Phenolic compounds and biological activities of grape (Vitis vinifera L.) seeds at different ripening stages: insights from Algerian varietiesABSTRACT
The surge in interest in bioactive plant compounds, particularly from grapes (Vitis vinifera L.), has led to this study on phenolic compounds at different ripening stages. Grapes were collected before veraison, at veraison, and during ripening. Phenolic compounds were extracted using 50% acetone and quantified through spectrophotometric assays for total phenolic, flavonoid, flavanol, condensed tannin, and hydrolysable tannin contents. LCMS/MS identified phenolics. Antioxidant activities were assessed via (DPPH), (ABTS), phenanthroline, reducing power, and silver nanoparticle assays. Additionally, anti-tyrosinase activity and photoprotective effects were evaluated. The results indicated that before veraison (BV) stages yielded the highest concentrations of phenolic compounds. The most abundant compounds at the berry veraison stage were catechin (72645.40, Sabel), gallic acid (1130.75, Red Glob), and luteolin (164.44, Cardinal). Antioxidant activities were consistent with phenolic content trends, with significant tyrosinase enzyme suppression and high SPF factor observed. Principal Component Analysis highlighted strong correlations between phenolic profiles and antioxidant activities. This study is the first to examine how grape ripeness affects phenolic content and biological activities in Algerian grape seeds. It identifies optimal harvest times for maximizing bioactive compounds and supports the use of grape seed extract in sunscreens and antioxidants.
Keywords:
repining stages; antioxidant; LCMS/MS; anti-tyrosinase; SPF factor
RESUMO
O aumento do interesse em compostos bioativos de plantas, especialmente de uvas (Vitis vinifera L.), levou a este estudo sobre compostos fenólicos em diferentes estágios de amadurecimento. As uvas foram coletadas antes do pintor, no pintor e durante o amadurecimento. Os compostos fenólicos foram extraídos com acetona a 50% e quantificados por meio de ensaios espectrofotométricos para os teores de fenólicos totais, flavonoides, flavonóis, taninos condensados e taninos hidrolisáveis. Os fenólicos foram identificados por LCMS/MS. As atividades antioxidantes foram avaliadas por meio de ensaios (DPPH), (ABTS), fenantrolina, poder redutor e nanopartículas de prata. Além disso, foram avaliados a atividade antitirosinase e os efeitos fotoprotetores. Os resultados indicaram que os estágios antes do veraison (BV) produziram as maiores concentrações de compostos fenólicos. Os compostos mais abundantes no estágio de veraison da baga foram a catequina (72645,40, Sabel), o ácido gálico (1130,75, Red Glob) e a luteolina (164,44, Cardinal). As atividades antioxidantes foram consistentes com as tendências de conteúdo fenólico, com supressão significativa da enzima tirosinase e alto fator SPF observado. A análise de componentes principais destacou fortes correlações entre os perfis fenólicos e as atividades antioxidantes. Este estudo é o primeiro a examinar como a maturação da uva afeta o conteúdo fenólico e as atividades biológicas nas sementes de uva da Argélia. Ele identifica as épocas ideais de colheita para maximizar os compostos bioativos e apoia o uso de extrato de semente de uva em protetores solares e antioxidantes.
Palavras-chave:
estágios de repintura; antioxidante; LCMS/MS; antitirosinase; fator SPF
INTRODUCTION
In recent years, there has been a notable increase in interest in exploring bioactive compounds obtained from plants. These agrochemicals and compounds have the potential to be used in various industries, such as food additives, pharmaceuticals, flavors, colors, and fragrances. Additionally, studies have revealed their protective effects on human health, safeguarding against a variety of degenerative diseases. These compounds are mainly classified into three groups: polyphenols, terpenoids, and alkaloids (Ben Khadher et al., 2022). Naturally occurring polyphenols are secondary metabolites mostly present in fruits, vegetables, cereals, and drinks. It was noticed that fruits like grapes, apples, pears, cherries, and berries contain up to 200-300mg polyphenols per 100 grams of fresh weight and that these chemicals are mostly involved in defense against pathogen aggression or UV radiation (Pandey and Rizvi, 2009). Vitis vinifera L., belonging to the Vitaceae family, is a widely abundant fruit crop globally, celebrated for its high polyphenol content (Kodeš et al., 2021).
