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Article

Study on Kinetics of Trans-Resveratrol, Total Phenolic Content, and Antioxidant Activity Increase in Vine Waste during Post-Pruning Storage

by
Alina Lenuța Crăciun
* and
Gheorghe Gutt
Faculty of Food Engineering, Stefan cel Mare University of Suceava, 720225 Suceava, Romania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(3), 1450; https://doi.org/10.3390/app12031450
Submission received: 24 November 2021 / Revised: 20 January 2022 / Accepted: 21 January 2022 / Published: 29 January 2022
(This article belongs to the Special Issue Selected Papers from the 8th International Conference of Biotechnologies, Present and Perspectives)
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Abstract

:
There is increasing evidence surrounding the health benefits of E-resveratrol; this has triggered interest in stilbenoids in grapes, wine, and by-products. On the one hand, there is an enormous amount of underutilized vine waste, rich in bioactive substances during wine production. On the other hand, there is a growing demand for promising phytochemicals, for dietary and pharmaceutical purposes. Vine shoots are promising sources of stilbenoids; they have economic potential because they are sources of high-value phytochemicals. Recent studies have shown that, due to biosynthesis pathway genes, especially STS (forming trans-resveratrol), which is abundant during storage periods of vine shoots—trans-resveratrol accumulates up to 40-fold. The objective of this research was to determine the most economical part of vine waste to be exploited, and to study the kinetics of resveratrol increase in a 90-day period, to determine the optimal storage period to reach a maximum trans-resveratrol content. Total phenolic content (TPC) and antioxidant activity (AA) were studied to determine possible correlations. In Fetească Neagră vine shoot varieties stored at laboratory temperatures, trans-resveratrol content increased to a maximum (2712.86 mg/kg D.W.) at day 70, and then slightly decreased until day 90. TPC remained constant and there was a slight increase in AA. Vine shoots contained the largest amounts of trans-resveratrol (1658.22 mg/kg D.W.), followed by tendrils (169.92 mg/kg D.W.), and leaves (43.54 mg/kg D.W.).

