The Utilization of Physiologically Active Molecular Components of Grape Seeds and Grape Marc

Nutritional interventions may highly contribute to the maintenance or restoration of human health. Grapes (Vitis vinifera) are one of the oldest known beneficial nutritional components of the human diet. Their high polyphenol content has been proven to enhance human health beyond doubt in statistics-based public health studies, especially in the prevention of cardiovascular disease and cancer. The current review concentrates on presenting and classifying polyphenol bioactive molecules (resveratrol, quercetin, catechin/epicatechin, etc.) available in high quantities in Vitis vinifera grapes or their byproducts. The molecular pathways and cellular signaling cascades involved in the effects of these polyphenol molecules are also presented in this review, which summarizes currently available in vitro and in vivo experimental literature data on their biological activities mostly in easily accessible tabular form. New molecules for different therapeutic purposes can also be synthesized based on existing polyphenol compound classes available in high quantities in grape, wine, and grape marc. Therefore an overview of these molecular structures is provided. Novel possibilities as dendrimer nanobioconjugates are reviewed, too. Currently available in vitro and in vivo experimental literature data on polyphenol biological activities are presented in easily accessible tabular form. The scope of the review details the antidiabetic, anticarcinogenic, antiviral, vasoprotective, and neuroprotective roles of grape-origin flavonoids. The novelty of the study lies in the description of the processing of agricultural by-products (grape seeds and skins) of industrial relevance, and the detailed description of the molecular mechanisms of action. In addition, the review of the clinical therapeutic applications of polyphenols is unique as no summary study has yet been done.


Introduction
Vitis vinifera grapes are extremely rich in bioactive components [1]. Grape marc is a mixture of grape seeds and skins, which remain as a by-product of the wine production   The main polyphenolic constituents in grape seeds are catechins (catechin, epicatechin, procyanidin [19]). Except for epicatechin, they are found in the outer, soft layer of the grape seed. The most physiologically important compounds of polyphenols isolated from grape seeds are summarized in Table 2. Table 1. Main groups of polyphenols.

Compound Group General Structural Formula Function Representatives
Flavonoids Anthocyanidins Figure 1. General structure of polyphenols.  Figure 1. General structure of polyphenols.

Compound Group General Structural Formula Function Representatives
Flavonoi Table 2. Most important physiologically active compounds of the polyphenol fraction isolated from grape marc (grape seeds and grape skins).

Source Compound Name Classification Structural Formula Function
Grape seed and skin

Review Methodology
Our aim was to prepare a scoping review to demonstrate that there is a significant amount of active substances in grapes, mainly in the seeds and pomace, which in many cases become waste. We also provide an overview of the wide range of physiological effects of these available active substances. Therefore, the extraction and use of these mole-

Review Methodology
Our aim was to prepare a scoping review to demonstrate that there is a significant amount of active substances in grapes, mainly in the seeds and pomace, which in many cases become waste. We also provide an overview of the wide range of physiological effects of these available active substances. Therefore, the extraction and use of these mole-

Review Methodology
Our aim was to prepare a scoping review to demonstrate that there is a significant amount of active substances in grapes, mainly in the seeds and pomace, which in many cases become waste. We also provide an overview of the wide range of physiological effects of these available active substances. Therefore, the extraction and use of these mole-

Review Methodology
Our aim was to prepare a scoping review to demonstrate that there is a significant amount of active substances in grapes, mainly in the seeds and pomace, which in many cases become waste. We also provide an overview of the wide range of physiological effects of these available active substances. Therefore, the extraction and use of these mole-

Review Methodology
Our aim was to prepare a scoping review to demonstrate that there is a significant amount of active substances in grapes, mainly in the seeds and pomace, which in many cases become waste. We also provide an overview of the wide range of physiological effects of these available active substances. Therefore, the extraction and use of these mole-

Review Methodology
Our aim was to prepare a scoping review to demonstrate that there is a significant amount of active substances in grapes, mainly in the seeds and pomace, which in many cases become waste. We also provide an overview of the wide range of physiological effects of these available active substances. Therefore, the extraction and use of these molecules as food supplements or possibly as novel pharmaceutical concepts such as dendrimer nano-bioconjugates could have a significant health-enhancing and disease-preventive effect on the population.
To access relevant articles the Web of Science and PubMed databases were used, augmented with the Google Scholar search engine. The "polyphenols" and "grape" keywords were applied, and 5981 results have been found on Web of Science, 3690 results on Pubmed, and more than 131,000 articles, dissertations, and scientific reports in Google Scholar.

Physical Properties
The most important physical properties of the main monomeric components of polyphenols, such as catechol, epicatechin (EC), and epicatechin-(3-O)-gallate (EGC) monomers are summarized in Table 3. Properties relevant for the separation of the components: Molecular weight: Based on the differences in molecular weight, it is possible to separate fractions by gel chromatography or membrane filtration.
Solubility: Several polyphenols are water-soluble, and many are lipid-soluble. In general, catechins are fat soluble and procyanidins are water-soluble. This allows their relatively easy separation by extraction.

Chemical and Biochemical Properties of Polyphenols
Hydrogen donor: Polyphenols have numerous hydroxyl groups, acting as hydrogen donor antioxidants, and scavenging singlet oxygen. Therefore, they can be classified as reducing agents. They form chelates with metals. They bind free radicals and stop radical chain reactions [20].
Stability: The antioxidants that can be extracted from grape seeds are very sensitive to oxygen, light, acidic, or alkaline environments, and variably sensitive to heat [21].
Polyphenol-protein interactions: The polyphenols in grape seed extract can form strong, specific bonds with protein binding units (e.g., proline-rich proteins). This binding is used in the extraction of polyphenols and the extraction of plant proteins (gluten removal). They can also inhibit or activate enzymes in grapes that protect the fruit from microbial attack. The interactions between polyphenols and proteins can be covalent, ionic, hydrogenbonded, or hydrophobic. Many proteins can precipitate polyphenols [22].

