Vitis vinifera (Vine Grape) as a Valuable Cosmetic Raw Material

This review refers to botanical, ecological and phytochemical characteristics of Vitis vinifera L. (vine grape)–a species, the valuable properties of which are widely exploited in the food industry and in recent times in medicine as well as in phytocosmetology. The general characteristic of V. vinifera, followed by the chemical composition and biological activities of different extracts obtained from the plant (fruit, skin, pomace, seed, leaf and stem extracts), are provided. A concise review of the extraction conditions of grape metabolites and the methods of their analysis are also presented. The biological activity of V. vinifera is determined by the presence of high contents of polyphenols, mainly flavonoids (e.g., quercetin, kaempferol), catechin derivatives, anthocyanins and stilbenoids (e.g., trans-resveratrol, trans-ε-viniferin). The review pays particular attention to the application of V. vinifera in cosmetology. It has been proven that V. vinifera possesses strong cosmetological-related properties, such as anti-ageing properties, anti-inflammatory properties and skin-whitening properties. Moreover, a review of studies on V. vinifera biological activities, which are of particular interest for dermatologic problems, are disclosed. Furthermore, the work also emphasises the importance of biotechnological studies on V. vinifera. The last part of the review is addressed to the safety of the use of V. vinifera.


Introduction
Modern phytocosmetology is an extremely prominent field that has recently drawn the attention of research centres. There is an increasing demand for natural cosmetics, the key task of which is to protect the skin from free radicals and oxidative stress with a low risk of side effects [1,2]. Scientists are constantly searching for innovative raw materials with potential applications in different cosmetic formulations [3]. Vitis vinifera L. (vine grape, Vitaceae) is one of the best-known fruit crops with wide applications in the food, pharmaceutical and cosmetic industries. The main cultivation regions of V. vinifera are located in Europe (France, Italy and Spain), Asia (China) and the Americas (United States, Argentina and Chile) [4].
V. vinifera is a rich source of secondary metabolites, particularly flavonoids (flavan-3ols, flavonols), phenolic acids, anthocyanins, fatty acids, amino acids and vitamins. It also contains the characteristic stilbene derivatives. In addition, the qualitative differences in the phytochemical content depend on the morphological part of the plant [5]. Due to the presence of the above-mentioned groups of compounds, V. vinifera is an object of specific scientific interest. It possesses antioxidant, antibacterial, anti-inflammatory and anticancer activities. Moreover, it exhibits cardioprotective, hepatoprotective and neuroprotective properties [6][7][8][9][10].

Botanical Characteristic
Vitis vinifera L. (vine grape, Vitaceae) is a climbing vine with shoots reaching ten to forty metres long in the natural environment. The main shoot is thick, woody and covered with brown or grey-brown bark. The shoot branching is multi-axis and often bare. The main shoot is pushed back by the side branches creating the tendrils which help the vine to attach to the support. The shoots can be short or long [24].
The leaves are simple, long-tailed and subalternate on the shoots. The pedicles are bare and 4-8 cm long. There are bracts at the root which fall fast. The length and width of the leaf blades are similar and range from 5-15 cm. The lobes vary in size and often overlap, and the sinuses which separate them are rounded. The leaf tooth is large, bare and glabrous, and the middle lobe has a pointed tip. The leaves are usually dark green and hairy but become bare over time. The underside of the leaf blade is light green and sparsely grey-haired [24,25].
The flowers are arranged into 10-20 cm long panicles. The inflorescences are 10-20 cm long and have spidery or bare 1-5 cm long peduncles. The flowers are small and inconspicuous. The flower calyx is five-sided and small. The corolla consists of five petals that are fused at the top. The petals are yellow-green and reach 1.5 mm in length. The staminal or monoecious flowers have five stamens. The flowers are self-pollinating or insect-pollinated [24].
V. vinifera fruits have elliptical or globose shape and are 1.5 to 2 cm long in diameter. The fruits are also variable in size and colour. Cultivated varieties can have fruits of red, pink, purple and light green colour. The fruits contain two to four seeds. The pulp is juicy, sweet or sour [24,25].
The seeds have an oval or pear shape with a pointed tip. They can reach up to 6 mm in length [24,25].