1. Introduction

Viticulture is one of the most important agricultural activities in the world; in 2018 approximately 7.4 million hectares were cultivated globally, of which 4.3 million hectares were cultivated in Europe. Statistics place Spain in first place regarding the areas cultivated with grape vines (969,000 hectares); in second place is China (875,000 hectares); in third place is France (793,000 hectares) [1]. The average number of byproducts harvested from annual vine trimmings is 1.3 kg of wood/live log, thus “accumulating” to 2 × 107 tons of wood in the world. The only use for this waste is to grind it and use it as a fertilizer, by distributing it on vineyards [2].
Recent studies have shown that vine waste from annual trimmings accumulate via a wide range of stilbenoids, from resveratrol to complex stilbene oligomers [3]. Thereby, due to the fact that vine waste contains valuable bioactive substances, with potential applications in medicine and agriculture (due to multiple pharmacological and phytopathogenic properties), these can represent promising bioresources [4].
Stilbenoids are produced by plant secondary metabolisms and are a part of the non-flavonoid phenolic compound family. They are synthetized by plants as defense mechanisms in response to biotic and abiotic stresses. There is increased interest surrounding stilbenoids due to their potential effects on human health [5].
Stilbenes are a reduced group of phenylpropanoids characterized by a 1,2-diphenylethylene general structure. Plant stilbenes are derived from the general phenylpropanoid pathway [6]. Only a few plant species are capable of producing stilbenes, although all higher plants can synthesize esters of cinnamic acid derivatives and malonyl-CoA. The first enzymes of the phenylpropanoid pathway are phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL). Stilbene synthase (STS) catalyzes, in a single reaction, the biosynthesis of stilbene’s general structure from one CoA-ester of a cinnamic acid derivative (p-coumaroyl-CoA—most (or frequently) cinnamoyl-CoA) and three molecules of malonyl-CoA, and is only characteristic to plants that are able to produce stilbene [7]. First, the purified protein of the STS gene was from the stressed cell suspension cultures of the peanut (Arachis hypogaea) [8]. Subsequently, Scots pine (Pinus sylvestris) and grape (Vitis vinifera) were used to clone STS genes [9]. STS genes are found in many plant species as families of related genes. The vine was the only plant capable of producing stilbene, whose genome was sequenced until recently. It has been demonstrated in early studies that more than 20 STS genes are contained by the grapevine genome [10]. The large size of the multigene family was confirmed by an analysis of the first sketch of the grapevine genome, with an estimated 20–40 STS genes. A minimum of 20 different STS genes are expressed in the grape after infection with Plasmopara viticola [11].
Resveratrol is synthesized in grape vines using three molecules of malonyl-CoA and one molecule of coumaroyl-CoA, by the catalysis of stilbene synthase (EC 2.3.1.95; STS, malonyl-CoA:4-coumaroyl-CoA malonyltransferase). Most stilbenes synthesized by plants are derived from the basic unit trans-resveratrol (trans-3,5,4′-trihydroxystilbene) [12].
Generally, plants synthesize trans-resveratrol (3,5,4′-trihydroxystilbene) as a phytoalexin, naturally, in response to exogenous factors, such as ultraviolet (UV) irradiation, traumatic damage, pathogen infection, and other stresses. Only a few dietary sources have been found to contain this compound; the main representatives being peanuts, grapes, hops, strawberries, blueberries, and the products derived therefrom [13].
Several reviews and research studies have shown that trans-resveratrol, due to its pharmacological activities, is an important anti-carcinogenic and anti-aging/anti-inflammatory agent, with neuroprotective and antioxidant proprieties, and that it could protect, to some extent, against cardiac and metabolic disorders. Therefore, a growing demand for trans-resveratrol is expected in areas such as nutraceuticals, health, cosmetics, and food, due to its pharmacological importance [14].
The majority of resveratrol supplements available on the market are made of extracts obtained from the roots of Japanese knotweed (Polygonum cuspidatum) and the degree of purity can vary widely. Research studies have shown that emodin, a compound that could have laxative effects, is found in unpurified (or partially purified) resveratrol extracts [15]. The Japanese knotweed is an invasive plant that grows in heavily polluted environments and is contaminated with heavy metals. Additional concerns about the safety of resveratrol supplements have been raised, due to the possibility of the root tissue of Japanese knotweed to cellular uptake of these contaminants [16,17].
Houillé et al. studied the biosynthetic origins of trans-resveratrol accumulation in grape canes during postharvest storage and they assayed the structural genes PAL, C4H, 4CL, and STS (forming E-resveratrol) through a quantitative real-time PCR. They demonstrated that, due to transcript abundance of the structural genes, the resveratrol content increases during storage time. In the first four weeks of storage, the STS gene was induced; during the 6 weeks of storage, the three genes (PAL, 4CL, and C4H) of the general phenylpropanoid pathway were constitutively expressed. These results confirm that vine shoots during the storage time is still transcriptionally active [14]. The results can vary widely, despite recent advances in the determination of stilbenoid compositions of vine shoots [18]. This variability can be attributed to the various factors influencing the stilbenoid composition, especially the provenance and the variety of the grape cane [19]. Gyongyi et al., (2017) reported a resveratrol content ranging from 0.95 ± 0.08 to 3.94 ± 0.21 g/100 g D.W. for 10 samples of vine waste of different varieties extracted with 60% ethanol [20].
The aim of this study was to characterize trans-resveratrol accumulation in pruned vine shoots, and particularly to determine the optimal storage period for vine variety Fetească Neagră, to identify which vine waste contains a higher amount of trans-resveratrol, to be exploited economically and to study possible correlations among the kinetics of resveratrol increase, total polyphenol content, and antioxidant activity of vine waste.

2. Materials and Methods

2.1. Vine Waste Samples

The samples of grape shoots, leaves, and tendrils of the grape variety Fetească Neagră were collected from the region of Cotnari, Iași (476,708,864;269,361,914) on 17 November 2020.

2.2. Reagent and Chemicals

The diethyl ether, trans-resveratrol standard (99% GC), sodium carbonate, and DPPH solution were purchased from Sigma-Aldrich Co (Burlington, MA, USA). Ethanol, methanol, and acetonitrile (LiChrosolv for HPLC) were obtained from Merck (Darmstadt, Germany). The aqueous solutions were prepared with twice distillated and demineralized water. The 0.22 µm PTFE membrane filters were purchased from Phenomenex (Torrance, CA, USA).

2.3. Standard Solutions

A stock solution of resveratrol with a concentration of 1 g/L was prepared. To guarantee a complete dissolution of the trans-resveratrol standard, it was initially dissolved in a small volume of methanol.
A set of standards was prepared with methanol, 99%, before the analysis, from the stock solution. To avoid degradation of the standard solutions, special care was taken, keeping them protected from exposure to light and air, and storing them in brown glass containers at −20 °C.