Analysis of Polyphenols
Several simple and inexpensive methods for the analytical determination of antioxidants have been developed (Table 4). Antioxidant activity can be determined simply by the FRAP method (FeCl 3 and using triazine) [23], and total antioxidant content can be determined by András Boór's determination of 2,4,6-Tris(2-pyridyl)-s-triazine [23], total polyphenol content can be determined by Folin Ciocalteu reagent [23]. Free radical scavenging activity can be measured using 1,1-Diphenyl-2-picrylhydrazine [23]. The anthocyanin content in hydrochloric acid ethanol can be determined spectrophotometrically (550 nm) [24], and leucoanthocyanins can be determined spectrophotometrically after heating with a hydrochloric acid-butanol mixture containing ferrous sulfate (II) in a 40:60 ratio [24]. Determination of catechol content in alcohol diluted solution reacted with sulfuric acid vanillin at 500 nm spectrophotometrically is simple [25], and resveratrol content can be determined directly in HPC by the Kállay-Török method [26] (Table 4). Free radical scavenging activity (antiradical activity) 1,1-Diphenyl-2picrylhydrazine [28] Determination of anthocyanin content Dilution at 550 nm with 96% ethanol containing 2% HCL at 2% v/v, followed by spectrophotometry [24] Determination of leucoanthocyanins spectrophotometrically after heating with a 40:60 mixture of hydrochloric acid and butanol containing ferrous sulphate [24] Determination of catechin content reacted with sulphuric acid vanillin in an alcohol-diluted solution at 500 nm by spectrophotometry Vanillin [25] Resveratrol content determination directly to HPLC [26]

The Beneficial Effects of Polyphenols on Health and Its Molecular Mechanisms
Polyphenols possess multifarious beneficial in vivo and clinical health effects, and their details are out of the scope of this review article. In summary, the evident antioxidant and reactive oxygen species inhibitory effects of polyphenols promote the following intracellular signal transduction and regulation process: they downregulate inflammatory proteins (for example IL-1, IL-6, TNFα, mTOR, Nuclear Factor-κB) and also oncogenes (for example C-MYC, RAS, NOTCH) and by the time upregulate tumor suppressor genes (for example SIRT, PTEN, P53) expression through epigenetic factors, such as histone acetylation, DNA methylation, and microRNA expression) [29][30][31][32]. These molecular effects and intracellular signals lead ultimately to the well-known clinically proven cardiovascular protective, anticarcinogen, antidiabetic, antimicrobial, etc. effects.

Antioxidant and Free Radical Scavenging Activity
The main physiological effects of grapeseed oil are its antioxidant properties and its ability to bind free radicals. The total polyphenol fraction in grape seed is characterized by these properties. The antioxidant effect of polyphenols is 20 times stronger than vitamin E and 50 times stronger than vitamin C. They protect LDL and cholesterol from oxidation and prevent platelet aggregation, thus they prevent coronary heart disease and maintain vas-cular integrity. Also polyphenols are capable of protecting the skin from sunburn [33,34], rejuvenating the skin, and preserving the flexibility and elasticity of joints, blood vessels, and tissues. Flavonols from grape seeds reduce alcohol-induced lipid peroxidation (lipofuscin formation) and thus protect the brain from the damaging effects of alcohol [35] Grape seed extract also protects against age-related DNA damage in the central nervous system, preventing DNA oxidation and the formation of DNA-protein bonds [36]. For a detailed mechanism of action broken down into molecules, see Table 5.

Anti-Atherosclerosis and Cardioprotective Effects
Grape seed extract does possess a cardioprotective effect, for example, it reduces the likelihood of heart attack [52]. Furthermore, it is also beneficial for numerous other cardiovascular diseases. Quercetin inhibits the oxidation of LDL and cholesterol and the clumping of platelets. Other beneficial effects are exerted by resveratrol, epicatechin, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), genistein, and daidzein, namely, they protect against atherosclerosis and alleviate arrhythmias of the heart [53]. Polyphenols reduce the risk of cardiovascular disease, for example, they decrease the risk of coronary heart disease. They act by dilating the blood vessel walls, thereby reducing blood pressure [54][55][56]. They also reduce women's blood pressure by regulating estrogen hormones [57] (Table 6).

Quercetin
Increase serum LDL-bound PON-1 10 levels HuH7 in human liver cell line In vitro [58] Induction of IFN-γ 11 gene expression Inhibition of IL-4 12 gene expression Peripheral blood in Human Peripheral-blood CD4+ T cells In vitro [59] Increase in intracellular GSH levels and activation of the γ-GCS 13 heavy subunit (GCS(h)) promoter Central neuron cell line In vitro [47] Genistein Daidzein They are incorporated into LDL, increasing its resistance to oxidation and its effectiveness in inhibiting cell proliferation

Neuroprotective Effects
Through the regulation of several enzymes, resveratrol protects nerve tissue from fibrosis, cell death in the case of prolonged damage, protects dopaminergic neurons, and prevents beta-amyloid deposition in prolonged inflammatory processes. EGCG, ECG, and myricetin inhibit the growth of tumors in the nervous system through the regulation of intracellular enzymes. Catechin and quercetin inhibit the progression of programmed cell death in the event of injury. For the molecular mechanisms of action of each fraction, see Table 7. Table 7. The protective effects of polyphenols isolated from grape marc (grape seeds and skins) on the nervous system.