Ecological Characteristic
It is accepted that V. vinifera is derived from a wild grapevine-V. vinifera spp. sylvestris. Wild grapevines still naturally occur in small populations in forest areas near rivers, and they climb trees. They are distributed from the Atlantic coast of Europe to Tajikistan and the Western Himalayas. The demand for grape fruits resulted in the domestication of V. vinifera spp. sylvestris to obtain fruits of better parameters. Due to the hybridisation of different varieties, nowadays, the determination of the point of origin and the spread of V. vinifera is complicated. It is assumed that the domestication of V. vinifera spp. sylvestris took place in the area between the Black Sea and Iran, where V. vinifera may have spread to the Near and Middle East and Central Europe [26,27].
Currently, V. vinifera is one of the most popular horticultural and crop plants in the world. There are around 10,000 V. vinifera varieties, which are planted in numerous countries and grow on six continents with large-scale cultivation areas in Europe, the Middle East and Asia. The main cultivation regions of V. vinifera are Spain, France, Italy, China, the United States, Argentina, Chile, Portugal, Romania, Australia, South Africa, Greece, Germany, Brazil and Hungary. The most popular crop varieties are Kyoho, Cabernet, Sauvignon and Sultana [4,28].
The major constituents of V. vinifera seeds are polyphenols (60-70%), which are mainly the flavan-3-ol derivatives, primarily catechin, epicatechin and epicatechin-3-O-gallate. Procyanidins are also found in V. vinifera seeds (e.g., procyanidin B1, B2, B3, B4, C1 and T2). The V. vinifera seeds are also a rich source of fatty acids, vitamins and minerals (Table 1) [5,16,32]. The V. vinifera seed oil is a raw material with a high nutritional value. It is characterized by the rich fatty acid profile, which is particularly plentiful in linoleic acid (≈70%). Other fatty acids presented in seed oil are oleic (≈15%), palmitic (≈7%), and stearic (≈3%) acids. The V. vinifera seed oil is also a rich source of tocopherols and tocotrienols. The most abundant vitamin E isomers are γ-tocotrienol followed by α-tocotrienol. The hydrophilic constituents, such as flavonoids, phenolic acids and tannins, are also found in V. vinifera seed oil. The composition of seed oil depends on the environmental factors of vine variety and the seed maturation degree [39][40][41]. Active ingredients present in seed oil can be extracted by various organic solvents. However, the residues of these solvents make the extracts obtained less valuable for the pharmaceutical or cosmetic industry because of their potentially hazardous properties [42]. What is more, the use of high temperatures in extraction processes may cause thermal degradation of active substances. Supercritical carbon dioxide (CO 2 ) extraction can be an alternative to conventional extraction with organic solvents. The density of CO 2 in the supercritical state is similar to organic solvents, which means that substances dissolve in them as in liquids, while the viscosity and surface tension are much lower. This allows for better penetration of the raw material. Since the obtained extracts do not contain chemical impurities, the process is environmentally safe and used CO 2 can be recycled and reused. CO 2 is considered a completely safe, non-toxic, non-flammable and relatively cheap eluent [43,44]. Grape seed oil constituents as non-polar compounds can be efficiently obtained using supercritical CO 2 as the extractor. Wenli et al., in their work, applied this solvent-free, green process to extract resveratrol [45], while Passos et al. evaluated the process conditions to obtain selected fatty acids and tocopherol [46].
The V. vinifera roots mainly contain stilbenoid compounds like hopeaphenol, ampelopsin A, vitisin A, isohopeaphenol and trans-resveratrol. There are no reports related to the other compounds present in the V. vinifera roots [5].
The sustainable exploitation of biological resources such as plant materials leads to the reduction of environmental impacts. The wastes obtained from plants could be potentially applied in the food industry to improve the nutritional food quality due to the presence of lipids, proteins and fiber or in the pharmaceutical field to retain the bioactive molecules [47,48]. Wastes obtained from V. vinifera, such as shoots, canes, stems, leaves and pomace are a rich source of bioactive compounds, e.g., stilbenes, flavan-3-ols, flavonols, hydroxybenzoic or hydroxycinnamic acids. Moreover, applications of V. vinifera wastederived bioactive compounds have been found in cosmetic formulations [49].

Methods of Extraction and the Identification of Selected Groups of V. vinifera Metabolites
The standard process of the efficient extraction of V. vinifera active metabolites is based on low-temperature processes in hydroalcoholic solutions. Ultrasound-assisted extraction (UAE) is commonly used as the process does not require the application of high temperatures, which is extremely important for heat-sensitive compounds [50]. Tannins can be extracted with the highest efficiency by the SPE (solid phase extraction) method. However, standard extraction with methyl alcohol and ethyl acetate 1:1 (v/v) is also commonly applied [31]. Stilbenoids are commonly extracted in aqueous-alcoholic conditions or aqueous-acetone solutions [51]. Oligomeric tannins, which are quite demanding metabolites according to their large molecular mass, are extracted by multistep processes, including lyophilisation, extraction, evaporation, liquid-liquid crude fractionation, solubilisation in water and second extraction. These multistep processes are aimed at obtaining tannins at high levels of purity because of the presence of particular lipophilic metabolites known as ballast compounds. Thus, liquid-liquid crude fractionation allows the removal of lipids and pigments [52,53].
The identification of the grape metabolites is practised mostly by the UPLC-MS method. Chromatographic and mass spectra data are given for analysed compounds [31]. Table 2 presents an overview of the methods of V. vinifera for the extraction and quantification of selected metabolite groups.