2.4. Sample Collection and Preparation

Vine shoots collected on 17 November, 2020, were analyzed in turn—one each day for 90 days. The last sample was prepared on 14 February, 2021. Meanwhile, the other samples were kept at laboratory temperature (22 °C) to increase the concentrations of trans-resveratrol; optimal parameters were as recommended by the latest research in the field. Each sample was previously dried in the oven at 45 °C to constant weight, after which they were crushed with a grinder and dried for an additional 24 h at the same temperature. The resulting powder was macerated for 72 h with ethanol–diethyl ether solution at a 4:1 ratio, and then filtered through filter paper. The extracts obtained were partially purified before HPLC analysis. The extracts were first evaporated using a rotary evaporator near to dryness and then redissolved in diethyl ether, and the solution was washed three times with a 5% sodium bicarbonate solution in a separatory funnel. The layer of diethyl ether was recovered, evaporated on a rotary evaporator, and redissolved in methanol. The obtained extract was filtered through a 0.22 µm PTFE membrane filter prior to HPLC analysis.
Another set of samples was processed on the day of collection, as follows: the vine shoots were cut into pieces of different dimensions: 2, 3, 4, 5, and 10 cm, and pieces smaller than 1 cm, cut in sections. One sample was left uncut. The samples were left for 6 weeks at laboratory temperature (22 °C) to determine the maximum increase of trans-resveratrol, depending on sample processing. Leaves and tendrils were left for 6 weeks at laboratory temperature (22 °C). The samples were prepared in the same way as the samples describe above. The extracts were kept in a refrigerator at 4 °C until used in the analyses.

2.5. Analytical HPLC Procedure

An HPLC instrument (Shimadzu, Kyoto, Japan) coupled with an SPD-M-20A diode array detector was used to determine the trans-resveratrol content of the samples. The system was equipped with a Phenomenex Kinetex 2.6 µm Biphenyl 100 Å HPLC Column 150 × 4.6 mm and thermostated at 20 °C.
The mobile phases were in pure water (solvent A) and acetonitrile (solvent B). Elution was carried out after a modified method described previously by Marshall et al., (2012), with a binary gradient as follows: linear gradient from 0% to 10% B in 42 min, 10–40% B in 42.6 min, 40–90% B in 46.5 min. Total time of running was 49.5 min [21].
For the determination of trans-resveratrol, the detection wavelength was set at 306 nm and the flow rate of the solvent was 0.5 mL/min.
A high degree of linearity was obtained for standard calibration curves (R2 > 0.9996). LC solution software version 1.21 (Shimadzu, Kyoto, Japan) was used to perform data collection and subsequent processing. The quantification was made based on the peak area, using the external standard method. The analyses were performed in duplicate.

2.6. Determination of Total Phenolic Content

Total phenolic content was determined by the Folin–Ciocalteu method proposed by Luque-Rodríguez et al., (2006) [22]. The Folin–Ciocalteu reagent was diluted with distilled water in a ratio of 1:10, and then 180 µL was added to 90 µL of the tested extract (10 mg/mL). A total of 730 μL of Na2CO3 (100 mM) was added, and then it was left in the dark for an hour for incubation. The absorbance of the samples was then measured with an Ocean Optics HR4000CG-UV-NIR spectrometer (SUA) at 765 nm. The calibration curve of gallic acid (0–1000 mg/L, R2 = 0.9856) was used to calculate TPC, and expressed in terms of gallic acid equivalents (GAE).

2.7. Determination of the Antioxidant Activity by the DPPH Method

To determine the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the extracts, we used a method proposed by Liu et al., (2015) and Brand-Williams et al., (1995), with some modifications [23,24]. Vine shoot extracts, at 10 mg/mL, were diluted in methanol (0.25 mL) after liquid–liquid extraction, and then the DPPH solution (60 μM) (9.75 mL) was added. The total volume of 10 mL was mixed properly and left to react for 60 min at 25 °C. The absorbance was measured at 515 nm on Ocean Optics QE65000 spectrometer (SUA), using absolute methanol as a blank control. The percentage of free radical scavenging by DPPH was calculated using the following formula:
DPPH (% inhibition) = ((absorbance of DPPH − absorbance of sample)/absorbance of DPPH) × 100. All determinations were performed in triplicate

2.8. Statistical Analysis

Statgraphics Centurion XIX software-trial version (Manugistics Corp., Rockville, MD, USA) was used to carried out ANOVA.

3. Results

3.1. Statistical Analysis of Variance

The tables presented bellow contain the mean values and standard deviation of resveratrol content, TPC and antioxidant activity for different samples of vine wastes. In the first table (Table 1) are shown the result for three types of vine wastes, leaves, shoots and tendrils. Table 2 presents the results for samples of vine shoots analyzed for 90 days in a row post-pruning. In Table 3 are summarized the results obtained after analyzing the samples of different length after 6 weeks of storage.