Resveratrol
Stimulates AMP kinase activity

Neuro2a in cells and primary neurons; MC3T3-E1 cells and primary osteoblasts
In vitro [64] Activation of phosphorylation of PKC Transcitrin selection to prevent Aβ1 aggregation 1 Rat hippocampal cell culture; endothelial cell culture In vitro [65] Protection of dopaminergic neurons Activation of the sirtuin family of NAD-dependent histone deacetylases Organotypic mid-brain slice culture; human umbilical vein endothelial cells In vitro [66] EGCG, ECG, Myricetin Inhibition of IL-6, IL-8, VEGF and PGE2 2 production Attenuation of COX-2 expression and NF-κB 3 activation Induction of MAPK phosphatase 1 expression Inhibition of phosphorylation of MAPK (p38 and JNK 4 ) Human astrocytoma U373MG cell culture In vitro [67] Attenuation of mitochondrial membrane potential rupture and release of CYT-C 5 Reducing caspase-9 and caspase-3 activity and increasing the BAX:BCL-2 ratio Rat PC12 cells; HeLa cell line In vitro [68] Epicatechin Protects neurons from programmed cell death induced by oxLDL 6 by inhibiting the activation of JNK, c-JUN and caspase-3 Primer neuron cell culture In vitro [69]

Anti-Inflammatory Effect
Polyphenols inhibit the action of several histamine-releasing enzymes and (among others) therefore have anti-inflammatory and anti-allergic effects. Grape seed extract promotes the healing of autoimmune rheumatoid arthritis [70]. Procyanidins, EGCG and EGC reduce the inflammatory activation of peripheral monocyte cells. Procyanidins also have anti-ulcer effects [71]. Resveratrol reduces inflammation in articular cartilage cells and prostate cells. Quercetin acts in vessel walls and macrophages, and anthocyanins in small blood vessels. They inhibit lipid peroxidation and DNA fragmentation in the liver and brain. For the molecular mechanisms of action of each fraction, see Table 8. Inhibition of iNOS mRNA and protein expression by inhibiting NF-κB activation Inhibition of NO production murine microglial cell line N9 In vitro [76] Activation of MAP kinase phosphatase Prostate cells In vitro [77] Quercetin Blocking the expression of ICAM-1 1 , VCAM-1, and E-selectin Inhibition of PG synthesis and IL-6, 8 productions

Mutation Reduction and Anti-Cancer Effect
Different types of procyanidins inhibit the proliferation of cancer cells and thereby inhibit metastasis formation, for example, prostate tumors [82,83]. Proanthocyanidins from grape seed extract also induce programmed cell death (apoptosis) in metastases of advanced breast cancer [84]. Furthermore, they inhibit colon cancer growth too [85]. For molecular mechanisms of action of each fraction, see Table 9.

Influencing Signal Transduction
In many cases, polyphenols in grape seeds affect interstitial, extra-and intracellular information transduction mechanisms through the cell membrane effects. Proanthocyanidins accelerate programmed cell death in cancer cells, quercetin enhances the functionality of primary cortical neurons through signal transduction. Resveratrol reduces inflammatory overactivation of monocytes via signaling and inhibits cardiac fibrosis (see Table 10). Table 9. Mutation-reducing/anti-cancer effects of the polyphenol content of grape marc (grape seeds and skins).

Polyphenol Name Molecular Mechanism of the Protective Effect Cell Culture Level
Ref.

Resveratrol
Inhibition of cell proliferation and reduction of telomerase activity Human cancer cell line HCT116 In vitro [86] Stimulation of the P53-dependent pathway of programmed cell death Human lung adenocarcinoma cells A549 In vitro [87] Inhibition of cell proliferation by interaction with the ERα 1 -related PI3K pathway

Effects on the Vascular Wall and Choroidal Cells
EGCG and quercetin reduce programmed cell death in the cells that build up the vascular wall. In calf vascular endothelial cell culture, Cy3G increases cell lifespan through cyclic guanosine monophosphate (cGMP) and nitric oxide (NO) regulation. In addition, EGCG increases vasodilator effects in calf aortic vascular endothelial cells. Catechins reduce the vascularization-inducing effect of angiogenin-like proteins in chickens. Proanthocyanidins reduce inflammation-induced cell damage in human choroidal cells. Proanthocyanidins and flavan-3-ols reduce the degradation of enzymes responsible for the relaxation of blood vessels (see Table 11).

Effects on Diabetes
EGCG, ECG, and (-)-EGC polarize the intestinal epithelial cells responsible for sugar uptake and inhibit sugar uptake in the rabbit's small intestine. Quercetin lowers blood glucose levels, tannins and anthocyanin inhibit the alpha-amylase enzymes responsible for glycogen degradation (which increases blood glucose levels) and the alpha-glucosidase enzymes in the intestinal wall responsible for sugar absorption (see Table 12). Quercetin Reduces blood sugar levels Inhibits SVCT1 1 and GLUT2 Intestinal cell model In vitro [110] Tannin, anthocyanin Inhibition of α-amylase and α-glucosidase In vitro [111]

Effects on the Cell Cycle
Resveratrol stops HepG2 liver cancer cells from dividing by stimulating the expression of the P21 protein, which inhibits the CDK cyclin-dependent kinase enzyme. In human skin tumor and human colon cancer cells, it inhibits the formation of complexes of cyclins involved in cell cycle regulation, reduces the phosphorylated form of the pRb enzyme responsible for initiating DNA synthesis during cell division and leads to cell cycle arrest in the G0/G1 phase through inhibition of the expression of the transcription factors E2F (1)(2)(3)(4)(5) and their heterodimeric partners DP1, DP2. Proanthocyanidins inhibit the expression of cell cycle regulators cyclin B1, D1, A1, and β-catenin, which accumulate in cancer cells and are responsible for inappropriate gene activity. They arrest the cell cycle in the G1 phase, reducing the expression of cyclins in human melanoma cells. In human skin cancer cells, they promote cell cycle arrest in the G1 phase, inhibit the function of cyclins and cyclin-dependent kinases (CDK), and promote the expression of CDK inhibitors (see Table 13). In the case of caries of the tooth root, proanthocyanidin-containing grape seed extract induces the re-crystallisation of the tooth enamel [118].

Antihyperlipidemic Effect
Grape seed extract has been shown in clinical trials to increase satiety, reduce energy intake from food intake, and increase fat breakdown in vitro [119]. Grape seed extract inhibits the enzymes involved in fat metabolism (pancreatic lipase, lipoprotein lipase), thus preventing the accumulation of fat in adipose tissue [120]. Mice fed grape seed extract have reduced tissue fat levels but not influenced body weight [121]. Polyphenols isolated from grape seeds and red wine inhibit intracellular cholesterol synthesis, and thereby reduce blood cholesterol level [122].