During agricultural practices this plant generates a substantial amount of waste, including skins, seeds, pulp, canes, stems, and grape leaves (Leal et al., 2020, Noviello et al., 2022). The utilization of agro-industrial wastes as natural sources for the manufacture of valuable chemicals has received significant attention in recent times. Grape seeds are a significant component of the entire fruit, accounting for around 60-70% of the total soluble phenolic contents. The principal phenolic chemicals found in grape seeds are proanthocyanidins and flavan-3-ols. The concentration of these phenols can be influenced by a range of factors, including grape varietals, seasonal fluctuations, and environmental circumstances (Pantelić et al., 2016).
Furthermore, due to their phenolic composition, which exhibited high antioxidant proprieties and other biological activities, grape seed has gained increased attention in recent decades, especially as a dietary supplements(Padilla-González et al., 2022, Ma and Zhang, 2017). The group of catechins ((+)-catechin, (-)-epicatechin and(-)-epigallocatechin-3-gallate), proanthocyanidins, flavanols such as quercetin and quercetin-3-glucoside, gallic acid, and resveratrol from the stilbenoid class presented in grape seed are considered potent antioxidant molecules (Chengolova et al., 2023). Additionally, the use of plant-based enzyme inhibitors is becoming popular in the pharmaceutical sector as essential part of the current prescription drug to treat numerous human disease (Dwibedi et al., 2022).
As far as we know, there has been no previous research conducted to examine how the maturity stage affects the phenolic composition and biological activities of Algerian grape seed extracts. The objective of this work was to investigate the alterations in the phenolic composition of V. Vinifera seed extracts during ripening, as well as the changes in their antioxidant, enzymatic, and photoprotective capabilities.
MATERIAL AND METHODS
Grapes were collected randomly from the region of Baghlia, Boumerdes, during the summer months (July to August) of 2022. They were handpicked at three distinct stages of maturation: before veraison (BV), at veraison (V), and repining (R) according to the maturation dates of each variety (Sabel, Red Glob, and Cardinal) (Fig. 1).
The maceration technique was used to extract the phenolic compound from seeds. After collection, grapes were washed using distilled water to eliminate any debris, then seeds were manually separated from the whole berry, air-dried at room temperature (25°C) for 48 hours, and ground using a coffee grinder to achieve a fine powder. A total of 50 grams of seeds were used. Subsequently, the specimens were stored under controlled conditions at a temperature of 4°C until the extraction process. The pulverized seeds were soaked in a 50% acetone solution (1:10 w/v) for 24 hours at room temperature. Subsequently, the mixture underwent filtration using Whatman No. 1 filter paper, and the acetone was then evaporated under reduced pressure utilizing a rotary evaporator at a temperature of 40°C. The desiccated extracts were stored at a temperature of -20°C until they were utilized in various spectrometric and chromatographic analyses.
The Folin-Ciocalteu reagent was utilized in a colorimetric test, in accordance with Muller et al. (2010)'s methodology, to measure the grape canes total phenolic content. The calibration curve of Gallic acid was utilized to quantify the total phenolic content, expressed as milligrams of gallic acid per gram of dry extract (mg GAE/g).
To determine the total flavonoid content a different colorimetric technique was employed according to Topçu et al. (2007). The total flavonoid content (TFC) of the various extracts was calculated using a quercetin calibration curve was utilized. The results were expressed as milligrams of quercetin per gram of dry extract (mg QE/g).
The determination of the total flavanol was assessed according to the method described by Bouzana et al. (2023). The calibration curve of quercetin with an equation of y = 0.007X+0.022 (R² = 0.998) was used to express the results as milligrams of quercetin equivalents per gram of dry extract (QE/g).
The total condensed tannin content was evaluated using the method described by Saci et al. ( 2020). The calibration curve for catechin was used, and the results were expressed as milligrams of catechin equivalents per gram of extract (mg CE/g extract).The quantification of the hydrolysable tannin content was carried out according to Esma’s et al. ( 2023) method, and the results were expressed as mg tannic acid equivalents (TAE)/g dry weight, using the standard equation (y = 0.0007x + 0.051;R² = 0.9975).
The phenolic compounds were identified following a method similar to that of Erenler et al. (2023). The data was processed using Mass Hunter software by comparing the retention time of the discovered substance with that of the standards. The quantification was performed using the calibration curves of the appropriate standard.
The DPPH free radical scavenging capacity of the grape extracts was assessed using the methodology described (Blois, 1958). The extracts were tested at various concentrations ranging from 3.125 to 200 μg/ml. The results were quantified as the IC50 value, which is the concentration of the extract needed to block 50% of the DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radicals, measured in μg/mL.