3.2. Kinetics of Resveratrol Increase during Post-Pruning Storage

Trans-resveratrol increase in vine shoots after pruning and storage at laboratory temperature (~20 °C) was analyzed for 90 days in a row and the results are displayed in Figure 1.

3.3. Total Phenolic Content

Total phenolic content of shoots after pruning and storage at laboratory temperature (~20 °C) was analyzed for 90 days in a row and the results are displayed in Figure 2.

3.4. Antioxidant Activity by the DPPH Method

Antioxidant activity in vine shoots after pruning and storage at laboratory temperature (~20 °C) was analyzed for 90 days in a row and the results are displayed in Figure 3.

4. Discussion

4.1. Analysis of Variance of Resveratrol Content, TPC, and Antioxidant Activity in Leaves, Tendrils, and Shoots

The cleaning of the vine generates considerable amounts of vegetable waste each year (tendrils, leaves, and shoots). This green waste could be an important source for high-value phytochemicals, as it (now) does not have any usage, and it could contribute to sustainability programs [25]. Trans-resveratrol is found in abundance in this vine waste, and its content can vary widely among the waste types [26].
Results for samples analyzed after 42 days are represented in Table 1. As it can be seen, the largest amount of resveratrol is contained in vine shoots (1658.22 mg/kg D.W.), followed by tendrils (169.92 mg/kg dry weight (D.W.) and leaves (43.54 mg/kg D.W.). Vine shoots contain up to 10 times more trans-resveratrol than tendrils and up to 40 times more than leaves. Although, besides vine shoots, this vineyard waste also contains trans-resveratrol—extraction and purification are not economically recommended.
These results agree with other studies, such as the study by Lachman et al., (2016), which determine the concentration of resveratrol on vine shoots, tendrils, and leaves. Vine shoots had the highest content of trans-resveratrol (12.5 mg/kg D.W.), followed by tendrils (0.51 mg/kg D.W.) and leaves (0.24 mg/kg D.W.) [26].
A study conducted on vines from the Bohemian and Moravian Region, Czech Republic, determined the resveratrol content in different parts of grapevines (leaves, rachis, and cluster stems). The results varied from 6 to 490 mg/kg D.W. [27]. Cluster stems were found as the richest sources of resveratrol; this is in accordance with the results of this study.
Gyongyi et al., (2017) reported that trans-resveratrol is undetectable in inflorescence and the highest content was found in cluster stems (344.5 mg/kg D.W.) after analyzing nine organs of Vitis vinifera cv. Merlot: canes, shoot tips, roots, buds, clusters at the veraison, inflorescences, mature berry skins, cluster stems, and seeds [20].
TPC values for the three types of waste did not vary widely, although vine leaves had the highest content (30.42 (±0.32) mg GAE/g D.W.).
The highest antioxidant activity recorded after the analysis of the extracts was found in the leaves (72.19%), although the highest amount of resveratrol was found in the vine shoots. The results are similar to those obtained by determining the total phenolic content. The lowest percentages were recorded in tendrils (14.69% DPPH inhibition) and vine shoots (52.72% DPPH inhibition).