EMA-HMPC
In 2010, the European Medicines Agency (EMA), by the decision of the Committee on Herbal Medicinal Products (HMPC), approved the use of Vitis viniferae folium (V. vinifera leaves) for the treatment of chronic venous insufficiency associated with swollen legs, varicose veins, a feeling of heaviness, tiredness, tension and pain in the calves. V. vinifera leaf preparations may also be helpful in the heaviness of legs that is associated with minor blood circulation problems in the veins or in the itching and burning sensations related to haemorrhoids. This opinion is based on scientific studies proving the effectiveness and safety of these preparations [11].

EFSA
The European Food and Safety Authority (EFSA) has approved V. vinifera seed and dry extracts as a water-flavouring additive for animals (except dogs) in a specified concentration. However, the EFSA concluded that the application of V. vinifera for the improvement of circulation or the reduction of swelling in the legs is not sufficient, and further analysis is required [13].

FDA
The U.S. Food and Drug Administration (FDA) has approved the use of V. vinifera fruits and leaves and their extracts as a component of the human diet. However, there are studies proving that V. vinifera var. Reiber and var. Tokays can cause an allergic reaction. The FDA has also listed V. vinifera extract as an ingredient in wound dressings [12].

CosIng
According to the CosIng database, V. vinifera fruit (Vitis viniferae fructus) can be used as a skin-conditioning agent. The seeds of V. vinifera (Vitis viniferae semen) are used as skin-protecting and conditioning components. The seeds have also shown indications of being anti-seborrheic, antimicrobial and antioxidant ingredients. The V. vinifera seed oil can be used as an emollient. The roots (Vitis viniferae radix) have skin-conditioning properties. The V. viniferae cauli (shoot) have skin-protecting and antioxidant properties. V. vinifera leaves (Vitis viniferae folium) are highly valued in cosmetic production as a skin-conditioning agent or fragrance [20]. A detailed description of V. vinifera with its functions based on the CosIng database is presented in Table 3. Table 3. Possible applications of V. vinifera in cosmetic production according to the CosIng database.

V. vinifera as the Ingredient of the Cosmetic Formulation
According to the FDA's Voluntary Cosmetic Registration Program (VCRP), from 2012, V. vinifera seed extract was used in 495 cosmetic formulations. V. vinifera fruit extract was used in 238 cosmetic formulations. V. vinifera leaf extract was reported to be used in 80 cosmetic formulations. The remaining V. vinifera-derived ingredients were used in fewer than 15 cosmetic formulations [16].
Nowadays, the production of cosmetics based on V. vinifera extracts is particularly popular in the countries of southern and central Europe, the United States, China and South Korea. It could be noted that V. vinifera extracts or oil demonstrate particular moisturising abilities as well as anti-ageing properties, which are confirmed by the increasing number of cosmetics containing these raw materials. Table 4 presents examples of cosmetic products containing V. vinifera-derived ingredients.

Anti-Aging and UV-Protection Activities
Letsiou et al. [23] evaluated the effect of V. vinifera leaf extracts on UV-stressed human dermal fibroblasts. Fresh leaves of V. vinifera var. Athiri extracted with a solvent system of glycine-H 2 O (4:1) were used. Primary normal human fibroblasts (NHDF) were isolated from adult human skin, incubated with V. vinifera extract (0.1 µg/mL) and incubated for 48 h. Cells were washed twice with phosphate-buffered saline (PBS) and exposed to UVA light. During analysis, the significant induction of sirtuin 1 (SIRT1) and heat shock protein 47 (HSP47) is demonstrated with the presence of V. vinifera extract under normal and UV conditions. In addition, DNA methylation changes were observed, which appear to have been induced by the V. vinifera extract. The results of the investigation clearly prove the protective effect of the V. vinifera extract, which is possibly associated with a transcriptional regulation of skin anti-ageing genes [23] (Table 5).
Cefali et al. [57] investigated the effectiveness of V. vinifera var. Benitaka skin extract with regard to sun protection, antioxidative activity and skincare formulation stability. The skins were extracted in ethanol and standardised with HPLC-DAD for the determination of flavonoid content. The results of the cell viability test showed that the extract had no effect on cell viability. In order to determine the effectiveness of the extract as a sun filter, in vitro SPF was determined and was equal to 18.56. The UVA protection factor determined by the spectral transmittance was 3.17, with a critical wavelength of 318 nm and a UVA/UVB rate of 0.9. The antioxidant activity was tested by DPPH and ABTS assays. In both assays, the extract exhibited antioxidant activity, reducing the DPPH and ABTS concentrations by 92.08% and 86.85%, respectively. The properties of the extract observed within a stable oil-in-water (O/W) emulsion support the potential use of the formula as a sunscreen. The emulsion was odourless, glossy and light pink with a characteristic desirable for skincare formulations (pH: 5.50, density: 1.001 g/mL, viscosity: 13,000.35 cP) [57] (Table 5).