The ability of the grape extracts to scavenge ABTS•+ radicals was evaluated following the method described by Re et al. (1999) with some modifications. The extracts were tested at various concentrations ranging from 3.125 to 200 μg/mL. The percentage inhibition was plotted against the concentration of the extract, and the IC50 value, which represents the concentration required to inhibit 50% of the 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) radicals, was calculated using linear regression analysis. The results were reported in μg/mL.
The iron reduction capacity of the various extracts in the phenanthroline-Fe3 complex was determined using the method outlined by Szydłowska-Czerniak et al. (2008) The results were given as the A0.5 value at a wavelength of 510nm.
The ability of the different extracts to reduce ferricyanide iron was also established as previously described by Oyaizu, (1986). with slight modifications. The results were displayed as the grapes extract concentration at an absorbance of 0.5 (A0.5) at 700 nm.
The silver nanoparticle test was performed according to the method previously described by Özyürek et al. (2012) to validate the antioxidant capacity of the various extracts by reducing silver ions (Ag+). The results were quantified in micrograms per milliliter (μg/ml) using the A0.5 value at a specific wavelength range of 400-450nm.
The method reported by Deveci et al. (2018) was used to determine the capacity of grape seed to inhibit the activity of mushroom tyrosinase. Cojik acid was used as standard, and results were expressed as inhibition percentage at a concentration of 12.5µg.
The method outlined by Mansur et al. (1986) for measuring anti-solar activity was applied to establish the photoprotective effect of the different extracts. The absorbance was read in a range of wave lengths from 290 to 320 nm. At least, the SPF factor was determined by applying Mansur's mathematical formula.
All statistical analyses were performed with the SPSS software package. The collected data were subjected to a one-way analysis of variance (ANOVA). The Tukey’s (HSD) multiple tests determined significant differences between means at P ≤0.05.
RESULTS
The study assessed the total polyphenol (TP) content and four sub-groups of polyphenols-total flavonoids (TF), total flavanols (TF-ol), total condensed tannins (TCT), and total hydrolysable tannins (TYT)-in seed extracts across three ripening phases using spectrophotometric assays, as detailed in Table 1. Significant variations (p < 0.05) in total phenolic compound content were observed among grape seeds (ranging from 527.4 to 716.1mg/g) across different ripening stages. All extracts exhibited high concentrations of Total Phenolic Content (TPC), with distinct trends observed for each phase: SB showed higher concentrations before veraison, RG peaked at veraison, and CR peaked at the ripening stage. In the BV and SB varieties, the highest concentrations were detected before veraison, decreasing to 650.7 and 669.5 mg GAE/g, respectively, at veraison, before increasing again to 595.1 and 527.4mg GAE/g. Conversely, RG displayed its highest amount around veraison (716.1mg GAE/g), with lower concentrations observed in the BV and R stages (554.8 and 574.2mg GAE/g, respectively), with R remaining higher than BV.
The TFC content ranged from 24.5 to 40.7mg QE/g, with the highest concentrations detected in the RG and CR varieties. The flavonoid content in the CR and SB varieties evolved similarly to the total phenols, showing the highest concentrations in the unripe stage, followed by a decrease at veraison, and then another increase until the ripe stage. In the RG variety, the flavonoid content followed the same pattern as the TPC, with the highest concentration detected at the veraison stage (716,1mg). Total flavanols also followed this trend, except in the CR variety, which showed a decrease from the unripe to the ripe stage.
In the case of total condensed tannin (TCT), each variety behaved differently. The RG variety showed an increase in TCT from unripe to veraison, followed by an obvious decrease until the ripe stage. Regarding the CR variety, the TCT showed a decreasing trend from the first to the last stage, whereas the SB variety followed the same trend as the TPC and TFC. In contrast, the highest concentration of TYT was detected at the veraison stage in all cultivars.