4.2. Kinetics of Resveratrol Increase during Post-Pruning Storage

Accumulation on trans-resveratrol during the storage period is reported on in many studies [5,28,29]. Recent research showed that increases in trans-resveratrol content in shoot samples were due to biosynthesis pathway genes (PAL, C4H, 4CL, and STS), present in abundance, especially the STS gene (forming trans-resveratrol), which was induced over the first 4 weeks of storage.
Vine shoot storage time after pruning exposed an “important” trans-resveratrol accumulation, with a maximum 181-fold induction on day 70, and a maximum average for days 71–75, as can be seen in Table 2. To highlight the increase in trans-resveratrol content over the 90 days, the average results were calculated for 5 consecutive days; the results are displayed in Figure 1.
Day 1 of the analysis was when the vine waste was collected from the vineyard; the first sample was dried and then extracted with an ethanol–diethyl ether mixture. The minimum value of trans-resveratrol content in vine shoots was registered on day one of the analysis (14.93 mg/kg D.W.) and the maximum value was registered on day 70 of the analysis (2712.86 mg/kg D.W.).
Cebrián et al., (2017) reported an increase in the concentration of trans-resveratrol and other non-volatile phenolic compounds on samples of vine waste for two Spanish varieties (Airèn and Cencibel) after different times of storage (one month, three months, and six months). After one month, the concentration of trans-resveratrol in the Airèn variety was 77.10 ± 11.29 mg/kg D.W. After three months, the concentration almost doubled (151.60 ± 9.42 mg/kg D.W.), and a slight increase was recorded after six months (170.44 ± 3.82 mg/kg D.W). The sample from the Cencibel variety followed the same way regarding increasing the concentration of resveratrol, with 50.41 ± 2.74 mg/kg D.W. after one month. After three months, the concentration was four time higher (224.83 ± 29.64 mg/kg D.W.); after six months of storage, a slight increase was recorded (227.00 ± 6.03 mg/kg D.W.) [28]. For the Airèn variety, the increase in resveratrol content from one month to three months was 196% and 448% for the Cencibel variety. According to the results of this study, the trans-resveratrol content increases only until days 70–75, after which, it decreases slightly. However, the content of trans-resveratrol per kilogram of dry matter was much higher for the Fetească Neagră variety than for the Spanish varieties, which reached up to 923.10 mg/kg D.W. after one month. Trans-resveratrol increase can vary widely due to the synthesis of the monomer in plants, if influenced by various factors (variety of vine, climate, growing conditions, temperature, exposure to fungal and bacterial infections, UV irradiation season of pruning).
These considerable increase in trans-resveratrol content in vine shoot samples can be attributed to the structural genes (PAL, C4H, 4CL and STS), which are responsible for the biosynthesis of trans-resveratrol. They are induced during the storage period, and are active until the vine shoots are dry. It is believed that the drying process during the storage period might be “sensed” to be a stress signal by living plant tissues, resulting in biosynthesis of this monomer, and trans-resveratrol accumulates until the tissues are dry [14].
This is confirmed by the results that show that, at temperatures below 20 °C, trans-resveratrol accumulation is delayed. If the vine shoot samples are stored at −20 °C, trans-resveratrol is not induced [14].
Houille et al., (2015) reported that, over the first six weeks of storage at 20 °C after pruning, the concentration of trans-resveratrol increased highly (approximately 106-fold) in eight different grape vine varieties, as it follows, the Sauvignon Blanc—48-fold induction (2908 mg/kg D.W.), Chardonnay—33-fold induction (3175 mg/kg D.W.), Côt—21-fold induction (3316 mg/kg D.W.), Grolleau—95-fold induction (3913 mg/kg D.W.), Chenin—106-fold induction (4631 mg/kg D.W.), Pinot Noir—19-fold induction (4725 mg/kg D.W.), Cabernet Franc—83-fold induction (4762 mg/kg D.W.), Gamay—51-fold induction (5100 mg/kg D.W.), and in Means—40-fold induction (4066 mg/kg D.W.) [14]. This study agrees with the present study concerning the increase of trans-resveratrol in the Fetească Neagră variety, which showed an 80-fold induction after 6 weeks (1205.11 mg/kg D.W.).
In the study by Ewald et al., (2017), the increase in trans-resveratrol concentration, depending on the storage time of the shoots, was studied. An increase between 400% and 1400% was observed after the first 6 months of storage. The values varied between 2096 mg/kg D.W. for the Regent variety and 7532 mg/kg D.W. for the Cabernet Sauvignon variety. The trans-resveratrol content dropped after 6 months in most of the varieties, except for the Regent variety, which continued rising [29]. The levels of increase in trans-resveratrol, obtained by Ewald et al., (2017), support our findings, with the mention that the increase of trans-resveratrol registered was between 52% and 18,063% compared to the amount of trans-resveratrol present in the sample from the first day (day of harvest).
A study conducted in Chile agrees with the results obtained in this work, regarding the increase of trans-resveratrol, depending on the retention time of the samples. Gorena et al., (2014) studied the trans-resveratrol increase in vine waste samples of the Pinot Noir variety harvested in 2012, for 8 months of storage, after cutting. A significant increase was registered after the second month. After the third month, the content of trans-resveratrol began to decrease; after that, it remained almost constant [5].
The grape variety also has a high influence on the amount of trans-resveratrol in vine shoots, according to Zhang et al., (2011), who analyzed 165 grape cane samples from large distribution centers and different major grape production regions. Trans-resveratrol content varied depending on the genotypes of vines; thus, V. labrusca and V. vinifera hybrids, as well as V. labrusca, had much lower content than V. vinifera. The content of this monomer can vary between the same genotype, depending on the purpose. Thus, in vine shoots from vine grapes for tables, the content of trans-resveratrol is much lower than from wine ones [13]. This could explain the large amounts of resveratrol obtained from extracts of the Vitis vinifera Fetească Neagră variety, which is a vine variety for wine.

