Antioxidant, antidiabetic, antiglaucoma, and anticholinergic e ﬀ ects of Tay ﬁ grape ( Vitis vinifera ): A phytochemical screening by LC-MS/MS analysis

: Grapes ( Vitis vinifera ), grape extracts, and grape products are known to possess bene ﬁ cial e ﬀ ects. Antioxidant activity and enzyme inhibition activities of Tay ﬁ grape ( Vitis vinifera ) extracts were studied and compared to standards. The IC 50 values of the ethanol extract of the Tay ﬁ grape ’ s scavenging abilities for ABTS ˙ + and DPPH ˙ radicals were found to be 5.9 and 16.1 μ g/mL, respectively, higher than those of positive controls. Also, the phenolic and ﬂ avonoid ingredients of the Tay ﬁ grape seed ethanol extract were measured to be 82.8 mg GAE/g and 91 mg QE/g. The Tay ﬁ grape seed water and ethanol extracts exhibited IC 50 values of 5.3 and 5.8 μ g/mL toward α -glycosidase, respectively; 385.2 and 567.6 μ g/mL toward α -amylase; 27.1 and 13.8 μ g/mL toward acetylcholinesterase (AChE); and 54.7 and 12.6 μ g/mL toward CA II. Twenty-two biomolecules were detected by LC-MS/MS. The four types of conventional antibiotics utilized by hospitals proved ine ﬀ ective against Staphylococcus aureus and Escherichia coli bacteria. The Tay ﬁ grape ethanol and water extracts had high AChE, α -glycosidase, and CA II inhibitions in addition to having antioxidant activity.


Introduction
The grape plant, Vitis vinifera L., belongs to the Vitaceae family and has been domesticated for over 7,500 years.Due to its extensive global cultivation, grapes are currently regarded as one of the most significant fruit crops [1].Active secondary plant metabolites, such as phenolics, have been isolated and used to treat a few medical conditions.Increasing demands and numerous studies have recently been made for the approval of the use of herb metabolites in medications to cure a variety of ailments [2].The Tayfi grape has a delicious flavor and lovely pink bunches.This cultivar is oriental and adores warmth and sunlight.
Reactive oxygen species (ROS) are different types of triggered oxygen and involve free radicals like hydroxyl radicals (OH˙), superoxide radicals ( − O 2 ), and non-free-radical species like singlet oxygen ( 1 O 2 ) and hydrogen peroxide (H 2 O 2 ) [3].Antioxidant system in organisms maintains an equilibrium between the production of ROS and its deactivation.Under pathological conditions, an excess generation of ROS causes oxidative stress (OS).When natural antioxidant defense is inadequate, ROSs are formed [4].Antioxidant substances and antioxidant enzymes are part of the immune system's defense mechanism.Carbohydrates, lipids, proteins, and nucleic acids found in living things are just a few of the biomolecules that they can fix or remove.Antioxidants slow down, stop, or prevent these biomolecules from oxidizing.They include phenols and polyphenols, which are potent inhibitors of ROS or substances that can effectively neutralize their harmful and undesirable effects [5].Reactive oxygen or nitrogen species can help cells survive and operate normally by eliminating and digesting infections or antigens.Proteins, lipids, carbohydrates, and nucleic acids can all be harmed by the excessive synthesis and buildup of reactive pro-oxidant species over time.This OS has the potential to exacerbate a number of age-related progressive disorders, including cancer, diabetes, cataracts, Alzheimer's, and Parkinson's diseases [6].A free radicals or ROS scavengers, many antioxidants found in plants have been identified so far.Searching naturally occuring antioxidants in plants to utilze in foods and medications has received far more attention recently since synthetic antioxidants have become increasingly unpopular because of their undesirable adverse effects, like carcinogenesis [7].Currently, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are the antioxidants that are most frequently utilized.However, there are severe restrictions on BHT and BHA after serious concerns were raised about their toxic effects.As a result, consumers' preference for natural antioxidants has boosted their interest in food and pharmaceutical applications [8].Antioxidants can be included in products to prolong longevity, particularly for lipids and foods that contain lipids.Scientific studies have recently confirmed the strength of antioxidants; moreover, grape seed extract has health advantages, and as a result, these products are now used as food additives and dietary supplements [9].Anthocyanins are found abundant in grape skin, while grape seeds are a great provider of extractable phenolic antioxidants like flavonoids, proanthocyanidins, and resveratrol [10].Antioxidant activity is present in grape seeds, which possess a shielding impact on pancreatic islets in diabetes by reducing the effects of OS [11].Vegetables, fruits, drinks, herbs, and spices all include polyphenols, which are naturally occurring substances.By donating an electron (e − ) or hydrogen atom (H) to different reactive oxygen, chlorine, and nitrogen species, phenolic compounds scavenge free radicals [12].
Among the primary important worldwide medical issues at the moment is Alzheimer's disease (AD).For AD, numerous treatment plans have been developed.Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is the most pertinent technique [13].In order to treat the symptoms of AD, various AChE inhibitors are used.Tacrine, donepezil, rivastigmine, and galantamine are a few examples of cholinesterase inhibitors that have recently been used in clinical AD treatment.Due to side effects like gastrointestinal disturbance and hepatotoxicity, the use of these drugs is also restricted [14].Memory loss and various cognitive problems that are progressing are the initial symptoms of AD.Acetylcholine (ACh) depletion, inflammation, and OS are other factors that might be linked.The course of AD and neurodegeneration may thus be slowed down by consuming plants that have antioxidant capabilities [15].
Bioactive molecules were frequently used in clinical trials such as AChE inhibitors (AChEIs), particularly in AD therapy.Phenolic substances contain more AChEIs and are the first drugs for AD treatment [16].
Diabetes is categorized as either insulin affiliate (Type 1) or insulin-independent (Type 2), depending on the amount of biological efficiency of the excreted insulin and the amount of insulin secreted by the pancreatic beta cells.Type-2 diabetic mellitus (T2DM) has a significant defect in which resistant insulin cells are developed, which interferes with the insulin signaling system and affects muscle and fat tissue's ability to absorb glucose [17].An imbalance between the generation of ROS and the body's antioxidant capacity is the hallmark of OS.Due to the increased ROS generation by multiple metabolic pathways and the corresponding decline in antioxidant defense mechanisms, it might develop problems in DM [18].Inhibiting hydrolyase enzymes of carbohydrates like α-amylase and α-glucosidase is one of the currently available approaches to treat T2DM.It is possible to reduce glucose levels in postprandial plasma, and postprandial suppression of hyperglycemia is also possible as a result of delaying the absorption of glucose units [19].α-Glycosidase is synthesized from epithelial cells of the intestine and hydrolyzes oligo-and poly-carbohydrates into glucose and fructose components of monosaccharide.In order to control T2DM and hyperglycemia for good health, the α-glycosidase inhibitors (α-GIs) are very important [15].
Carbonic anhydrases (CAs), which have Zn 2+ and are metalloenzymes, are a family of enzymes that can reversibly hydrate carbon dioxide (CO 2 ) to produce bicarbonate ( − HCO 3 ) and protons (H + ) [20].Numerous metabolic pro- cesses, including ureagenesis, lipogenesis, and gluconeogenesis, are carried out by these isoenzymes.In addition, there are areas of use of CA inhibitors in curing a number of chronic illnesses, such as cancer, glaucoma, and infections [21].
This research aimed to examine the polyphenolic contents and the antibacterial and antioxidant properties of Tayfi grape seeds, skin extract, and ethanol extract to evaluate the relation among compounds' ingredients, and antioxidant activities, and to determine the potential antioxidative potency of Tayfi grape seeds and juice used in the conventional treatment of many diseases.The antibacterial properties of the extracts were evaluated toward S. aureus and E. coli.
Inhibition studies are performed on the AChE enzyme to show the anti-Alzheimer efficacy of Tayfi grape extracts.The CA II enzyme inhibition was studied to determine its relation to glaucoma.The antidiabetic effect of fruit extracts against α-amylase and α-glucosidase is investigated in similar studies, and the IC 50 values for the extracts are calculated.
Additionally, FRAP, ferric ions (Fe 3+ ), copper ions (Cu 2+ ) reduction and DPPH˙, and ABTS˙+ scavenger assays are carried out, and the total polyphenolic quantities of fruit extracts are determined to determine the antioxidant capabilities of Tayfi grapes (Vitis vinifera).Our aim in this study is to increase the scientific awareness of the Tayfi grape, which grows as a common ecotype in the region.Additionally, to identify the chemical components that underlie the fruit's biological activity, a phenolic analysis is conducted using the LC-MS/MS assay.The other important aim of this research is to show how the Tayfi grape ethanol and water extracts inhibit the AChE, CA II, α-amylase, and α-glycosidase enzymes, which are linked with widespread health problems.It is proposed that the information obtained from the current study will support the need for additional research aimed at creating novel diets and food supplements.