Anti-Inflammatory Activity
Sangiovanni et al. [58] evaluated the ability of an aqueous extract of the leaves of V. vinifera var. Teinturiers inhibit inflammation in human keratinocytes (HaCaT cells) caused by the mediators of inflammation or oxidative stress, which are released in psoriasis. Human keratinocytes were cultured using Dulbecco's Modified Eagle Medium (DMEM) supplemented with penicillin, streptomycin, L-glutamine, and 10% heat-inactivated Fetal bovine serum (FBS) and cultured in twenty-four-well plates. It was then treated with inflammatory mediators: tumour necrosis factor-α (TNF -α) and lipopolysaccharide (LPS). Human keratinocytes cells were plated and transfected with plasmid NF-kB-LUC (nuclear factor Kappa B luciferase) or native IL-8-LUC (IL-8 Luciferase) promoter, which contains sequences responsive to several transcription factors, both at 250 ng per well. The cells were treated with increasing concentrations of V. vinifera-leaf extract in the presence of inflammatory mediators and after the luciferase assay was performed. It was demonstrated that the extract inhibited the interleukin-8 (IL-8) secretion induced by TNF-α (IC 50 = 2.60) or LPS (IC 50 = 14.04). In addition, it was also associated with the inhibition of the nuclear factor-κB (NF-κB)-driven transcription which indicates the presence of the anti-inflammatory properties of grape extracts [58] (Table 5). Table 5. Biological activity of V. vinifera with the direct application in cosmetology.

Skin-Whitening Activity
Lin et al. [22] tested the effectiveness of V. vinifera leaf extract on the tyrosinase inhibitory activity. The presence of gallic acid, chlorogenic acid, epicatechin, rutin and trans-resveratrol in the extracts was detected with the HPLC method. It was demonstrated that V. vinifera leaf extract reduced the tyrosinase activity in a dose-dependent manner (IC 50 = 3.84 mg/mL). The kinetic study showed the tyrosinase inhibitory activity using a competitive mechanism [22] (Table 5).
Malinowska et al. [50] investigated the rejuvenating effect of five selected varieties of V. vinifera (Villard Noir, Sauvignon, Savagnin, Riesling and Magdeleine Noire des Charentes) cane extracts by tyrosinase inhibition and the delaying of cell ageing. The skin whitening potential of V. vinifera cane extracts was compared to pure trans-resveratrol and ε-viniferin. The HPLC-MS analysis determined the main polyphenols presented in the ethanol-water (60/40 v/v) extract, namely catechin, epicatechin, piceatannol, trans-resveratrol, ampelopsin, ε-viniferin, hopeaphenol, isohopeaphenol, miyabenol C and vitisin B. The SIRT1 activity was determined using the SIRT1 assay kit. Most of the extracts showed relatively high SIRT activation. Among all the varieties, Riesling was the most potent, with 171% SIRT activation. The tyrosinase inhibition was performed with a tyrosinase inhibition assay. All the tested extracts are relatively efficient tyrosinase inhibitors. The highest results were obtained for ε-viniferin (76%) and trans-resveratrol (75%). Riesling and Villard Noir extracts showed the highest inhibition activity (62.5% and 58.5%) [50] (Table 5).