4.3. Total Phenolic Content

Total phenolic content of the vine shoots was estimated using the Folin–Ciocalteu colorimetric method. The mean values calculated for 5 days in a row for TPC of vine shoots samples analyzed for 90 days are displayed in Table 2. As seen in Figure 2, the total phenolic content (TPC) values remained constant, between a 20 and 30 mg gallic acid equivalent (GAE)/g D.W., except for 5 days when higher values were recorded. The highest value was recorded on day 54 (82.42 mg GAE/g D.W.), and the lowest on day 57 (14.61 mg GAE/g D.W). Total polyphenol content remained constant, which could be attributed to polyphenols, other than trans-resveratrol, whose content may increase, decrease, or remain constant during the 90 days (piceatannol [14,17], apigenin, ellagic acid [30], viniferin, trans-vitisin, trans-piceid [31], gallic acid, ellagic acid, p-coumaric acid and others [32].
Cetin et al., (2011) reported that a TPC for grape canes ranged from 25.36 to 36.56 mg GAE/g D.W. in different vine varieties from Turkey: Alphonse Lavallée, Atasarısı, Cardinal, Hafızali, Horoz, Karası, Isabella, Italia, Sultani, Cekirdeksiz, Tekirdag, Cekirdeksiz, and Trakya Ilkeren [33]. Alexandru et al., (2014) reported that the values for total phenolic content for vine shoots ranged from 18.23 to 198 mg GAE/g D.W. for vineyards from Cuneo, Italy [34].
The TPC of the Portuguese vine shoot extract varieties Touriga Nacional and Tinta Roriz were analyzed by Moreira et al., (2018), using the three different extraction techniques—subcritical water extraction, microwave-assisted extraction, and conventional extraction. The vine shoot variety Tinta Roriz had the highest total phenolic content (32.1 ± 0.9 GAE/g D.W.) and antioxidant activity [34].
Farhadi et al., (2016) reported higher values for the vine shoots (around 200 mg GAE/g D.W.) compared to the leaves (61 mg GAE/g D.W.) after analyzing the total phenolic content of different parts of the grape and vine (seed, skin, pulp, leaf, and vine shoots) for five different varieties native in West Azerbaijan (Ghara Shira, Hosseini, Agh Shani, Ghara Ghandome, and Ghara Shani) [35].
The lower amount of TPC in the extracts analyzed in this study may be attributed to the extracts being washed with sodium bicarbonate solution, and only the diethyl ether layer was analyzed, so the water-soluble polyphenols were not count. This theory is confirmed by the study by Angelov et al., (2016), where they analyzed the content of polyphenols in both fractions, “ethanol” fraction (with 295.1 mg·g−1 TPC content) and an “aqueous” fraction (with 103.0 TPC content), with a 143.7 mg·g−1 GAE of dry total extract [2].

4.4. Antioxidant Activity by the DPPH Method

Resveratrol is a phenolic compound that can act against free radicals, and it can act as a promoter of cellular antioxidant enzymes, such as peroxidase, glutathione, glutathione reductase, and glutathione-transferase, to induce neutralization of radical peroxides [36]. It is important to determine the antioxidant activities of vine shoots, due to future correlations with phenolic content, and to establish the important impacts of plant components to human health [22].
The antioxidant activities of the extracts were measured using the DPPH radical scavenging assay, as described by Liu et al., (2015) [23]. Several research studies were conducted to determine the antioxidant activity of vine shoots, via different methods (Barros et al., 2014; Ruiz-Moreno et al., 2015; Ju et al., 2016; Moreira et al., 2018; Karacabey and Mazza, 2010; Rajha et al., 2015); however, the most common was the DPPH method [32,37,38,39,40,41,42]. Although much data have been reported, antioxidant activity was reported in µM Trolox equivalent/mg extract or inhibition percentage (IC50), and a comparison of the results is not possible. Furthermore, even if the vine shoots are from the same variety, the analytical methods for extractions employed can widely vary, as reported in most studies (Ruiz-Moreno et al., 2015; Ju et al., 2016; Rajha et al., 2015; Farhadi et al., 2016) [35,37,40,41,42].
The storage times of the shoots after cutting revealed a slight increase in antioxidant activity starting on day 1 (31.90% DPPH inhibition), reaching a maximum of 76.52% DPPH inhibition on day 85, followed by a sharp decrease, as can be seen in Figure 3. To highlight the increase of DPPH inhibition over the 90 days, the average results were calculated for 5 consecutive days; the results are displayed in Figure 3. The increase in antioxidant activity follows the same trend as the increase in resveratrol, with the difference being that the maximum concentration of resveratrol is recorded 10 days before the maximum antioxidant activity, followed by a decrease in both cases. The different paths, regarding the increase of antioxidant activity toward trans-resveratrol, can be due to other polyphenols with antioxidant activities than this monomer, whose content can increase, decrease, or remain constant in vine shoot samples [14,32]. In any case, W=when the samples are dry, in both cases, a decrease is registered. That finding can be correlated to the findings of Houillé et al., who reported that trans-resveratrol and piceatannol accumulate until the tissues are dry [14]. This ascertainment is in agreement with the work by Guerrero et al., (2016), who reported that no correlation between antioxidant activity and total stilbene content was observed, due to the fact that non-phenolic antioxidants could be extracted from the vine shoot, and they could contribute to antioxidant activity [42].