Plant material
Dr. Mehmet Fidan (Biology Department, Siirt University) defined the Tayfi grape (Vitis vinifera L.) as the Siirt ecotype.The plants were obtained from Pirinçli village (Şirvan district, Siirt province) in October at altitudes of 2,080 ft (634 m).Tayfi grape seeds and skin parts were dried in the shade and stored at 25 o C.They were protected from the sunlight and light.

Ethanol and water extract preparation
As previously mentioned, the extraction procedure followed that given in the study of Gulcin et al. [8].Water extracts of the samples were prepared with 300 mL of distilled and deionized water, and 30 g of dried Tayfi grape seeds and skins were ground in a mill.On a magnetic stirrer, this mixture was boiled for 20 min.At a pressure of 5 mmHg and a room temperature of −50°C, the extracts of the filtration were frozen and lyophilized (Labconco Lyophilizator, Freezone).About 30 g of dried Tayfi grape seeds and skins were crushed before being mixed with 300 mL of ethanol and stirred for an hour to prepare the ethyl alcohol extracts of the specimens.The filtrate solution of the extracts was obtained following filtration.The ethanol was eliminated using an evaporator (RE 100 Bibby rotary, Stone Staffordshire, England) set to a temperature of 50°C.All of the extracts were maintained in an opaque cup at −20 °C previously being utilized in experiments [8].

Total phenolic contents
The phenolic content in the Tayfi grape skins and seed ethanol and water extracts were analyzed using Singleton and Rossi's method [22] by adopting minor modifications [8].About 500 µL of each extract sample was transferred to the Folin-Ciocalteu reagent (1,000 µL).Following an elaborate blending, the carbonate solution (500 µL, 1%) was transferred to the mixture to neutralize it.After incubation in the dark for 2 h at 25 o C, the absorbance was measured at 760 nm in contrast to a blank sample made up of distilled water.The gallic acid curve was constructed, and the phenol contents were calculated utilizing the linear regression equations of this slope.The phenolic content is given as milligrams of GAE (gallic acid equivalents) per gram of Tayfi grape extracts.