Antioxidant Activity
Antioxidant activity plays a significant role in the maintenance of good skin condition as well as in the prevention of numerous skin diseases and dysfunction. The main protective mechanisms of antioxidative molecules contained in grape extracts are free radical scavenging abilities. This simple mechanism ensures DNA damage repair, the modulation of gene expression in proliferation, metabolism, and cell survival, as well as the antioxidant defence [62]. It was proven that grape phytochemicals' in vivo molecular mechanisms lead to health promotion by avoiding oxidative stress-related pathologies.
Tzanova et al. [19] evaluated the antioxidant activity of the commercial V. vinifera skin extracts of different red varieties obtained from separate Bulgarian regions. The antioxidant potential and the total phenol content were measured by UV methods. All the tested extracts have a similar radical scavenging capacity ranging from 23.2 ± 1.7 to 48.7 ± 5.1 mmol/kg Trolox equivalent (TE), depending on the variety. The highest antioxidant activity was observed for the Syrah variety (48.7 ± 5.1 mmol/kg) from the Mogilovo vineyard. The total phenolic content ranged from 33.4 ± 4 in Merlot to 202 ± 19 mmol/kg gallic acid equivalent (GAE) in the Syrah variety [19].
Zielonka-Brzezicka et al. [59] tested the antioxidant activity of fresh and frozen V. vinifera fruits and leaves of an unspecified red variety. The antioxidant activity was established by ABTS and DPPH assays in ethanol, methanol, isopropanol and water extracts. The methanolic extracts of fresh leaves showed the highest activity in the DPPH assay: 3.12 AAE (ascorbic acid equivalent, mg AA/g of raw material). Moreover, the highest antioxidant capacity was indicated for the frozen leaves extracted with isopropanol in the ABTS assay (26.94 AAE) [59] (Table 5).
Llobera [60] confirmed the antioxidant activity of V. vinifera stems. Some 80% and 70% acetone extracts of the red variety Manto Negro and white variety Prensal Blanc were used. The free radical scavenging activity was determined by the DPPH assay. The values of EC 50 (half maximal effective concentration) of extracts obtained from red-grape variety Manto Negro extracts were 0.14 g dm (dry matter)/g DPPH and 0.20 g dm/g DPPH for the acetone and the ethanol extracts, respectively. The white grape variety Prensal Blanc extracts showed 0.26 g dm/g DPPH and 0.37 g dm/g DPPH, respectively. Studies showed that the antioxidant activity of V. vinifera stem extracts significantly correlated with the total content of polyphenols and flavanols [60] (Table 5).
Chidambara et al. [61] evaluated the antioxidant activity of V. vinifera pomace ethyl acetate, methanol and water extracts using different methods. The methanol extracts showed the highest antioxidant activity (87%) in the DPPH assay. Ethyl acetate and water extracts showed 76% and 21.7%, respectively. The methanol extract demonstrated the strongest activity and was selected for further analysis using the thiobarbituric acid method, hydroxyl scavenging activity and LDL oxidation. The methanolic extracts showed inhibition levels of 71.7, 73.6, and 91.2%, respectively. The in-vivo study demonstrated that treatment with a single dose of 1.25 mg/kg of CCl 4 decreases the activity of peroxidase (89%), catalase (81%) and superoxide dismutase (49%) in albino rats. The pre-treatment of the rats with 50 mg/kg grape pomace methanolic extract followed by the treatment with CCl 4 resulted in catalase, peroxidase and SOD restoration at the level of 43.6, 54.0 and 73.2%, respectively. The histopathological studies of the liver of the different groups confirm the protective effect of the V.vinifera pomace methanolic extract, which contributed to the restoration of normal liver structure [61] (Table 5).