We detected two flavanols: catechin and epigallocatechin. Catechin was the most abundant flavanol in all cultivars at different ripening stages. The BV stage contained the highest concentration of catechin in all cultivars, with 72645.40a in Sabel (SB), 24586.40a in Red Glob (RG), and 31148.85a in Cardinal (CR). The evolution of catechin in the SB and RG varieties showed a similar trend, shifting from the BV to the R stage. In the SB variety, catechin decreased from 72645.40 at BV to 18245.33 at R. In the RG variety, catechin decreased from 24586.40 at BV to 4537.68 at R. In the CR variety, catechin decreased from 31148.85a at BV to 13700.78 at V, then increased again to 20337.11 at R, though still lower than at BV. Epigallocatechin was found in lower concentrations than catechin. In the SB cultivar, epigallocatechin decreased from 12.00 at BV to 4.99 at R. The RG variety showed similar quantities of epigallocatechin at BV (4.58) and R (3.97), but none during veraison. The CR type only identified epigallocatechin during the R phase (5.26). (Table 2).
Gallic acid and o-coumaric acid were detected in all cultivars, with gallic acid being more abundant than o-coumaric acid. During the green stage (BV), the Red Glob (RG) variety exhibited the greatest concentration of gallic acid with 1130.75. The Sabel (SB) cultivar had the highest level of o-coumaric acid with 72645.40 during the ripening phase. The levels of gallic acid showed a similar pattern in both the SB and Cardinal (CR) cultivars, peaking at 275.16 and 387.54 respectively during the initial phase, declining during veraison to 124.25b and 119.13c, and then rising again to 271.70 and 698.61 at maturation. The RG variety exhibited a fluctuating pattern, with values of 1130.75 at BV, 365.95 at veraison, and 119.13 at R.
Polydatin showed no specific trend: the SB variety had none in the first stage and low concentrations at veraison and ripening; the RG variety had low concentrations in the first and second phases and decreased at ripening; and the CR variety detected polydatin only during ripening with the highest concentration among the three varieties. Only the CR cultivars exhibited low quantities of resveratrol during the ripening stage. Luteolin concentration ranged from 85.06 to 164.44 mg/kg in all cultivars. The SB and CR cultivars showed a decrease in luteolin concentration from BV to V, followed by an increase from veraison to ripening. In contrast, the RG cultivar showed an increase from BV to V, followed by a decrease from veraison to ripening (Table 2).
Isoquercitrin was the only flavanol detected in grape seeds of different varieties, found in low concentrations across different ripening stages. The SB variety had the highest amount of isoquercitrin. We noted no significant differences between ripening phases, except for the CR variety, where we only detected isoquercitrin at the ripening phase. Only the CR variety had a low concentration of diosgenin at the veraison stage (Table 2).
Grape seed extracts demonstrated potent antioxidant activity across all ripening stages, with higher activity noted at BV and R stages (Table 3). The extracts showed effective scavenging activity against ABTS free radicals and strong iron reduction in the phenanthroline test, surpassing the efficacy of DPPH assays. CR and SB extracts from the BV stage exhibited higher antioxidant activity than the standard BHT in the DPPH and phenanthroline assays, respectively. In the SNP test, all samples showed higher potency than the standards BHA and BHT.
The seed extracts' antioxidant activity varied with the ripening stage and changes in phenolic content. IC50 values exhibited a trend similar to that of TPC and TFC. In the DPPH assay, the scavenging ability was most potent at the BV stage, decreased at veraison, and increased again at ripening in all cultivars. The ABTS test showed that the CR and SB cultivars had similar patterns. However, the RG variety had lower IC50 values and stronger activity at the BV and V stages compared to the R stage. The reducing power test showed that the SB variety was most active at veraison, while the RG and CR varieties were more active at the BV and R stages. For all cultivars, the phenanthroline assays showed close IC50 values across different ripening stages. The SNP test revealed high activity at BV and V for RG and CR, as well as at BV and R for SB.
A Principal Component Analysis (PCA) was conducted to elucidate the associations and correlations between seed development stages, phenolic profiles, and antioxidant activities in the studied seed varieties. The PCA analysis revealed a total variance of 100% in all cultivars. For the SB variety, PC1 represented 58.73% of the variance, while PC2 represented 41.27%. There was a positive correlation between most variables and PC1, but negative correlations were seen for catechin (C), epigallocatechin (EGC), gallic acid (GA), luteolin (Lut), total condensed tannin (TCT), and total flavonoid (TF-OL). Key markers for the BV stage included C, EGC, TPC, TF-OL, TCT, and GA. Higher amounts of Iso, Pol, and TYT characterized the veraison stage, while TFC Lut and O-CA marked the ripening phase. All tests positively correlated with PC1 in terms of antioxidant activity, and the veraison stage area showed lower antioxidant capacity, as indicated by higher IC50 values (Fig. 2A).
