171], aiming at achieving low limits of detection and quantification.

Grape Infusions as a Way of Extracting Nutraceutical and Antimicrobial Compounds
If an herbal formulation should generate far less or even zero wastes, otherwise the very essence of sustainability is canceled, an infusion made of grape berries or leaves should follow the same principle. In this regard, a solid-liquid extraction of bioactive substances from medicinal plants should embrace methodologies characterized by using water as a solvent. That is the case with grape infusions. Dried non-fermented/semi-fermented and even fermented grapes, skins, and seeds can be used in the preparation of infusions or tisanes [24].
To prepare the dried vegetal by-product for infusion preparation, the most utilized drying methods are hot-air/oven drying, low temperature-air drying, and freeze-drying [24,180,181]. Grape by-products can simply be placed into trays and dried in ovens with controlled temperature and air circulation. Then, the dry material (non-fermented/semifermented or fermented grapes, skins, and seeds) is crushed using a mincer and the minced material can then be used to prepare infusions by adding water at a temperature of about 95-100 °C ( Figure 8A-D). The drying temperature of the grapes influences the infusion's chemical and sensory characteristics, including color [24] (Figure 8E,F). Yet, the optimal drying conditions for the storage of grape pomace, for further application, needs to be more investigated in order to optimize the extraction of phenolics. Gerardi et al. [132] performed the drying by oven at 50 °C which allowed the storage of grape skin from pomace, making it a fast and reproducible method.
Recently, Goulas et al. [182] studied the development of a functional infusion from the "Commandaria" grape pomace. They varied the brewing parameters such as the ratio of water to grape pomace powder, infusion time, and infusion temperature. Interestingly, brewing 200 mL of water per g of dried material for 12.2 min at 95 °C was the optimum method for preparing the infusions. The infusions also presented antimicrobial activity against Listeria monocytogenes serotypes S. enterica, S. aureus, and E. coli bacteria. Moreover, minimum inhibitory concentration (MIC) of 0.25-0.5 mg mL −1 , concentrations such as a cup of infusion, was able to prevent the growth of some Listeria serotypes.
Bekhit Ael-D et al. [183] aiming at studying the antioxidant activities, sensory and anti-influenza activity of grape skin tea infusion found that low antioxidant recovery was obtained using hot water, however, the extracts maintained their antiviral activity. They concluded that grape skin extracts could be incorporated in different functional healthpromoting beverages. Moreover, the use of ingredients such as green tea improved the antioxidant activity and the final infusion sensory profile.
Vine leaf infusion may also be an alternative for the valorization of vine leaves after the grape harvest. The leaves are usually left on the vine; in the autumn they fall and are used as organic material for natural fertilization. The possible use of vine leaves in herbal infusions depends on their aroma profile, as well as on their mouth-feel attributes, and, of course, their potential benefit for human health. According to HMPC 184 for the grapeleaf extract preparation (Extractum Vitis vinifera foliae aquosum siccum, 4-6:1), dried leaves of red varieties of Vitis vinifera L. (Vitis vinifera folium) which comply with the monograph described in Pharmacopée Française for "Vigne rouge" are used. Thus, the herbal substance consists of the dried leaves of the black to pulp-red grape which finally undergoes a specific production process resulting in defined flavonol content in the dry extract preparation. While the whole dry extract preparation as such is considered as an active agent, it is particularly characterized by its content of flavonol glycosides and glucuronides, i.e., quercetin-3-O-β-D-glucuronide, quercetin-3-O-β-glucoside, and kaempferol-3-glucoside. These flavonoids are considered to contribute predominantly to pharmacological effects.

Final Remarks
The "green chemistry" concept is strictly connected to the prospect of a cleaner and more sustainable world and includes any practice that aims to reduce the use of hazardous and polluting substances and any waste released into the environment, and thus supports any action that enables the reuse, treatment, and disposal of such materials.
The natural products obtained from the grapevine and its by-products proved to be of great value, due to the wide variety of compounds present with therapeutic properties, specifically phenolic compounds, such as anthocyanins, trans-resveratrol, quercetin, and proanthocyanidins. The production of these extracts has been recognized as a profitable way of valuing low-value and most abundant grape by-products, in foods, agrochemical, cosmetics, and pharmaceutical industries.
In the field of grape and wine by-products valorization, a few techniques can be applied, at laboratory and industrial scales. Nevertheless, the utilization of naturally deep eutectic solvents (NADESs) represents a promising environmentally friendly extraction technique. Additionally, the application of enzymes for the grape-marc phenolics recovery has sought not only quantitative results in terms of extract yield but also can lead to the obtention of extracts with highly valued bioactive characteristics.
Given the complex chemistry of the grape/wine by-products, liquid chromatographic methods, and its derivatives with low detection limits (HPLC-DAD, UPLC-ESI-MS, TLC, HPLC-ESI-MS/MS, UHPLC-Q-TOF-MS, and UHPLC-ESI-Q-TOF-MS) have been used aiming to identify or quantify specific molecules that are present in minimal amounts.
For centuries, the therapeutic benefits of grapevines and other by-products have been empirically explored. Recently, interest has grown in the health benefits of vine leaves and grape by-products (dried non-fermented/semi-fermented and even fermented grapes, skins, and seeds) and infusions, in water, prepared with dried leaves or dried grape byproducts can be a sustainable way of valorizing these products and, also, a sustainable way of extracting nutraceutical compounds directly from a pleasant infusion cup.
Author Contributions: A.V. and T.P. equally contributed to the work. All authors have read and agreed to the published version of the manuscript.