Antimicrobial Activity
Oliveira et al. [63] tested the antimicrobial activity of V. vinifera pomace extracts of Merlot and Syrah varieties. The extracts were obtained by a supercritical CO 2 extraction method, and CO 2 was added with co-solvent (ethanol) extraction at pressures of up to 300 bar and temperatures of 50 and 60 • C. The constituents of the extracts were identified using the HPLC method. The dominant compounds were gallic acid, p-hydroxybenzoic acid, vanillic acid and epicatechin. The antibacterial activity and antifungal activity were assessed against the Bacillus cereus, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa bacterial strains and the Candida albicans, Candida parapsilosis and Candida krusei fungal strains. The supercritical fluid extracts showed high antimicrobial activity (inhibition > 9 mm), particularly against Gram-positive bacterial strains. The SFE SC-CO 2 (supercritical fluid extraction obtained by supercritical CO 2 ) extracts of the Merlot variety were effective against C. albicans and C. krusei with MIC (minimum inhibitory concentration) of 500 µg/mL [63] (Table 6). Fruit skin E. faecallis, S. aureus, E. aerogenes [65] Anti-inflammatory Dried fruit -interleukin IL-8, (NF)-κB inhibition [7] Seed -albumin denaturation assay [66] Filocamo et al. [64] evaluated the antimicrobial activity of white grape juice extract derived from a mixture of white grape juice containing Catarratto, Grillo and Insolia V. vinifera varieties. The antimicrobial activity was tested against S. aureus, Listeria monocytogenes, Staphylococcus epidermidis, Enterococcus hirae, Streptococcus pneumoniae, Bacillus subtilis, Streptococcus pyogenes, Enterococcus durans, Streptococcus mutans, Moraxella catarrhalis Grampositive bacteria strains, Salmonella typhi, Serratia marcescens, E. coli, P. aeruginosa, Proteus mirabilis, Klebsiella pneumoniae Gram-negative bacteria strains and Aspergillus niger and C. albicans fungal strains. The extract was obtained by passing the juice through the mustmute columns equipped with adsorbent resins, which retain polyphenols. The molecules were eluted with 4% NaOH and passed through the cationic resins. The products were then collected, filtered and sprayed in order to obtain a dry powder. The dominant polyphenols determined in V. vinifera juice extract were quercitin-3-glucuronide, procyanidin B1, quercetin-3-glucoside, catechin and trans-coutaric acid. The V. vinifera juice extract inhibited all tested Gram-positive bacteria (MIC = 3.9-1000 µg/mL −1 . The best results were observed for S. aureus [64] (Table 6).
Yadav et al. [65] accessed the antibacterial and antifungal activities of the Sharad variety of V. vinifera seedless-fruit-skin extracts against antibiotic-resistant pathogenic bacteria and toxin-producing moulds. Among the tested bacteria strains were Enterococcus faecallis, S. aureus, Salmonella typhimurium, Enterobacter aerogenes and E. coli. The antifungal activity was tested for the following strains: Penicillum expansum, Penicillum chrysogenum, A. niger and Aspergillus versicolor. The V. vinifera skin was extracted using different solvents: water, acetone, ethanol and methanol. The antibacterial activity was determined by the agar well diffusion method. The antifungal activity was evaluated as a percentage of conidia germination inhibition. The methanolic extracts possessed strong antibacterial and antifungal activity. The maximum zone of inhibition was determined for S. aureus (22 mm), followed by E. faecalis (18 mm) and E. aerogenes (21 mm) [65] (Table 6).

Anti-Inflammatory Activity
Di Lorenzo et al. [7] tested the anti-inflammatory activity of extracts from raisins of different varieties: Early Gold (Portugal) and Sultana (Turkey). Attention was focused on the interleukin (IL-8) and nuclear factor (NF)-κB pathways. The composition of raisin extracts was evaluated by the HPLC-DAD method and screened for the ability to inhibit IL-8 release induced by a tumour necrosis factor (TNF-α) and promoter activity in human gastric epithelial cells. The Turkish variety (Sultana) inhibited the release of IL-8 affected by the impairment of promoter activity. The researchers also tested the seed extract, which showed slightly higher inhibitory activity against IL-8 and (NF)-κB than the raisin extract. The results suggest that the consumption of selected raisins (for instance, the Sultana variety) could be beneficial against gastric inflammatory diseases [7] (Table 6).
Chopra and Geetha [66] studied the anti-inflammatory effect of V. vinifera seed extract using the albumin denaturation assay. The extract was tested in different concentrations from 10-50 µg/mL. The results showed that the V. vinifera seed extract possessed better anti-inflammatory activity in comparison to diclofenac sodium used as a reference. The extract also had fewer side effects. It is suggested that polyphenols are responsible for this effect [66] (Table 6).