Antibacterial and Antifungal Effect
The antibacterial activity of polyphenols covers both Gram-positive and Gram-negative bacteria. It also enhances antifungal effects, for example in the case of Candida albicans infection (candidiasis) it increased the efficacy of medicines [123].

Anti-HIV Effect
Proanthocyanidins inhibit the expression of HIV-secreting coreceptors in normal peripheral mononuclear cells [124].

Sensory Effect
Proanthocyanidins and resveratrol enhance the expression of vascular endothelial growth factor (VEGF) in pigment cell culture [124,125] and animal model (hamsters) [126].

Hepatoprotective Effect
Novel proanthocyanidins IH636 increase the expression of the mitochondrial signal transduction enzyme BCL-xL and attenuate acetaminophen-induced hepatic DNA damage and programmed and necrotic destruction of liver cells in an engineered mutant (ICR) mouse strain. In rat liver, daidzein improves the growth of d-galactosamine-induced malondialdehyde-protein adducts and cytoplasmic superoxide dismutase (SOD) activity. In rats, genistein reduces experimental liver damage caused by CCl 4 by preventing lipid peroxidation and enhancing the antioxidant system (see Table 14). In vitro [127] 4.11. Anti-SARS-CoV-2 Effect Beneficial effects of tannins against "cytokine storm" of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are also effectively investigated recently. It has been discussed that tannins have both preventive and therapeutic potential against SARS-CoV-2 infections [13,128].

Risks Associated with Polyphenols
To date, no adverse effects of polyphenols have been reported at low concentrations [129]. The adverse effects of polyphenols are observed at high doses. Adverse effects on the body may include carcinogenesis/genotoxicity, thyroid damage, the estrogenic activity of isoflavones, dietary effects, and drug interactions [130].
Grape seed extract may contain isoflavones at a concentration of 50 mg/dose or proanthocyanidins at a concentration of 100-300 mg/dose. In rats and mice, 0.5-2.0 g of proanthocyanidin extracted from grape seeds per kg body weight does not cause acute toxicity [131]. In rats, 60 g/kg of ellagitannin does not cause acute toxicity [132]. However, chronic kidney damage has been observed in rats when given high doses (2% or 4% per kg body weight) of quercetin in their diet [133]. A reduction in life expectancy has been observed even when 0.1% quercetin was added to the diet of mice [134].
Some polyphenols are carcinogenic and genotoxic at higher concentrations [135]. For example, caffeic acid at 2% in the diet induces gastric and renal tumors in mice and rats [136]. High levels of quercetin can cause tumor formation [137]. Catechins in high doses promote the division of tumor cells in the colon, but quercetin reduces the division of tumor cells [138,139].
At high doses, proanthocyanidins (also known as procyanidin oligomers) can cause liver damage, hemophilia, tumors of the female reproductive organs and inflammation of the intestine [140].
Some flavonoids were reported to inhibit thyroid hormone synthesis [141,142]. Among the isoflavones (flavanols), a decrease in thyroid hormone levels has been observed in rats at high doses of genistein [143], as well as disruption of some female hormones [144]. However, isoflavones are present in grape seeds only in negligible amounts.
Polyphenols are also known to inhibit nutrient absorption. For example, they impair iron absorption [145], but vitamin C, which may be present in nutrient sources alongside polyphenols, promotes iron absorption [146]. In addition, proanthocyanidins (condensed tannins) may also have an inhibitory effect on nutrient absorption as they can interact with proteins and inhibit the function of several enzymes. However, these properties are only seen at extremely high doses (10 g/kg body weight in the diet) and not at lower doses [147]. Diets high in tannins may show a protein absorption-reducing effect [148]. It should be noted that such high polyphenol content is not found in Western diets.
Finally, polyphenols can affect the bioavailability and mechanism of action of drug substances. Some drugs, such as benzodiazepines and terfenadine, may have a threefold increase in bioavailability in the presence of polyphenols (due to CYP3A4 inhibition) [149].