4.5. Influence of Sample Sectioning on Trans-Resveratrol Increase, TPC, and Antioxidant Activity

The high increase of trans-resveratrol during the post-pruning period has been displayed in many studies in the field. Due to stilbene synthase gene transcription inducted by stilbenoid metabolism, this occurrence in vine shoots is explicit [14].
Notably, it was also found that the induction level was modulated by control storage temperatures. Based on STS induction properties, it was hypothesized that the growth of stilbenoid content could be raised by varying the external stress factors [43].
The effect of mechanical stress was tested by cutting the freshly-pruned vine shoots at different section lengths from pieces smaller than 1 cm, cut in sections to 10 cm and an uncut sample, over 42 days of post-harvest storage. Mechanical injury to freshly cut vine waste did not allow excessive accumulation of trans-resveratrol compared to the cut samples. It can be seen that only the sample cut into pieces came close in value to the uncut sample (Table 3). This result is confirmed by the results obtained by Billet et al., (2018), who also tested the effects of mechanical stress on trans-resveratrol accumulation by cutting freshly-pruned vine shoots into pieces from 0.5 to 10 cm. Only the sample cut into 0.5 cm allowed a higher accumulation of the monomer [17].
For the samples cut in different sizes, the highest TPC was found in the 4 cm sample section (35.55 mg GAE/g D.W.), and the lowest TPC content was found in the pieces of vine shoots (22.03 mg GAE/g D.W.).
The samples that were cut into pieces showed the highest antioxidant activity, of 79.24% DPPH inhibition. The uncut sample (52.72% DPPH inhibition) had almost the same antioxidant activity as the 1 cm sample (53.26% DPPH inhibition). The lowest antioxidant activity was for the 4 cm sample of 22.74% DPPH inhibition.
The 4 cm long sample had the higher mean value for TPC (3.555 mg GAE/g D.W.); the mean values for all samples for TPC did not vary widely.

5. Conclusions

The interest in resveratrol commercial supplements has increased due to its proven health benefits (e.g., anti-aging, anti-inflammatory, anti-carcinogenic, neuroprotective, and antioxidant agents), and that it could protect against, to a certain extent, heart and metabolic disorders.
The majority of resveratrol supplements available on the market are made of extracts obtained from the roots of Japanese knotweed (Polygonum cuspidatum); some studies showed that emodin, a compound that could have a laxative effect, was found in partially purified extracts of resveratrol. Research studies show that resveratrol could be extracted from vine waste, which is abundant in this compound, without contamination of emodin.
Moreover, vine waste resulting from annual trimming is utilized as fertilizer by grinding or burning. Agricultural vine waste is an important source for phytochemicals, although economically it is unevaluated.
During the vineyard trimming process, a considerable amount of green mass (portions of shoots, leaves, tendrils) is removed. Vine shoots contain up to 10 times more trans-resveratrol than tendrils, and up to 40 times more than leaves. Although, besides vine shoots, the vineyard waste also contains trans-resveratrol, extraction and purification are not economically recommended.
To increase the extraction yield in the pruned vine shoots, these should be kept for 9–10 weeks at a controlled temperature, at around 20 °C. The increase is 18.063% on day 70 of storage compared to the first day of harvest.
Mechanical stress did not influence the increase of resveratrol content in vine shoots. A small difference was recorded between the sample that was cut in pieces smaller than 0.5 cm (1641.64 mg/kg D.W.) and the uncut samples (1667.49 mg/kg D.W.).
A correlation among the resveratrol content, TPC, and DPPH % inhibition could not be established, due to the fact that polyphenols and other compounds with antioxidant activity present in vine shoots, other than trans-resveratrol, can increase, decrease, or remain constant during the 90 days. The only similarity was that resveratrol content and DPPH % inhibition increased, with the difference being that the resveratrol content significantly increased until day 70, and then dropped from day 76; DPPH % inhibition slightly increased until day 85, and then decreased 50% over the next 5 days.