The Applications of V. vinifera In Vitro Cultures in Cosmetology
In recent years, natural cosmetics with innovative ingredients have been in demand. The particular interest of the cosmetic industry is focused on V. vinifera stem cells, which have applications mainly in anti-ageing creams and essences [1]. The Mibelle Biochemistry company (Switzerland) has developed a new biotechnology technique under the name PhytoCellTech™, which is used to generate plant stem cells. The growth of V. vinifera callus cells was induced under special conditions. The undifferentiated callus cells, i.e., stem cells, are involved in further cultivation in special bioreactors to obtain a sufficient amount of plant cells. The technology is highly sustainable and enables the production of large amounts of high-quality active ingredients. The PhytoCellTech™ Solar Vitis is a stem cell obtained from the Gamay Teinturier Fréaux variety of V. vinifera of French origin, which was characterised as a high-polyphenol line [67].
Due to the recent high interest in in vitro cultures, including at the Faculty of Pharmacy of Jagiellonian University Collegium Medicum, Cracow (Poland), the V. vinifera in vitro culture of different varieties has been performed ( Figure 2). The aim of the study is the qualitative and quantitative analysis of the extracts obtained from V. vinifera in vitro cultures followed by the determination of the total phenolic content (Folin-Ciocalteu method), the evaluation of the antioxidant activity using different methods (e.g., DPPH, ABTS, FRAP) and the future application of the most potent varieties in cosmetic formulations [unpublished]. Solar Vitis is a stem cell obtained from the Gamay Teinturier Fréaux variety of V. vinifera of French origin, which was characterised as a high-polyphenol line [67].
Due to the recent high interest in in vitro cultures, including at the Faculty of Pharmacy of Jagiellonian University Collegium Medicum, Cracow (Poland), the V. vinifera in vitro culture of different varieties has been performed ( Figure 2). The aim of the study is the qualitative and quantitative analysis of the extracts obtained from V. vinifera in vitro cultures followed by the determination of the total phenolic content (Folin-Ciocalteu method), the evaluation of the antioxidant activity using different methods (e.g., DPPH, ABTS, FRAP) and the future application of the most potent varieties in cosmetic formulations [unpublished]. Before for production of secondary metabolites, Bonello et al. [68] studied the V. vinifera var. Ġellewża callus cultures. Callus was incubated in an MS medium with plant growth regulators (PGRs) (6-benzylaminopurine (BAP); 3-indoleacetic acid (IAA); kinetin (KIN); 1-naphthaleneacetic acid (NAA); IAA+BAP; IAA+KIN; NAA+BAP; NAA+KIN) to determine its best combination needed for the production of metabolites. Some 0.5 g aliquots of callus extracts were analysed by UV-Vis spectrophotometry and HPLC method. The best callus production was obtained when the MS medium was enriched with IAA and IAA+BAP. The high content of flavonoids, mainly anthocyanins, was associated with the presence of cytokinins (especially BAP). Catechin, luteolin, myricetin, naringenin and quercetin-3-O-glucoside were identified among the flavonoids. Transresveratrol and polydatin were the most abundant among the stilbenoid compounds. Two coumarin derivatives were identified (aesculetin and aesculin) [68].
Mewis et al. [69] studied the production of polyphenolic compounds in callus cultures of V. vinifera var. Gamay Fréaux. The cell cultures were cultivated on B5 medium and were transferred to the fresh sterile medium after twenty-eight days. The red callus cultures were selected for future cultivation. The cultures were transferred every three weeks to fresh Erlenmeyer flasks containing the B5 medium. The obtained samples were frozen and lyophilised. The HPLC method was used for the analysis of 70% methanol extracts. The HPLC analysis revealed the presence of phenolic acid derivatives such as 3-O-glucosylresveratrol and 4-(3,5-dihydroxyphenyl)-phenol and cinnamoyl derivatives, including cyanidin 3-O-p-coumaryl glucoside and peonidin 3-O-p-coumaryl glucoside. The major anthocyanins identified in callus cultures were cyanidin 3-O-glucoside and peonidin 3-O-glucoside. The anthocyanins levels were significantly increased after cultivation for four days in the new medium [69]. Before for production of secondary metabolites, Bonello et al. [68] studied the V. vinifera var.Ġellewża callus cultures. Callus was incubated in an MS medium with plant growth regulators (PGRs) (6-benzylaminopurine (BAP); 3-indoleacetic acid (IAA); kinetin (KIN); 1-naphthaleneacetic acid (NAA); IAA+BAP; IAA+KIN; NAA+BAP; NAA+KIN) to determine its best combination needed for the production of metabolites. Some 0.5 g aliquots of callus extracts were analysed by UV-Vis spectrophotometry and HPLC method. The best callus production was obtained when the MS medium was enriched with IAA and IAA+BAP. The high content of flavonoids, mainly anthocyanins, was associated with the presence of cytokinins (especially BAP). Catechin, luteolin, myricetin, naringenin and quercetin-3-O-glucoside were identified among the flavonoids. Trans-resveratrol and polydatin were the most abundant among the stilbenoid compounds. Two coumarin derivatives were identified (aesculetin and aesculin) [68].

Safety of Use
Mewis et al. [69] studied the production of polyphenolic compounds in callus cultures of V. vinifera var. Gamay Fréaux. The cell cultures were cultivated on B5 medium and were transferred to the fresh sterile medium after twenty-eight days. The red callus cultures were selected for future cultivation. The cultures were transferred every three weeks to fresh Erlenmeyer flasks containing the B5 medium. The obtained samples were frozen and lyophilised. The HPLC method was used for the analysis of 70% methanol extracts. The HPLC analysis revealed the presence of phenolic acid derivatives such as 3-O-glucosylresveratrol and 4-(3,5-dihydroxyphenyl)-phenol and cinnamoyl derivatives, including cyanidin 3-O-p-coumaryl glucoside and peonidin 3-O-p-coumaryl glucoside. The major anthocyanins identified in callus cultures were cyanidin 3-O-glucoside and peonidin 3-O-glucoside. The anthocyanins levels were significantly increased after cultivation for four days in the new medium [69].