In Vivo Investigations of Grape Seed Extract and Its Components
The number of in vivo investigations using a wider range of immunohistochemistry to examine various molecular mechanisms of action is growing exponentially (Table 15).
By using MTT assay, flow cytometry, and immunoblot analysis, lipophilic grape seed proanthocyanidin (LGSP) was assessed for its anti-prostate cancer activity against the PC3 cell line in vitro. A mouse xenograft model generated from PC3 was used to test LGSP's anti-prostate cancer impact in vivo. In tumor tissues, immunostaining tests for Ki67 and cleaved caspase 3 were carried out. By triggering apoptosis, LGSP had a potent inhibitory effect on PC3 cell proliferation [150].
Treatment with LGSP caused G1 phase cell cycle arrest in PC3 cells, which was further validated by increased expression of the tumor suppressor p21 and p27 and decreased expression of cyclin D1 and CDK 4. Additionally, it was demonstrated that LGSP-induced apoptosis is caspase-dependent by the activation of cleaved fragments of caspases 3, caspase 9, as well as PARP.
LGSP boosted the release of cytochrome c in the cytoplasm upstream of the caspase cascade. In PC3 cells following LGSP administration, the Bcl-2/Bax ratio likewise fell [150].
Considering tumor research, LGSP inhibited the proliferation of PC3-derived mouse xenografts and induced apoptosis [150].
Nude mice with a human liver cancer cell (HepG2)-derived xenografts were treated with grape seed proanthocyanidins (GSPs). According to the findings, GSPs triggered autophagy, and inhibiting autophagy caused an increase in apoptosis in HepG2 cells. Since stimulating the phosphorylation of mitogen-activated protein kinase (MAPK) pathwayassociated proteins, p-JNK, p-ERK, and p-p38 MAPK, and decreasing the expression of survivin, GSPs at 100 mg/kg and 200 mg/kg significantly inhibited the proliferation of HepG2 cells in nude mice without manifesting toxicity or autophagy [151].
Grape seed procyanidin reverses the change in pulmonary hemodynamics in the cigarette smoke-induced pulmonary arterial hypertension model applied in rats. According to mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular hypertrophy index, wall thickness, and wall area data grape seed procyanidin decreases the inflammation by the PPAR-γ/COX-2 pathway [152].
In monocrotaline-induced pulmonary arterial hypertension rats, mean pulmonary arterial pressure, pulmonary vessel resistance, right ventricular hypertrophy index, percentage of medial wall thickness, percentage of medial wall area, and lung weight of wet and dry tissue ratio all decreased. The expression of endothelial nitric oxide synthase in lung tissue and plasma NO levels were raised up; the Ca 2+ level in pulmonary arterial smooth muscle cells (PASMC) was lowered; the transcription of inflammatory factors including myeloperoxidase, interleukin (IL-1, IL-6), and tumor necrosis factor-alpha (TNF-alpha) was down-regulated in lung tissue; the nuclear factor-B pathway was also inhibited [153].
Neuroprotective effect of red grape (Vitis vinifera) seed and skin extract (GSSE) was determined in a mice model of Parkinson's disease induced by neurotoxin 6-hydroxydopamine (6-OHDA), which causes oxidative damage and mimics the degeneration of dopaminergic neurons observed in Parkinson's disease (PD). It was found that GSSE was effective in protecting dopamine neurons from 6-OHDA toxicity by reducing apoptosis, the level of reactive oxygen species (ROS), and inflammation; reducing the cleaved caspase-3 activity that helps inhibit 6-OHDA-induced mDA neuron death in a cellular model of PD; decreases ROS production induced by 6-OHDA in ESC-derived DA neurons; decreases phospho-NF-kB p65 activation induced by 6-OHDA in dopaminergic neurons; rescues motor deficits induced by 6-OHDA; prevents the loss of midbrain dopaminergic neurons (mDA) in a 6-OHDA mouse model of PD; prevents the loss of SOD1 level induced by 6-OHDA lesion. The biomarkers were for immunostaining: MAP2, AB5622, r tyrosine hydroxylase, caspase-3, and phosphorylated NF-kB p65 [155].  [154] Red grape seed and skin extract GSSE was effective in protecting dopamine neurons from 6-OHDA toxicity by reducing apoptosis, the level of reactive oxygen species (ROS) and inflammation; reducing the cleaved caspase-3 activity that helps inhibit 6-OHDA-induced mDA neuron death in a cellular model of PD; decreases ROS production induced by 6-OHDA in ESC-derived DA neurons; decreases phospho-NF-kB p65 activation induced by 6