Measurement of total flavonoid content
The most prevalent class of polyphenolic compounds in the typical diets of humans are flavonoids, which are also widely distributed in plants.The colorimetric test of Gülçin et al. [23] was employed to calculate the total amount of flavonoid in the Tayfi grape ethanol and water extracts.Then, 1,500 µL of methanol and 0.5 mL of the ethanol extract of Tayfi grape (EE) or the water extract of Tayfi grape (WE) samples were combined.The samples were then vortexed and 1,500 µL of Al (NO 3 ) 3 (10%), 500 µL of CH 3 COOK (1.0 M), and 2,300 µL of distilled water were added.The vortexed samples are then incubated in the dark for 40 min at room temperature.Then, the absorbance readings were measured at 415 nm.Distilled water was utilized as a blank and control.The quercetin calibration curve was constructed, and the expression for the calibration curve's linear regression was used to estimate the flavonoid content.Quercetin equivalents (QE) are given as μg per g of Tayfi grape extracts.
2.6 LC-MS/MS analysis 2.6.1 Preparation of the samples About 100 mg of each Tayfi grape water and ethanol extract were mixed in 5 mL of ethanol/water solution (50:50 v/v) in a volumetric flask, and 1,000 µL of this mixture was added into a different volumetric container with a total volume of 5 mL.Thereafter, 100 μL of Vitis vinifera skin, seed water, and ethanol extract were added and the volume was adjusted with (50:50 v/v) ethanol/water.A 1,500 µL aliquot from the last mixture was transferred to a covered flask, and 10 μL of the specimen was inserted into the LC-MS/MS.Throughout the assay, specimens in the autosampler were maintained at 15°C [24].

Preparation of the LC-MS/MS test solution and chromatography conditions
Phytochemical quantification of water and ethanol extracts of the Tayfi grape was carried out by a previously developed, validated, and comprehensive LC-MS/MS method [25].
The LC-MS/MS method contained 53 fingerprint phytochemicals.The crude ethanol and water extracts of Tayfi grape were dissolved in appropriate solvents to get a final concentration of 1 g/L prior to the LC-MS/MS analysis.Table 1 lists the names, molecular ions, daughter ions, and uncertainty coefficients of the phytochemicals that belong to the utilized LC-MS/MS analytical method and the quantity of the phenolics in Tayfi grape seed extracts (μg/g extract).Additionally, the extracts were prepared in triplicates and their mean values were calculated.Results were given with their uncertainty values.

UHPLC and tandem mass spectrometry conditions
Quantitative assessment of bioactive phenolic compounds was conducted using a triple quadrupole mass spectrometer (LCMS-8040 model) coupled to an ultra-high performance liquid chromatograph (Shimadzu-Nexera model).The reverse-phase UHPLC consisted of a column oven (model: CTO-10ASvp), a degasser (model: DGU-20A3R), binary pumps (model: LC-30AD), and an autosampler (model: SIL-30AC).A reverse-phase Agilent Poroshell 120 EC-C18 model analytical column (150 mm × 2.1 mm, 2.7 µm) was utilized to perform the chromatographic separation.The temperature of the column was adjusted to 40°C.The elution gradient consisted of mobile phase A (water, 0.1% formic acid, 5 mM NH 4 HCOO) and mobile phase B (methyl alcohol, 0.1% HCOOH, 5 mM NH 4 HCOO).The gradient was as follows: 0-25 min linear gradient from 20 to 100% B, held at 25-35 min at 100% B, and held at 35-45 min at 20% B to re-equilibrate the column for the next injection.Moreover, the injection volume and the flow rate of the solvent were adjusted to 5 µL and 0.5 mL/min, respectively.A Shimadzu LCMS-8040 endowed with an ESI source running in both negative and positive ionization modes was used for mass spectrometric detection.LabSolutions software was used to capture and process the LC-ESI-MS/MS data (Shimadzu).The phytochemicals were measured using multiple reaction monitoring (MRM) modes.Based on evaluating specific precursor phytochemical-to-fragment ion transitions, the MRM approach was designed to identify and quantify phytochemicals with high specificity.For obtaining the best phytochemical disintegration and the most significant amount of the intended product ions, the collision energies were tuned.
The MS operating parameters were 15 L/min of drying gas (N 2 ) flow, 3 L/min of nebulizing gas (N 2 ) flow, 250°C for the DL temperature, 400°C for the heat block temperature, and 350°C for the interface temperature.

Reducing ability assays 2.7.1 Fe 3+ -reducing ability assays
The capability of the Tayfi grape extract in reducing Fe 3+ was calculated in accordance with Oyaizu [26].In a nutshell, various concentrations of EE and WE (10-30 μg/mL) were poured into identical phosphate solution (1,250 µL, pH 6.6; 200 mM) and a K 3 Fe(CN) 6 mixture (1%).After 30 min of incubation at 50°C, the mixture was acidified with trichloroacetic acid (TCA, 10%, 1,250 µL).Finally, 0.5 mL of 0.1% FeCl 3 mixture was poured before measuring the absorbances of the EE and WE at 700 nm.The phosphate buffer solution was used as a blank sample.