Safety of Use
According to the Voluntary Cosmetic Registration Program (VCRP), data obtained from FDA in 2012, ingredients derived from V. vinifera can be used in different cosmetics formulations, depending on the raw material, but in relatively low concentrations. For instance, V. vinifera leaf extract could be present in leave-on formulations at levels of up to 3%. V. vinifera fruit extract and V. vinifera juice could be used in skin-cleansing products and masks at levels of up to 2%. Other V. vinifera-derived ingredients are included at levels of up to 1% in formulations. Grape skin extract contains enocianine, which is approved as a food colour additive with no required certification. According to the evaluation of the Joint Food and Agriculture Organization of the United States and the World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA), the acceptable daily intake (ADI) of grape skin extract varies from 0-2.5 mg/kg bw (body weight) [2,16].
The cosmetics formulations containing the ingredients from V. vinifera could be applied to the eye area or mucous membranes and could also accidentally be ingested. Furthermore, the majority of V. vinifera extracts from different parts of the plant (fruit, leaf and seed), as well as V. vinifera juice and V. vinifera fruit water extracts presented in the cosmetics, could possibly be inhaled [16].

Skin Irritation and Sensitisation
In a dermal irritation test on human skin, products containing 3% V. vinifera fruit extract are non-irritant. According to the Epiderm MTT viability assay, products containing 10% of V. vinifera fruit extract are either non-irritant or minimally irritant. In in vitro assay, the hydrolysed grape skin did not demonstrate the stimulating potential of the monocytes and macrophages mediated cellular immune response. As reported by the human two-week use study, the product containing 0.15% of V. vinifera seed extracts was also non-irritant. In a clinical test conducted using patch tests, extracts from V. vinifera fruits, juice and seeds at a maximum concentration of 1% also did not demonstrate any irritating or sensitising potential [16].

Eye Irritation
The ocular irritation of a product containing 3% V. vinifera fruit extracts is predicted to be minimal in an EpiOcular assay. The ocular irritation potential was evaluated using the ocular irritation test for a single sample of a product with 3% V. vinifera fruit water extract. The irritation Draize equivalent (IDE) score ranged from 4.5-6.4. A product containing 10% V. vinifera fruit extract was classified as a non/minimal irritant. Hydrolysed grape skin extracts are predicted to be ocular non-irritating in a cytotoxicity assay. In in vitro testing, a product containing 0.15% V. vinifera seed extracts was found to be a mild ocular irritant [16].

Conclusions
V. vinifera is one of the most popular fruit crops around the world. It is a useful species which is cultivated within all continents, especially in Europe, the Middle East and Asia. Its valuable properties have been known since ancient times and used for a number of ailments, including cancer, eye infection, sore throat and nausea [4,28,70]. Currently, V. vinifera is intensively exploited primarily in terms of sustainable development. The waste matter of the vine grapes (e.g., stems, pomaces and seeds) are desirable raw materials which contain valuable bioactive compounds [49]. Recently, V. vinifera has been an extremely preeminent plant used in the food and pharmaceutical industries.
Numerous scientific studies have proven the valuable chemical composition of V. vinifera, which is dominated by phenolic compounds. The main group of metabolites present in V. vinifera are flavonoids, stilbenoids, phenolic acids, anthocyanins, catechin derivatives, procyanidins, fatty acids and vitamins [5,30]. The obtaining and the quantitative analysis of grape extracts are well established in the literature, and the most challenging metabolites for quantification and purification are condensed tannins [54].
Despite the properties of V. vinifera, it is not mentioned in any pharmacopeia. Nevertheless, there are monographs with a positive opinion provided by respected organisations such as the EMA, the FDA and the EFSA [11][12][13].
In the food industry, V. vinifera is used mainly to produce wine, juice and raisins [18]. The phytochemical composition of V. vinifera determines the antioxidant, antibacterial and anti-inflammatory activities as well as the cardioprotective, neuroprotective and hepatoprotective properties. These V. vinifera activities are especially important for the pharmaceutical industry [6][7][8][9][10].
Due to the widespread application of V. vinifera in the cosmetics industry, it deserves special attention. Raw materials obtained from V. vinifera are highly valued in cosmetics, particularly due to their antioxidant, anti-ageing, skin-whitening and UV-protection properties. The proven safety of V. vinifera also contributes to its extensive use. There is a wide range of cosmetics based on V. vinifera-derived ingredients [20]. Nowadays, the production of cosmetics based on V. vinifera is particularly popular in the countries of southern and central Europe, the United States, China and South Korea.
V. vinifera is also a research subject in terms of biotechnological studies. There is growing interest in V. vinifera in vitro stem cells as well as tissue cultures [1,67]. It is expected that V. vinifera in vitro culture extracts will be proposed as innovative and effective cosmetic ingredients in the future.