Clinical Studies of Grape Seed Extracts
Recently, there has been a significant increase in clinical trials, which further confirm the efficacy of grape seed extract in the treatment of major diseases such as various cancers, diabetes, hypertension, neurodegenerative disorders (e.g., Parkinson's disease), etc (Table 16).  [163] Resveratrol (RSV), a naturally existing polyphenol, has been shown to have significant antioxidant, anti-inflammatory, and anticancer properties. Regarding inflammation in a mouse model of collagen-induced arthritis, RSV was newly identified as a new treatment drug for suppressing said condition. Nonetheless, the medical advantages of RSV in the therapy of rheumatoid arthritis (RA) have not been established. The purpose of this randomized controlled clinical trial is to offer insight into the therapeutic advantages of RSV in the treatment of RA in patients at various stages of disease activity. In this randomized controlled clinical trial, 100 RA patients (68 females and 32 males) were randomly assigned to one of two groups of 50 patients each: an RSV-treated group that received a daily RSV capsule of 1 g in addition to routine care for 3 months, and a control group that received only basic care. Both groups' clinical and biochemical markers of RA were evaluated. Clinical markers (such as the 28-joint count for swelling and tenderness) and disease activity score assessment concerning 28 joints were reported to be considerably lower in the RSV-treated group. Furthermore, serum levels of certain biochemical markers like C-reactive protein, erythrocyte sedimentation rate, undercarboxylated osteocalcin, matrix metalloproteinase-3, tumor necrosis factor-alpha, and interleukin-6 levels were considerably lower in RSV-treated individuals. The present study proposes that RSV be used as an adjuvant to standard antirheumatic medications [156].
In a clinical trial meta-analysis of grape seed extract (GSE) on diabetes and blood lipid levels, data were pooled using a random-effects model and weighted mean difference (WMD) was considered as the overall effect size. The grape seed had no implications on HbA1c levels, HDL cholesterol levels, or anthropometric parameters. The meta-analysis showed that consuming GSE lowered FPG, TC, LDL cholesterol, triglycerides, and CRP levels considerably [157].
The effect of grape seed extract ointment on wound healing was also investigated in cases of Cesarean section. A total of 129 women participated in this double-blind, randomized, controlled clinical trial. Participants were chosen through the convenience sampling method and were randomly assigned into three groups: 2.5% grape seed extract ointment, 5% grape seed extract ointment, and petrolatum. The REEDA scale was used to examine CS wound healing indices beforehand, 6 and 14 days after the treatment (redness, edema, ecchymosis, discharge, and approximation).
The extract increased the synthesis of vessel enclosure growth factor (VEGF) along the wound's edge. Furthermore, GSE demonstrated anti-inflammatory action via cytokines (TNF, IL-1, IL-6, IL-14), as well as antibacterial and antioxidant activities. On days 6 and 14 following intervention, the mean scores were 2.02, 0.52 and 0.98, 0.61 in the 5% ointment group, 2.83, 0.54 and 1.58, 0.67 in the 2.5% ointment group, and 2.91, 0.51 and 1.55, 0.74 in the petrolatum group. Whilst the 5% ointment group's mean score was significantly dissimilar from the 2.5% ointment and petrolatum groups (p < 0.001), the 2.5% ointment group's mean score was not statistically different from the petrolatum group on days 6 and 14 following intervention (p = 0.38 and p = 0.79, respectively) [158].
GSE can be successfully applied for cancer prevention, e.g., in preventing lung cancer. A modified phase I, open-label, dose-escalation clinical study was conducted to evaluate the safety, tolerability, MTD, and potential chemopreventive effects of leucoselect phytosome (LP), a standardized GSE complexed with soy phospholipids to enhance the bioavailability, in heavy active and former smokers. Bronchoscopies with bronchoalveolar lavage and bronchial biopsies were performed before and after 3 months of LP treatment. Hematoxylin and eosin stain for histopathology grading and IHC examination for Ki-67 proliferative labeling index (Ki-67 LI) were carried out on serially matched bronchial biopsy samples from each subject to determine responses to treatment. Such a treatment regimen significantly decreased bronchial Ki-67 LI by an average of 55% (p = 0.041), with concomitant decreases in serum miR-19a, -19b, and -106b, which were onco-miRNAs (oncomIRs) previously reported to be downregulated by GSE, including LP, in preclinical studies. In spite of not reaching the original enrollment goal of 20, our findings nonetheless support the continued clinical translation of GSE as an antineoplastic and chemopreventive agent against lung cancer.
It has been reported that GSE acts on downregulating well-known oncomIRs namely miR-19a, -19b, and -106b. Decreases in miR-19a and -19b upregulated insulin-like growth factor II receptor (IGF2R), PTEN mRNA expressions, and their respective protein products. Furthermore, GSE increased PTEN activity and decreased phosphorylation of AKT-a key procarcinogenic driver in lung cancer. Both PTEN and IGF2R are tumor suppressors and predicted targets of miR-19a and -19b. Downregulation of miR-106b resulting in upregulation of its downstream target, the tumor suppressor cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNA and protein (p21) levels, further contributed to the antineoplastic effects of GSE [159].
In the continuation of the study, it was concluded that one month of leucoselect phytosome treatment significantly increased eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the omega-3 polyunsaturated fatty acids (n-3 PUFA) with well-established anticancer properties. Leucoselect phytosome also significantly increased unsaturated phosphatidylcholines (PC), likely from soy phospholipids in the phytosome and functioning as transporters for these PUFAs. Furthermore, 3-month leucoselect phytosome treatment significantly increased serum prostaglandin (PG) E3 (PGE3), a metabolite of EPA with anti-inflammatory and antineoplastic properties. Such increases in PGE3 correlated with reductions of bronchial Ki-67 LI (r = −0.9; p = 0.0374). Moreover, posttreatment plasma samples from trial participants significantly inhibited the proliferation of human lung cancer cell lines A549 (adenocarcinoma), H520 (squamous cell carcinoma), DMS114 (small cell carcinoma), and 1198 (preneoplastic cell line) [160].
According to another randomized, double-blinded, placebo-controlled clinical trial, GSE reduces significantly the risk of cardiovascular diseases in obese patients. In this trial the effects of GSE supplementation along with a restricted-calorie diet (RCD), on changes in blood lipid profile, visceral adiposity index (VAI), and atherogenic index of plasma (AIP) are investigated. Forty obese or overweight individuals (25 ≤ body mass index < 40 kg/m 2 ) were randomly assigned to receive GSE (300 mg/day) or placebo, plus RCD, for 12 weeks.
Levels of high-density lipoprotein cholesterol (HDL-C) and HDL-C/low-density lipoprotein cholesterol (LDL-C) significantly increased in the GSE group as compared with the placebo group at week 12 (p = 0.03 and 0.008, respectively, The investigation adjusted for age, sex, energy and saturated fatty acid intake). VAI, AIP, total cholesterol and triglyceride significantly decreased in the GSE group compared with the baseline (p = 0.04, 0.02, 0.01, and 0.02, respectively).
TNF and interleukin-6 may contribute to increased TG and VLDL levels and also decreased HDL-C levels. LDL-C reduction following GSE intake compared with the placebo did not remain significant, after adjusting for inflammatory markers. So it can be assumed that LDL-C more than HDL-C might be affected by inflammatory markers. GSE protects against atherosclerosis also [161].
GSE protects against insulin resistance in metabolic syndrome through the increasing of glucose transport. Participants were divided into grape seed extract (GSE) and placebo groups (n = 24 each) and received 100 mg/day of GSE or placebo and were placed on a weight loss diet for 8 weeks. The MD (mean difference ± SEM) of HOMA-IR between the GSE group (−1.46 ± 0.45) and the placebo group (−0.48 ± 0.47), (p = 0.020), and the MD of insulin between the GSE group (−7.05 ± 2.11) and the placebo group (−1.71 ± 2.12), (p = 0.024), were significant [162].
According to another randomized double-blind placebo-controlled clinical trial, red grape seed extract (RGSE) reduces serum paraoxonase activity and hyperlipidaemia. For 8 weeks, 70 MMH patients were given a placebo or the therapy (200 mg/day of RGSE). In the instances, there were significant elevations in the blood concentrations of apo-AI (p = 0.001), HDL-C (p = 0.001), and PON activity (p = 0.001) as well as significant reductions in the concentrations of TC (p = 0.015), TG (p = 0.011), and LDL-C (p = 0.014). The activity of PON was shown to be significantly correlated with apo-AI (r = 0.270; p < 0.01) and HDL-C (r = 0.45; p < 0.001). Significant differences between the RGSE and control groups (before and after treatment) for TC (p = 0.001), TG (p = 0.001), PON (p = 0.03), apo-AI (p = 0.001) and LDL-C (p = 0.002) were observed.
RGSE had the ability to significantly elevate HDL-C apo-AI levels while lowering TC, TG, and LDL-C levels. RGSE had the capacity to significantly increase the concentration of HDL-C apo-AI and lead to decreased TC, TG, and LDL-C levels. Following two months of RGSE treatment, a significant correlation between the changes in HDL-C and apo-AI values was also noted [163].

Grapeseed Oil and Polyphenols
In the grocery business, grapeseed oil is utilized for frying, salad dressings, vinegar marinades, hot oil frying, flavoring oils, and cereal frying. It is used in the cosmetics sector to make body lotions, hand creams, lip balms, body oils, sun lotions, and hair care products. Although France, Spain, and Argentina are all significant producers, Italy is where the majority of grapeseed oil gets produced.