Cu 2+ -reducing ability assays
The Cu 2+ reducing effects of Tayfi grape extracts are determined according to Apak et al. [27] with slight modification [28].To achieve this, identical volumes of 250 µL of the grape extracts (10-30 µg/mL) in a glass cup were added to the same concentrations of the neocuproine (7.5 mM), CuCl 2 solution (10 mM), and acetate buffer (1.0 M, 250 µL).The combination volumes were calibrated to a total of 2 mL.The glass tubes were then sealed and kept at 25°C until they used in experiments.Finally, spectrophotometric measurements of their absorbances at 450 nm were taken after 30 min.Acetate buffer solution was employed as a control sample.A greater absorbance of the reaction demonstrates a greater capability for Cu 2+ conversion [29].

Radical scavenging activities 2.8.1 DPPH˙scavenging activity
The DPPH˙free radical scavenging capability of the Tayfi grape extracts was calculated according to the method of Blois [31], originally used by Gülçin [32], with slight modification.When the specimen was present, the whitening of a steady free radical, DPPH, was observed at a certain wavelength.The DPPH mixture was prepared a day prior to the measurement.The mixture in the beaker was covered with aluminum foil and placed in darkness at 4°C for 16 h, with continuous swirling.Tayfi grape extracts were dissolved in ethanol at various concentrations (10-30 g/mL), and 500 µL of a 0.1 mM DPPH solution was quickly poured into 2 mL of this mixture.After vortexing, the Tayfi grape extracts were incubated at 30°C in the dark for 30 min.In comparison to blank samples, absorbance was recorded at 517 nm [4].The decrease in absorbance shows that DPPH is actively displayed to radical scavengers.

ABTS˙+ scavenging activity
ABTS, a rather steady free radical, discolors when it is not a radical.The procedure used by Re et al. [33] was utilized to assess the spectrophotometric analysis of ABTS˙+ scavenging activity.An antioxidant was added to a previously prepared ABTS radical mixture using this method, and the residual ABTS˙+ was then detected spectrophotometrically at 734 nm after a predetermined length of time [34].In order to prepare ABTS˙+, potassium persulfate (K 2 S 2 O 8 , 2.45 mM) was mixed with ABTS (2 mM) in water.ABTS˙+ was subsequently rested for 6 h at room temperature in the dark.However, it took more than 6 h for the absorbance to peak and was steady after ABTS began to oxidize.The radical cation was stable in this state for 2 days when kept at 25 o C and in complete darkness.The pH of the mixture was adjusted to 7.4 by phosphate buffer.The absorbances were measured at 734 nm.Finally, 3 mL of Tayfi grape solutions in ethanol at 10-30 g/mL were mixed with 1 mL of the ABTS˙+ mixture.The radical scavenging percentage was determined for all concentrations in contrast to a blank which included no scavenger.The absorbance of the mixture was measured after stirring for 30 min.The amount of discoloration was assessed using the percentage decrease in absorbance.

Enzyme inhibition studies 2.9.1 Acetylcholinesterase inhibition study
Tayfi grape extract activities on cholinergic enzymes were carried out using Ellman's methodology [35].This was achieved by employing the AChE serum of electric eels.In a nutshell, Tayfi grape extracts at 10-30 g/mL in a buffer (1.0 M Tris/HCl, 200 μL, pH 8.0) are added to the AChE mixture (100 μL, 5.32 × 10 −3 EU).The solution was incubated at 20°C for 10 min.Following that, 100 μL of DTNB (500 μM) and AChI were added.The reactions were then initiated, and the absorbances of the combination were determined at 412 nm spectrophotometrically [36].

α-Glycosidase inhibition study
The inhibition results of the Tayfi grape extracts on the αglycosidase enzyme were investigated in accordance with Tao et al. [37].Various amounts of both extracts were mixed in the phosphate (150 μL, pH 7.4) buffer.Following that, 40 μL of the α-glycosidase mixture is added to the identical buffer and incubated for 20 min.The final mixture is mixed with an aliquot (100 μL) of p-nitro-phenyl-D-glycopyranoside (p-NPG) dissolved in the buffer.The solution was then incubated at 25°C, and the absorbances were estimated at 405 nm in contrast to a blank, which was composed of phosphate solution.

α-Amylase inhibition study
The inhibitory results of Tayfi grape extracts on α-amylase were performed according to the previous study [38].In a nutshell, 1.5 g of starch was first disintegrated in 60 mL of 0.4 M alkaline medium; afterward, it was heated for 30 min at 80°C.After cooling, the pH of the combination was adjusted to 6.9, and deionized water was used to make up the total volume to 200 mL.Next, the identical volume (70 μL) of starch (pH 6.9) was combined with various quantities of the EE and WE.The resulting mixture was then transferred to a 40 μL container containing α-amylase solution, which was then incubated at 35°C for 30 min.As a last step, 100 μL of HCl (100 mM) was added to terminate the reaction.The samples' absorbance was then measured at 580 nm in contrast to a blank specimen composed of a phosphate buffer mixture.Then, an identical volume (70 μL) of starch and phosphate buffer (pH 6.9) were mixed with various concentrations of the sample extracts.The final mixture was then added to 20 μL of the α-amylase solution, which was left at 35°C for 1 h.The reaction was finally terminated by adding 100 μL of HCl (100 mM), and the absorbance of the specimens was estimated at 580 nm in contrast to a blank specimen that included the phosphate buffer mixture.

CA II inhibition study
Human specimens of blood were utilized to isolate and purify the CA II isozymes using the Sepharose-4B-L-tyrosine sulfanilamide affinity method, as previously reported [39].Following the CA II purification of the enzymes, the quantity of protein was determined at 595 nm using a former assay [40].The esterase activity assay was performed at 348 nm utilizing a spectrophotometer (Shimadzu, UVmini-1240 UV-VIS) [41].Acetazolamide (AZA) was employed as a standard as described previously.