In the CR variety, PC1 and PC2 accounted for 61.82% and 38.18% of the total variance, respectively. Higher amounts of C, GA, Lut, TPC, TCT, and TF-OL were found in the BV stage. TCT and TF-OL were linked negatively to PC1. We detected no specific markers in the veraison stage except for Dios and TYT and identified six markers in the ripening stage. The veraison area had the least effectiveness in terms of antioxidant activity, as indicated by the Phen, ABTS, and DPPH tests. Conversely, the ripening phase area demonstrated the most effectiveness, as shown in Fig. 2B.
In the case of the RG variety, the first principal component (PC1) accounted for 59.65% of the overall variability and showed a positive association with most variables. On the other hand, the second principal component (PC2) explained 40.35% of the variability. Key markers for the BV stage included C, EGC, GA, O-CA, TF-OL, TCT, and TYT. The veraison stage was characterized by higher amounts of Pol, Lut, Iso, TPC, TFC, TYT, and TCT. The ripening stage did not exhibit any specific markers. The distribution of antioxidant tests indicated higher IC50 values and lower effects in the ripening stage (SNP, ABTS, Phen) and the veraison stage (DPPH, RP), in contrast to the BV stage, which exhibited potent antioxidant activity, likely due to the quality and concentration of phenolic compounds in this stage (Fig. 2C).
Principal component analysis of the different seed extracts grape varieties. TPC: Total phenolic, TFC: flavonoid, TF-OL: flavanol, TCT: condensed tannin, TYT: hydrolysable tannin, O-CA: O-coumaric acid, GA: gallic acid, Res: resveratrol, Pol: polydatin, CAT: catechin, EGC: epigallocatechin, Iso: Isoquercetin. A: Sabel, B: Red Glob, C Cardinal.
We assessed the anti-tyrosinase activity of the three extracts during the different ripening phases. The results showed that the last phase represented the highest inhibition percentage in all cultivars (Fig. 3). The CR variety demonstrated the most potent inhibition percentage (45%), followed by SB, which exhibited a non-significantly lower activity (44%), while RG showed the lowest activity with a percentage of 38%. We found that the standard cojik acid had an inhibition percentage of 36.29%.
Percentage inhibition of tyrosinase activity by seed extracts of different grape varieties during repining. CR: Cardinal, RG: Red Glob, SB: Sabel, BV: Before veraison, V: Veraison, R: Repining.
The current study established the photoprotective effect of each extract through its SPF factor. The results revealed no significant differences between the varieties or between the same variety at different ripening phases, except for the CR variety at the ripening stage, which showed the highest SPF factor (Fig. 4). The correlation between SPF factor and resveratrol (Table 4) explains why the CR variety had the highest SPF factor in the latest stage compared to other varieties. The SPF factor of most extracts was around 30, a value considered to have high protective effects according to the Recommendation of the Commission of the European Communities (2006).
Sun protection factor (SPF) of the different seed extracts grape varieties during the different repining phase.
DISCUSSION
The study found significant variations in total phenolic compound (TPC) content among grape seeds across different ripening stages. All extracts showed high concentrations of TPC, with distinct trends for each phase: SB had higher concentrations before veraison, RG peaked at veraison, and CR peaked at ripening. Variety is one of the main factors influencing grapes' phenolic content. Furthermore, factors such as maturity, environmental stress, agronomic techniques, geographic regions, irrigation, the presence of plant pathogens, extraction techniques, and solvents have an impact on grapes' phenolic content (Du et al., 2021, Dudoit et al., 2020). This difference based on variety is consistent with previous studies. Labri et al. (2020) used 80% methanol as an extraction solvent to find out how much TPC was in Algerian grape seeds while they were ripening. They found that the Red Globe variety had 398.01 mg GAE/g and the Valenci variety had 335.11 mg GAE/g. Elagamey et al. (2013) used the same extraction method to look at the total phenolic compounds in six Egyptian seeded grapes. They found that the TPC levels were different between the varieties, with Black Rose, Red Globe, and Roumi Ahmer having the highest levels.