The Composition of Grape Seeds
Grape seeds contain 13-19% oil, which is rich in essential fatty acids, about 11% protein, 60-70% digestible fiber, and non-phenolic antioxidants such as tocopherols and beta-carotene. In several grape varieties, 60-70% of polyunsaturated fatty acids are present in grapeseed oil. Tocopherols and β-carotene, which are non-phenolic antioxidants, are concentrated in grapeseed oil (mainly α-tocopherol and tocotrienol). In addition, phytosterols, which have been shown to have a strong antiatherosclerotic effect (β-sitosterol, stigmasterol, campesterol, and sitostanol), are present in significant amounts in grapeseed oil.

Location of Polyphenols in Grape Seed Cells
Polyphenols in grapes are found in the grape seed oil droplets. The oil is located within the cell walls in the form of discrete oil droplets, 0.6-2.0 µm in size. The oil droplets, which are composed of triglycerides, are covered with a phospholipid layer that binds to proteins. The stability of the droplets is ensured by the repulsive effect of proteins and the negative surface charge.
The methods for the extraction of polyphenols from grape seeds are summarized in Appendix A.

Conclusions and Future Perspectives
Grape polyphenols exert cardioprotective, anti-cancer, anti-diabetic, anti-obesity, antiosteoarthritis, anti-neurodegenerative and anti-microbial effects both through direct antioxidant properties and antioxidant enzyme stimulating effects, and via modulating other signal transducers, for example inducing SIRT-1 gene, and inhibiting NFkappaB and mTOR gene expression, among other inflammatory genes (COX-2, MMPs). Thus, several patented products with high grape polyphenol content for therapeutic application and disease prevention too were developed. The present review article may contribute to further studies, therapeutic approaches and even the development of new compounds and products, too.
The physiologically active molecules of the polyphenol fraction can be selectively delivered to diseased cells by binding to dendrimers. Dendrimer binding can increase their chemiluminescence and antioxidant properties. Thus further in vivo and clinical studies are warranted to elucidate their beneficial effects in combination with active pharmaceutical ingredients or food supplements.
The number of studies on the binding of polyphenols to drug carriers, in particular, the formation of nanoscale conjugates, has increased significantly in the last decade. Effective use of polyphenols requires their substitution at higher concentrations than usual. As a major component of grape seed extract, resveratrol plays a central role in the synthesis of anti-cancer polyphenol-containing nanobioconjugates (e.g., dendrimers, polymer nanoparticles, liposomes, nanotubes, micelles, etc.) [164]. Dendrimers, as nanocarriers, can play a prominent role in this, because their multifunctional surface area allows them to contain a high local concentration of an active ingredient in a small volume, which makes their application more efficient [165].
Polyphenol dendrimers are used to enhance chemiluminescence [164,166]. Dendrimers made from gallic acid produce singlet oxygen in the presence of hydrogen peroxide. They also have chemiluminescent properties, allowing the presence of singlet oxygen to be detected in chemical systems [166]. Second-generation polyphenol dendrimers were synthesized with various core molecules and chemiluminescence was measured upon reaction with H 2 O 2 at basic pH. High chemiluminescence was measured for all types of polyphenol dendrimers, which was 120 times higher than that of gallic acid. The intensity of chemiluminescence is strongly dependent on the distance of each branch in the structure of the polyphenol dendrimers [167]. Stilbene dendrimers have also been prepared, which also have increased photochemical activity [167,168].
The antioxidant activity of polyphenols from green tea could be significantly enhanced by enzymatic polymerization (polycatechin) or coupling to polyamino-amide dendrimers (PAMAM-catechins) [169]. First, second, and third generation dendrimers containing two, four, and eight tannic acid groups, respectively, were synthesized. The antioxidant property of the dimer is more than four times that of the monomolecular tannic acid, the tetramer more than twice that of the monomolecular tannic acid, and the octamer one and a half times [170]. The antioxidant activity of the vitamin E analog (tonox) bound to gold nanoparticles is increased eightfold compared to the free molecule [171]. Naturally occurring polyphenols bind strongly to both proteins and cell membranes. Taking advantage of this, dendrimers with a catechol-modified surface can deliver a wide variety of bioactive proteins and polypeptides into cells. Recent experimental results demonstrate that catechol dendrimers can also kill tumor cells in vivo, e.g., by transporting the enzyme alpha-chymotrypsin into the tumor cell-matrix [36].
Because of their general anti-inflammatory and free radical scavenging properties, polyphenols are likely to be widely used as medicines [172]. The studies presented demonstrate that their therapeutic and preventive role can be significant against the most common pathologies (lung cancer, atherosclerosis, hypertension, diabetes, microbial infections, etc.) [173]. The efficacy and/or targeted therapeutic use of polyphenols can also be achieved by using various nanocarriers such as dendrimers [174]. Active substances (e.g., from grape seed extract), which can be produced cheaply and in an environmentally friendly way on an industrial scale [175], could significantly replace synthetic drugs, which are not only expensive to produce but also are more toxic for the living organisms than polyphenols in general [165]. Combined with the latest therapeutic technologies (e.g., gene therapy), polyphenols could well complement and make the medicine of the future more effective [165,176].  Acknowledgments: The authors express their special thanks to Peter Szabó for his outstanding work in correcting the manuscript and interpreting it into English.

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

Appendix A. Extraction of Polyphenols from Grape Seeds
The procedure consists of the following steps. First, the grape seeds are separated from the grape skins in the grape pomace. The cleaned grape seeds are dried and then ground into grape seed flour. The grape seed flour is pressed and the grape seed oil is removed. Polyphenols are extracted from grape seed oil by various methods. The bitter flavoring substances (small molecule polyphenols) are removed from the polyphenol fraction and the remaining polyphenol fraction is added to the grape seed flour to obtain a functional food raw material with high antioxidant content.