Microorganisms used in the study
This investigation used microorganisms that could be harmful to people.

Statistical analysis
Each experiment was repeated three times.The measurements are shown as mean ± SD.Tukey post hoc test was used after the one-way ANOVA; variations were assessed and significant when p < 0.05.

Results and discussion
Phenolic compounds and flavonoids, which have been separated from a variety of plants, have antimicrobial, antioxidant, and other beneficial properties as well as the ability to block light.Since flavonoids can both prevent the free radical's production and the removal of free radicals, their antioxidant activity has drawn a lot of attention [43].Most of the phenolic compounds in grape seed extracts, including catechin, epicatechin, flavonols, and others, have pharmacological effects that are anti-inflammatory, anticancer, and antioxidant [44].The phenolic and flavonoid contents in the Tayfi grape seed ethanol extracts were found to be 82.8 mg GAE/g and 91 mg QE/g.However, phenolic and flavonoid contents in Tayfi grape skin ethanol extracts were found to be 2.3 mg GAE/g and 13.0 mg QE/g.The use of grape extracts as nutritional antioxidant supplements has attracted increasing attention in recent years.Grapes contain a high number of polyphenols, of which 8% or less are found in the pulp, 46-69% in the seed, and 12-50% in the skin [45].The total phenolic and flavonoid contents in methanol extractions of Vitis vinifera fruit cultivated in Algeria were found to be 23.06 ± 0.17 and 14.37 ± 0.04 mg GAE/g [46].The phenolic content of Quebranta (Vitis vinifera) grape seeds varied from 27.9 ± 2.3 to 167.6 ± 10.4 mg GAE/g dw, as shown by impacts of extracting subcritical water and cultivar region.The antioxidant capacity values by DPPH ranged from 174.7 ± 26.2 to 1628.2 ± 80.3 µmol TE/g dw [47].The fact that grape seeds contain a lot of galloylated flavanols, and, in an aqueous medium, galloylated flavanols have a higher antioxidant effect than their nongalloylated homologs, as well as the activity of the grape seeds was higher than for the skins, could both be attributed to the greater phenolic compound ingredients of seeds [48].Herein, it was found that Tayfi grape seed extracts have higher phenolic and flavonoid contents than grape skin, which would also affect the antioxidant capabilities of these extracts directly.Tayfi grape extracts have shown, in this study, to have a comparable effective number of polyphenolics.
The LC-MS/MS technique was employed to determine the main ingredients in Tayfi grape extracts, using 53 phenolic compounds as standards (Figure 1 and Table 1).Comparing the chromatographic characteristics, UV spectra, and MS data of the phenolic compounds with those of standard molecules allowed for the elucidation of these metabolites, and 22 molecules were investigated (Table 1).In agreement with the results of the LC-MS/MS testing, the table displays the mean levels for each compound.The main compounds found in the ethanol extract of Tayfi grape seeds were epicatechin (4711.946μg/g), catechin (1518.961μg/g), gallic acid (76.101 μg/g), quinic acid (16.674 μg/g), epicatechin gallate (6.26 μg/g), cyranoside (5.179 μg/g), fumaric acid (2.089 μg/g), pieceid (1.56 μg/g), vanillin (1.488 μg/g), protocatechuic acid (1.322 μg/g), isoquercitrin (1.305 μg/g), astragalin (1.172 μg/g), and tannic acid (1.102 μg/g).The other detected compounds in lower amounts were protocatechuic aldehyde, quercitrin, cosmosiin, p-coumaric acid, coumarin, miquelianin, naringenin, coumarin, and aconitic acid.Only epicatechin was found in higher amounts (39.528 μg/g) in the water extract of Tayfi grape seed extract.However, the other standards of the LC-MS/MS method were not recorded in ethanol and water extracts of Tayfi grape extracts.Natural antioxidants have been used in extracts made from grape seeds and pomace.High concentrations of monomers of phenolic substances are found, such as (−)-epicatechin, (+)-catechins, and (−)-epicatechin-3-O-gallate, along with procyanidins [49].A mixture design experiment will combine them in the most effective way possible to create a new, secure, multi-target antidiabetic formulation, which will be effective in managing both diabetes and its adverse effects.Epicatechin, catechin, and rutin all possess strong hypoglycemic effects; their combined effect promotes an original notion that has the potential to compete successfully with existing medications [50].Epigallocatechin gallate is a flavanol that is most common and is around 59% of the total catechins.Epigallocatechin gallate has positive benefits, one of which is a reduction in the risk of T2DM due to its influence on metabolism [51].The pharmacological effects of astragalin include antidiabetic, neuroprotective, cardioprotective, anti-inflammatory, antioxidant, and anticancer properties [52].Examples of polyphenols with antioxidant characteristics that guard against DNA oxidative destruction and suppress LDL oxidation in vitro include resveratrol, catechin, quercetin, and gallic acid [53].By causing apoptosis-mediated cellular toxicity in breast cancer cells, the antioxidant quinic acid has shown anticancer potential [54].Tannic acid has homeostatic and antioxidant properties.Additionally, tannic acid has the power to fight against ROS, which are assumed to be the underlying cause of many ailments, including diabetes, cardiovascular, and neurodegenerative diseases.Anticancer properties of tannic acid have also been previously demonstrated.Since it reveals bioactive characteristics and improves the qualities of components for applications in biomedical, it is nowadays considered an organic polymer compound [55].
The ability of a chemical to reduce may be a key indicator of the compound's possible antioxidant action.Antioxidant chemicals can transform reactive radicals into stable and inert forms [56].The Tayfi grape's effectiveness as an antioxidant may be attributed to its variety, abundance of components, and high phenolic ingredient.Three distinct reduction systems, involving the reducing capabilities of Fe 3+ , CUPRAC, and Fe 3+ -TPTZ ions, were used to determine the reducing abilities of the phenolic compounds extracted from the Tayfi grape.By using the DPPH˙and ABTS˙+ scavenging analyses, the antiradical abilities of the Tayfi grape were investigated.
The Tayfi grape natural compounds may possess reducing qualities that neutralize oxidants and ROS.The direct transformation of Fe 3+ (CN − ) 6 to Fe 2+ (CN − ) 6 and the absorbance caused by the formation of Perl's Prussian Blue behind the insertion of too many additional ferric ions (Fe 3+ ) were utilized for evaluating the Tayfi grape extracts' capacity to reduce Fe 3+ .The ferric-reducing technique of Oyaizu [26] was employed to evaluate the reducing capability of Tayfi grape extracts.
The Cuprac antioxidant technique is particularly efficient in determining the reducing capabilities of bioactive compounds.First, the FRAP test utilizes the sample's antioxidants as reducing agents in a redox-linked colorimetric process.Second, the FRAP test process is very simple and easy to verify [30].The FRAP analysis was designed to evaluate how well biological fluids and pure compound aqueous solutions reduce ferric ions.Additionally, the capability of polyphenols acting as antioxidants has been evaluated [58].Depending on how powerful the reducing ability of an antioxidant samples is, in this assay, the test mixture's initial yellow hue changes to a range of blue and green hues.The prospective antioxidant impact of a substance may be well predicted by the compound's reducing capability.The reducing potential of Tayfi grape seeds, skin extracts, and standards were calculated using the Fe 3+ -TPTZ reducing test.In accordance with the results, the reduction capabilities of the analyses diminished in the order as follows (Table 2 and Figure 2c): α-tocopherol (2.434 ± 0.103, r 2 : 0.8714) > grape seed ethanol extract (2.195 ± 0.010, r 2 : 0.9796) > BHA (2.151 ± 0.020, r 2 : 0.9367) > Trolox (2.108 ± 0.026, r 2 : 0.9291) > BHT (2.031 ± 0.190, r 2 : 0.9670) > grape seed water extract (1.955 ± 0.041, r 2 : 0.9916) > grape juice (1.824 ± 0.123, r 2 : 0.9993) > grape skin water (0.631 ± 0.016, r 2 : 0.9720) > grape skin ethanol extract (0.537 ± 0.010, r 2 : 0.9304).These results were found with the 30 µg Tayfi grape extracts.According to this method, test samples' capacity to reduce is directly correlated with how high the absorbance measurements are.