Regarding maturity, our results align with those of Dudoit et al. (2020), who found higher seed TPC values at the veraison stage. Ivanova et al. (2011) reported the highest TPC for some varieties at veraison and for others at physiological ripening. Benbouguerra et al. (2021) also noted that the highest TPC content in three grape skin varieties occurred at the first stage, with subsequent stages showing a decrease. Studies by Kurt-Celebi et al. (2020) and Prakash and Kudachikar (2020) demonstrated an increasing trend in TPC from the unripe to the ripe stages of grape berries. These results confirm that grape maturity influences the TPC content of different varieties. The partial oxidation of phenolic compounds during the ripening phases may be responsible for the decrease in TPC content (Dudoit et al., 2020). Additionally, the continual transformation of phenolic compounds into other substances, the increase in fruit weight, and the reduction in bitterness by polyphenols, which serves to protect young fruits from animal consumption, can explain this decrease (Zhang et al., 2022).
The study found that total flavonoid content (TFC) in various grape varieties increased from the unripe to the ripe stages, with the highest concentrations observed in the RG and CR varieties. However, the CR variety had fewer total flavonoids. The TFC values we obtained were higher than those reported by Derradji-Benmeziane et al. (2014), who estimated the TFC content in five Algerian grape varieties. Kurt-Celebi et al. (2020) also observed a similar trend in TPC and TFC during maturity, noting an increase in concentrations from unripe to overly ripened berries. Fang et al. (2013) tracked the evolution of total flavanols during grape berry development and identified two accumulation peaks: the first peak appeared about 20 days after full bloom, followed by a significant decrease until veraison, when they detected another accumulation peak.
Analyzed the quantity of total condensed tannins (TCT) in the seeds and skins of the Algerian grape variety Cardinale at the ripening phase and found amounts ranging from 537.25mg/g to 1332.90 mg/g of berries. These results were higher than our findings. Several factors can affect the quantity of TCT in grapes and explain the differences between results, such as variety, climate conditions that vary from year to year, number of seeds, berry size, and harvest year (Benmeziane and Cadot, 2019).
Vineyard practices such as irrigation, vigor, vintage, altitude, and shading, along with climate conditions, are responsible for the decrease in tannin content during ripening. These activities also aid in the ongoing expansion of tannin polymers, resulting in the formation of longer chains. Additionally, they facilitate the bonding of tannins with other chemicals in the berry as it ripens (Kennedy et al., 2007). The impact of climatic and maturity conditions on the development of seed tannins over two different years during maturation has been confirmed. Additionally, the amount of condensed tannins (CT) differed between two studied vineyards, and the seeds' soluble CT content exhibited an overall decreasing pattern during ripening (Wang et al., 2023).
The detection of catechin as the most abundant flavanol aligns with findings by others, indicating catechin as a predominant flavonoid in grape skins and seeds (Du et al., 2021, Obreque-Slier et al., 2010). The evolution pattern of catechin and epigallocatechin suggests that ripening stages significantly impact flavanol content, which might be due to phenolic oxidation, polymerization, and interactions with other substances like proteins and polysaccharides (Andjelkovic et al., 2013, Liu et al., 2010). The significant differences in gallic acid content between varieties and the impact of ripening stages corroborate findings by those who observed variations in phenolic acids during grape ripening and maturation (Liang et al., 2011, Topalovic and Mikulic-Petkovsek, 2010).
The low concentration of polydatin and resveratrol detected, primarily in the skin, is consistent with Benbouguerra et al. (2021) and Moreno et al. (2008), indicating that stilbenes are generally present in low amounts in grape seeds. The absence of stilbenes in seeds during ripening stages, as noted by Dudoit et al., 2020, supports these findings. The changes in luteolin content during ripening stages, with variations between cultivars, align with observations by Fang et al. (2013), who reported slight modifications in luteolin content during grape berry development.
Isoquercitrin was the only flavanol found in grape seeds, and quercetin-glucoside levels rose during maturation, as reported by Ivanova et al. (2011) and Topalovic and Mikulic-Petkovsek (2010). This shows that flavanols play a big part in grape development. Diosgenin was only found in the CR variety at the veraison stage, which suggests that this secondary metabolite is only made in that cultivar during certain stages of ripening.