Appendix A.1. Oxidative Polymerisation of Polyphenols during Separation
The polymerization of polyphenols may be of interest in reducing bitterness, as recent studies have shown that the bitter taste is caused by low molecular weight polyphenols. Higher molecular weight polyphenols are less bitter, the strength of the bitter taste sensation being inversely proportional to the molecular weight of the polyphenol components [177]. However, small polyphenols readily polymerize to oligomers, which are no longer bitter to any significant extent, but retain their antioxidant properties and several physiologically important properties [147]. Most proanthocyanidins are identified as oligomers, as larger polymers are difficult to identify [178]. The average degree of polymerization of proanthocyanidins extracted from grape seeds of Vitis vinifera L. is more than 85 [179]. Spontaneously: during polyphenol separation operations. Polyphenols are sensitive to light, oxygen, acid, or alkali and can oxidize and polymerase [20].
Enzymatically: by polyphenol oxidase during plant maturation. Enzymatic biosynthesis of polyphenol oligomers is known, More recently, it has also become possible to regulate polyphenol synthesis in plants, e.g., to produce higher amounts of flavonoids and stilbene from glucose by regulating malonyl-CoA levels [180]. spontaneously, e.g., in acid-butanol [181]. Under highly acidic conditions, proanthocyanidins are converted to anthocyanidins by cleavage of the C-C interflavanil bond; ii. and may also degrade enzymatically during storage [182]. If they are composed of only catechol and epicatechol subunits, the products of hydrolysis are only cyanidins, then proanthocyanidins are called procyanidins. Procyanidins are the most abundant proanthocyanidins in plant-derived food [147,183].
Procyanidins may also be degraded in living organisms before absorption. Degradation within the body starts with depolymerization [184]. In artificial humidity (pH = 2-4), proanthocyanidins isolated from cocoa are degraded to monomers and dimers [185,186].

Appendix A.2. Extraction of Grape Seed Oil from Grape Seed Flour
Before the grape seed oil is extracted, the grape seeds and skins are separated from the pomace and the grape seeds are dried at 60 • C for 8 h. To remove the grape seed oil more effectively, the dried grape seeds are ground. The removal of grapeseed oil from grapeseed flour can be done by (a) extraction Grape seed oil is obtained from grapeseed flour by extraction with petroleum oil at 60-70 • C for 6 h. De-oiled flour can be obtained by removing the residual oil (acetone: water: acetic acid, followed by methanol: water: acetic acid extraction at 90:9.5:0.5 for 8 h) [123]. The extraction of polyphenols can be divided into two main steps. Firstly, the total polyphenol fraction is dissolved at the level of the plant cell matrix, followed by the dissolution of the polyphenol fraction in an external solvent. The main components of the polyphenol fraction are then separated [187].

Appendix A.2.2. Direct Extraction of Vitamin E from Grape Seed Flour
There are several publications on the antioxidant extraction of alpha-tocopherol (vitamin E) directly from grape seed meal. The extraction can be performed by classical extraction with hexane (20 h with Soxhlet apparatus) [188], mechanical pressing (Bovenau) for 72 h, liquid extraction under pressure [189], and high-pressure supercritical carbon dioxide [190]. (Alpha-tocopherol is not a polyphenol but has antioxidant properties).

Appendix A.3. Extraction of Polyphenols from Grape Seed Oil
To make antioxidant-rich flour, the flavonoid components, which are usually antioxidant (see Section A.2.1), are separated from the grapeseed oil and mixed with the flour. The grapeseed oil is first stripped of its phenolic acids. For example, 5 mL of crude grapeseed oil is extracted with 10 mL of deionized water and 10 mL of 0.01% v/v hydrochloric acid [191]. The total polyphenolic fraction can be selectively separated from the carboxylic acid (after extraction with olive oil) by diluting the grape seed oil in hexane (50 mL hexane by extraction with 20 mL methanol/water 60:40 v/v slurry), the carboxylic acid fraction is removed to the aqueous phase, after separation on silica gel the residual hexane is removed in vacuum [192]. Note that separation with hexane is obsolete. Deacylation can also be performed by molecular distillation [192,193].

Appendix A.3.2. Separation of Polyphenols from Grape Seed Oil
In the separation of the polyphenol fraction, the whole grape seed oil is first extracted from the powdered grape seed. Flavonoids are then removed from the grape seed oil by extraction with 20 mL ethyl acetate [194].

Appendix A.3.3. Enzymatic Pretreatment Effect
Grape seed oil can be extracted more efficiently if the grape seeds are treated with cell wall-degrading enzymes. Enzymatic treatment is most optimal when carried out for 24 h at pH 4, 30-40 • C, using enzymes cellulase, protease, xylanase, and pectinase [195].
In a different research, cell walls' polysaccharides were broken down using the Novoferm 106 and Cellubrix L enzyme complexes. The extract was separated from the solid residue by pressing. Once the pectin-degrading and cellulose-degrading enzymes were present during the 2 h treatment at a concentration of 4500 mg/kg dry weight at 40 • C, pH = 4, the extraction of the polyphenolic component was at its highest [196].

Appendix A.4. Methods for the Determination of Polyphenol Content
It is simple and affordable to determine total polyphenols using the Folin-Ciocalteu technique [197] and antioxidant activity using TEAC [196]. The process relies on reducing the radical cation 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS •+ ) to ABTS. It is also feasible to determine antioxidants using FRAP [197] based on the development of a tripyridyl-S-thiazine Fe 2+ complex (TPTZ-Fe 2+ ) (absorbance at 593 nm). On Table A1, the concentrations of the most significant physiologically active polyphenolic chemicals found in grape marc are listed [198,199]. Table A1. Amounts of the most important active substances in grape marc (in seed and skin). Polyphenols are also found in the residue of grape seed flour from which the grape seed oil has been extracted. The pressed grape seed also contains phenolic acids. The residual polyphenol content of the pressed grape seed is quite significant, with 57-79% of the total polyphenols remaining in the grape seed after pressing [200] (see Table A2).