The antioxidant properties of compounds, drinks, foods, and plant preparations are frequently assessed using spectrophotometric techniques based on radical scavenging.The peak of absorption for a freshly made DPPH solution is at 517 nm, and it has a deep purple hue.When there is an antioxidant in the solution, the violet hue usually disappears.Because of the antioxidant action, the free DPPH shows a decrease in absorbance [59].If Tayfi grape extracts have the capability to scavenge DPPH˙, they are regarded to have natural antioxidant capacity.Tayfi grape extracts were tested for their ability to neutralize DPPH˙, and the IC 50 value was calculated (Table 3, Figure 3a).As the concentration of the extracts of Tayfi grapes in the medium changed, their radical scavenging activities changed.The Tayfi grape seed ethanol and water extracts revealed comparable and stronger antiradical activities (IC 50 s: 21.0 and 21.6 μg/mL) like BHA (IC 50 : 17.3 μg/mL) and BHT (IC 50 : 53.3 μg/mL); also near and likely to other reference compounds like α-tocopherol (IC 50 : 15.7 μg/mL) and Trolox (IC 50 : 12.6 μg/mL).As seen in this study, the Tayfi grape seed EE and WE have a comparable better antioxidant potential when compared with standard antioxidants and the results of different studies given.In another study, IC 50 of Vitis vinifera, fruits macerated with methanol, DPPH, and ABTS scavenging activity were found to be 0.270 and 0.040 mg/mL, respectively [46].Each sample was measured in triplicate.
ABTS radicals were obtained in an ABTS/K 2 S 2 O 8 system.The test uses a discoloration method in which the radical is produced straight in a steady state before being exposed to potential antioxidants.In the developed assay for ABTS˙+ described here, potassium persulfate reacts with ABTS to directly yield green/blue chromogenic ABTS˙+ [8].One of the spectrophotometric techniques used to measure the total antioxidant activity of components, aqueous medium, and soft drinks is via the production of the ABTS radical cation [60].
The IC 50 values are 6.7 μg/mL for the Tayfi grape seed ethanol extract, 9.5 μg/mL for BHT, 8.8 μg/mL for BHA,    8.6 μg/mL for trolox, 8.9 μg/mL for α-tocopherol, 9.1 μg/mL for grape seed water extract (Table 3 and Figure 3b).The findings unambiguously demonstrate that Tayfi grape has approximated effective ABTS˙+ scavenging activity as compared to positive controls.The samples of Tayfi grape extracts demonstrated a greater level of radical scavenging activity than reference standard antioxidants.More ABTS˙+ scavenging activity is suggested by a lower IC 50 value.α-Glycosidase inhibitors may be a key component of the curative strategy for T2DM.Controlling blood glucose level elevation may decelerate the development of secondary issues-linked diabetic mellitus ailments, which are associated with postprandial hyperglycemia, a remarkable and prior complication in diabetes [61].For enzyme glycosidase, the Tayfi grape seed ethanol extract has IC 50 values of 5.8 μg/mL, aqueous extract has IC 50 of 5.3 μg/mL (Table 4).Based on the outcomes, the inhibitory effects of the Tayfi grape ethanol extracts are comparable to acarbose, a typical α-glycosidase inhibitor.
Choosing Vitis vinifera L. cultivars and interbreeding hybrids were studied in vitro, and it was shown that the inhibitory action against α-glucosidase was greater when compared to α-amylase in all seeds, the majority of skins, and the majority of flesh samples [62].The inhibitory impacts of grape parts on two antidiabetic enzymes, α-glycosidase and α-amylase, obtained in this study followed a similar course.The α-amylase enzyme's inhibition aids in preventing and managing postprandial hyperglycemia since α-amylase is crucial for the ingestion of polysaccharides.The inhibition effects of herb extracts on α-amylase has been the subject of several investigations in the last few decades, and this inhibition has been reported in the study of Bashkin et al. [63].The ethanol extract of Tayfi grape extracts was adjusted to α-amylase inhibitory activity, and results are given in Table 4.For α-amylase, the IC 50 values of the Tayfi grape seed water and ethanol extracts were 385.2 and 567.6 μg/mL, respectively.The IC 50 values of the Tayfi grape skin water and ethanol extract were 116.5 and 268.5 μg/mL, respectively, higher than seed inhibition values.Another study found that the inhibitory activity toward α-amylase (IC 50 , mg of dried sample/mL) varied from 0.27 (Pinot gris skins) to 1.13 (Hibernal seeds) [62].
For therapy of AD, AChEIs are employed; nevertheless, they only offer transient relief.Historically, cholinesterase inhibitors have been abundant in medicinal plants.Medical herbs' inhibition of cholinergic enzymes is mostly caused by phenolic compounds [64].The Tayfi grape seed ethanol and water extract inhibition levels are lower, but near to the reference value when compared to tacrine, a prevalent AChE's standard inhibitor.Table 4 outlines the IC 50 values of various parts of Tayfi grape extracts for enzyme inhibition.The IC 50 value of the Tayfi grape seed ethanol extract against AChE enzyme inhibition was 13.8 μg/mL compared to Tacrine (Table 4).The IC 50 was 5.97 µg/mL for tacrine, and it served as the control in the experiment to determine how the AChE enzyme would be inhibited.
Based on the research, the biologically active fractions of grapes, grape seeds, skins, and flesh have the ability to inhibit acetylcholinesterase in vitro (AChE).Among them, the grape skin var Rondo exhibits the highest 55.33% AChE enzyme inhibitory activity [62].In rats treated with Al for Alzheimer's disease, Vitis vinifera treatment significantly improved cognitive function.Because it contains a lot of phytoconstituents, V. vinifera reduced the mRNA expression of amyloid-β precursor protein in rats and eliminated tau tangles.The histological studies provided additional proof of this [65].In this study, as shown in Table 4, Tayfi grape extracts effectively inhibited the AChE enzyme.
The reduced − HCO 3 production and aqueous humor synthesis as a result of CA II inhibition decrease intraocular hypertension [66].The multivariate visual illness, referred to as glaucoma, is defined by the deterioration of the optical nerves, which are primarily linked to elevated intraocular pressure (IOP) that leads to blindness.Innovative therapeutic approaches are necessary since hCA inhibitor drugs such as AZA, dorzolamide, and brinzolamide are successful in decreasing IOP following topical therapy [67].Furthermore, CA II has a history of being related to a variety of diseases, such as osteoporosis and glaucoma.The inhibitory effects of Tayfi grape extracts on the enzyme were examined and the results are shown in Table 4.The IC 50 values were found to be 12.6 and 18.6 μg/mL for the Tayfi grape seed and Tayfi grape skin ethanol extracts toward CA II enzyme, respectively, and efficient in comparison with its reference standard.The IC 50 was 8.37 μg/mL for AZA, which was used to determine the suppression of CA by isoenzymes [68].A contribution to the literature has been made about the inhibition of CA II enzyme by V. vinifera fruit via this study.According to the study's findings, Tayfi grape extracts can be useful in the treatment of glaucoma when appropriately used.A common Gram-positive bacterium that induces poisoning of foods is S. aureus.The reason for this is not the food itself, primarily humans who contaminate food after they have been prepared.E. coli, which is a normal component of human flora, is a representative of Gramnegative bacteria.But in order to stop the spread of an enterohemorrhagic variant of E. coli that has been connected to serious instances of food poisoning, chemical preservation agents are necessary [69].S. aureus and E. coli strains that are becoming resistant to many antibiotics are seen emerging in recent years.These have grown to be a significant issue for global public health, reviving the demand for new antimicrobial substances [70].Since it is very challenging to get rid of these microorganisms, we chose to test Tayfi grape's effectiveness against them because they are resistant to the majority of antimicrobial substances.The results are given in Table 5.
The potential antimicrobial properties of Tayfi grape extracts were investigated in this study using the microbial species E. coli and S. aureus.According to Table 5, two bacterial species are sensitive to the antimicrobial activity of extracts.Amc 30, amoxycillin/clavulanic acid disks, per disk-30 µg; Sxt 25, trimethoprim/sulfamethoxazole, per disk-25 µg; Cip 5, ciprofloxacin, per disk-5 µg; Cn 10 and gentamicin, per disk-10 µg were used as a positive control for bacteria.All four of the standard antibiotics failed to kill E. coli and S. aureus.In some, the zone diameter is 10 mm, etc.Although it failed to meet the required sensitivity sizes of CLSI criteria, it was considered resistant.Because no zone diameter (ND) was formed, the extracts contained resistant (R) ones.On the other hand, in certain extracts, zones with 7-10 mm gaps, or regions wherein the extract has an antibacterial action and the bacteria are eliminated, have been noticed.When contrasted with conventional antibiotics, the 7-10 mm discs created at 50 μg (concentration to be calibrated) rates to every extraction disc are excellent.It was seen in extracts that had no zones at all, or that were resistant to antibiotics.Table 1 demonstrates that ethanol extract had excessively greater phenolic and flavonoid concentrations than water extract.According to LC-MS/MS analysis, which was consistent with these findings, ethanolic extract had greater phenolic contents when compared to water extract.This difference in phenolic acid solubility across solvents may be the cause of the higher phenolic content of ethanol extract when compared to water extract.This also shows why the ethanol extract showed effective antioxidant, reducing power, and antibacterial activities.