Several biological or dietary systems have investigated the antioxidant properties of grape extracts and related compounds. Grape extracts and related products are widely recognized for their ability to decrease oxidative stress in biological systems and inhibit the deterioration of food. Therefore, the extracts from grape seeds are a promising antioxidant for dietary supplements. They are considered the grape part, which displayed the highest antioxidant activity, followed by the skin and the flesh (Xia et al., 2010). The antioxidant activity observed is similar to the findings of Ghouila et al. (2016), Hanaa et al. (2015), and Sochorova et al. (2020), which means grape extracts may help lower oxidative stress and keep food from going bad. The higher antioxidant activity at the BV and R stages corresponds with higher polyphenol concentrations, indicating a direct relationship between phenolic content and antioxidant capacity. Differences in antioxidant compounds such as condensed tannin, catechin, epigallocatechin, gallic acid, o-coumaric acid, and stilbenes account for variations in antioxidant capability. The study conducted by Benbouguerra et al. (2021) found that the antioxidant activity of grape skins decreases when the total phenolic content (TPC) decreases during ripening stages. This finding aligns with the results of the current investigation. Du et al. (2021) also noted significant differences in the antioxidant capacity of grape skins during ripening, with trends in the scavenging activities of DPPH, ABTS, and reducing power assays similar to those of TPC and TFC. This suggests that phenolic profile changes are crucial in determining the antioxidant activity of grape seed extracts.
The PCA results demonstrate that the initial stage (BV) of seed development is characterized by higher concentrations of TPC, TCT, C, and GA. These molecules are potent antioxidants and are highly correlated with various antioxidant tests, explaining the highest antioxidant capacity observed during this stage. The veraison and ripening stages show different phenolic profiles and antioxidant activities, reflecting the dynamic changes in phenolic compounds and their concentrations during seed maturation. The observed variations among the different stages and varieties provide insights into the phenolic profiles and antioxidant potentials, which can inform breeding and cultivation practices for improved antioxidant properties in seeds.
Grape seeds are considered an important source of antioxidants, which protect skin from UV damage and enhance the value of cosmetic formulations such as sunscreens (Baroi et al., 2022). A study conducted by Rauniyar et al. (2014) revealed that the inhibition of mushroom tyrosinase by grape seed extract showed a lower effect than our results (7%). In contrast, the UAE extracts of the same part also demonstrated high inhibition activity of tyrosinase in the study by Michailidis et al. (2021), represented by a maximum inhibition percentage of 75% at a concentration of 500µg/mL. Tyrosinase inhibitors have dual significance in safeguarding the skin and improving the quality of fruits and vegetables. By impeding the enzymatic browning process produced by tyrosinase, these inhibitors render plants a reliable, powerful, and harmless reservoir of these compounds (Michailidis et al., 2021, Rauniyar et al., 2014). Flavonoids, such as kaempferol and quercetin, and stilbenes like resveratrol and piceatannol have been reported to exhibit inhibitory activity against tyrosinase. This result is supported by the high coefficient of correlation between anti-tyrosinase activity, catechin, and stilbene polydatin. Lower correlations were also found with o-coumaric acid, epigallocatechin, and resveratrol (Dwibedi et al., 2022).
Due to heightened UV radiation levels reaching the Earth's surface and contributing to an increase in skin-related disorders, there has been significant interest in exploring natural chemicals as photoprotective agents (Nunes et al., 2017). Grapes and their derivatives are natural sources with potential photoprotective qualities against UV radiation, as explored in natural cosmetic formulations (Nunes et al., 2017). Studies such as those by Hübner et al. (2020) have shown that the addition of grape pomace to sunscreen formulations increases the SPF factor, confirming its photoprotective effect. Also, Katiyar (2009) showed that grape seed proanthocyanidins can stop experimental photo-carcinogenesis with low toxicity. Consequently, they have the potential to serve as components in sunscreens.
CONCLUSION
The study innovatively explores the variation in polyphenolic compounds and antioxidant activities in grape seeds across different ripening stages, using a comprehensive suite of assays including DPPH, ABTS, phenanthroline, and LC-MS/MS. Key findings include significant fluctuations in total phenolic content and antioxidant efficacy, with notable variations in specific compounds such as catechin, gallic acid, and luteolin. The research also highlights the high photoprotective potential (SPF) of grape seed extracts, especially in the CR variety at the ripening stage. Future research should focus on elucidating the mechanisms behind these activities, exploring additional grape varieties, and assessing the long-term stability and practical applications of these extracts in skincare products.
ACKNOWLEDGEMENT
The authors express their sincere appreciation to the Algerian minister of high education for providing the necessary material through free access to research laboratories and the Researchers Supporting Project number (RSP2024R112), King Saud University, Riyadh, Saudi Arabia.
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Publication Dates
-
Publication in this collection
17 Jan 2025 -
Date of issue
Jan-Feb 2025
History
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Received
05 Aug 2024 -
Accepted
16 Sept 2024