Conclusion
Tayfi grape (Vitis vinifera), which is a main nutritious fruit that has been traditionally consumed by people for years Biological activity of Tayfi grape (Vitis vinifera)  13 in nature, attracts attention for its smell, color, and appearance, especially because it provides energy easily with its intense sugar content and is known to strengthen immunity with its vitamin content, as in daily consumption.It is an indispensable fruit for a healthy life, with many products: raisins, molasses, fruit pulp, vinegar, and wine.In the last few decades, the limitations to the use of artificial antioxidants have led to alternative research.In vitro, cell cultures, and animal experiments also show that when different extraction methods are applied, the polyphenolic compounds in their ingredients might be released and can be utilized in medicine.These extracted compounds can be used alone as well as with other compounds.When the seed and skin of the Tayfi grape are compared, we experimentally observed that the secondary metabolites in the seeds are rich, but the compounds found in the skin are also important in enzyme inhibition studies.We found that the Tayfi grape, which is an ecotype, has antioxidant, antidiabetic, anti-Alzheimer, antiglaucoma, and antimicrobial effects.According to the LC-MS/MS, the principal elements found in Tayfi grape ethanol extracts were quinic acid, fumaric acid, aconitic acid, gallic acid, protocatechuic acid, catechin, protocatechuic aldehyde, tannic acid, epicatechin, vanillin, epicatechin gallate, piceid, p-coumaric acid, coumarin, cyranoside, miquelianin, rutin, isoquercitrin, ellagic acid, cosmosiin, quercitrin, astragalin, and naringenin.Furthermore, the Tayfi grape ethanol and water extracts had high AChE, α-glycosidase, and CA II inhibitions in addition to antioxidant and antiradical activities, reducing power, and phenolic content.The use of Tayfi grape extracts for pharmacological purposes in individuals with the diseases we have mentioned can be sustained by clinical pharmacology studies.The effect of fruit, which is the most important source of energy and nutrients in non-pharmacological use, is also an important subject of clinical pharmacology.

Table 1 :
Numbers, names, molecular ions, daughter ions, and uncertainty coefficients of the phytochemicals that belong to the LC-MS/MS analytical method used and quantification results of the compounds in Tayfi grape seed extracts (μg/g extract) [8]ibacterial activities of Tayfi grape extracts were determined by the disk-diffusion technique.The agar culture of test microorganisms was made according to Gülçin et al.[8].Several bacterial strains were cultivated on Oxoid blood agar (CM55, Basingstoke, Hampshire, UK) media.Pathogens for the study were inserted into the Oxoid Tryptone soy broth (CM129, Basingstoke, Hampshire, UK) for evaluation.Tryptone soy broth is a very nutritive multipurpose medium that is recommended for everyday laboratory usage and is employed for culturing facultative anaerobes and aerobes, involving particular types of fungi.At 37°C, the prepared cultures were cultured for 24 h.For the antimicro- [42]ureus-ATCC 25923 and E. coli-clinical strain were employed for the evaluation of the antibacterial function.Stock cultures (standard strains and clinical isolates) of the Medical Microbiology Department (Faculty of Medicine, bial test, sterile, 6 mm-diameter filter paper discs were filled with 50 μL of Tayfi grape extracts, and the diffusion technique was used to assess the susceptibility on the Mueller Hinton agar (Oxoid CM337, Basingstoke, Hampshire, UK) medium.The growth inhibition zones around the discs consisting of antibiotics and plant extracts were measured and recorded.Clear zones of inhibition encircling the discs indicated the presence of antibacterial action[7].Standard antimicrobial discs were compared to grape extracts: clavulanic acid-amoxycillin (10/20 μg/disc), gentamicin (10 μg/disc), sulbactam-ampicillin (10/10 μg/disc), and ciprofloxacin (5 μg/ disc, BD BBL™ Sensi-Disc™).The Clinical and Laboratory Standard (CLSI 2019) references were utilized to evaluate the outcomes of antimicrobial tests[42].