Author Contributions

Conceptualization, A.L.C. and G.G.; methodology, A.L.C.; validation, A.L.C. and G.G.; formal analysis, A.L.C.; investigation, A.L.C.; resources, A.L.C. and G.G.; data curation, A.L.C.; writing—original draft preparation, A.L.C.; writing—review and editing, A.L.C.; visualization, G.G.; supervision, G.G.; project administration, G.G.; funding acquisition, G.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data related to this study are explicitly plotted in the figures in this article.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 12 01450 g001 550
Figure 1. Trans-resveratrol increase in vine shoots for 90 days.
Figure 1. Trans-resveratrol increase in vine shoots for 90 days.
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Figure 2. Total phenolic content on grape canes during the 90-day post-pruning.
Figure 2. Total phenolic content on grape canes during the 90-day post-pruning.
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Figure 3. DPPH % inhibition in vine shoots during the 90-day post-pruning time storage.
Figure 3. DPPH % inhibition in vine shoots during the 90-day post-pruning time storage.
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Table
Table 1. Analysis of variance of resveratrol content, TPC, and antioxidant activity in leaves, tendrils, and shoots.
Table 1. Analysis of variance of resveratrol content, TPC, and antioxidant activity in leaves, tendrils, and shoots.
ParametersWaste TypeF-Ratio
LeavesShootsTendrils
Resveratrol content, mg/kg D.W.43.54 (±0.08)1658.22 (±0.17)169.92 (±0.03)131,472,834.15 ***
TPC mg GAE/g D.W.30.42
(±0.32)		29.36
(±0.41)		28.65
(±0.81)		50.43 ***
DPPH %73.19 (±0.04)52.72 (±0.22)14.19
(±0.05)		95,811.06 ***
(Mean values ± standard deviation). ns not significant (p > 0.05), *** p < 0.001.
Table
Table 2. Analysis of variance of resveratrol content, TPC, and antioxidant activity in vine shoots for 90 days.
Table 2. Analysis of variance of resveratrol content, TPC, and antioxidant activity in vine shoots for 90 days.
ParametersStorage Days
51015202530354045505560657075808590F-Ratio
Resveratrol content, mg/kg D.W.86.58
(±1.24)		163.56
(±2.31)		282.53
(±3.01)		386.44
(±5.12)		557.92
(±7.89)		923.10
(±13.05)		1190.79
(±16.84)		1236.28 (±17.48)1349.59 (±19.09)1401.91 (±19.24)1510.93 (±18.62)1524.73 (±19.40)1563.39 (±22.24)1605.61 (±22.70)1753.50 (±24.80)1532.31 (±21.67)1335.31 (±18.89)1135.43 (±16.05)2044.65 ***
TPC mg GAE/g D.W.35.86
(±0.53)		24.75 (±0.34)26.45 (±0.37)35.43
(±0.58)		28.16 (±0.45)28.48 (±0.41)23.53
(±0.34)		20.64 (±0.35)25.86 (±0.43)27.09
(±0.41)		35.38
(±0.56)		20.30
(±0.32)		20.45
(±0.35)		29.37
(±0.42)		21.97 (0.36)23.24
(±0.37)		23.70
(±0.31)		29.18
(±0.43)		3380.74 ***
DPPH %31.90
(±0.45)		39.84 (±0.56)44.09 (±0.62)35.16 (±0.50)41.77
(±0.59)		35.81 (±0.51)44.19 (±0.62)58.06 (±0.82)48.09 (±0.68)58.22
(±0.82)		59.53 (±0.84)56.34
(±0.80)		60.72
(±0.86)		50.85
(±0.72)		51.38
(±0.73)		48.44
(±0.69)		76.52 (±1.08)35.97 (±0.51)518.39 ***
(Mean values ± standard deviation). Ns not significant (p > 0.05), *** p < 0.001.
Table
Table 3. Analysis of variance of resveratrol content, TPC, and antioxidant activity in vine shoot samples of different lengths.
Table 3. Analysis of variance of resveratrol content, TPC, and antioxidant activity in vine shoot samples of different lengths.
ParametersShoot Length
<1 cm1 cm2 cm3 cm4 cm5 cm10 cmUncutF-Ratio
Resveratrol content, mg/kg D.W.1667.49 (±23.82)231.6
(±3.31)		509.92
(±7.28)		525.285
(±7.50)		618.67
(±8.84)		999.03
(±14.27)		1406.09
(±20.09)		1641.64
(±23.45)		2619.29 ***
TPC mg GAE/g D.W.22.03
(±0.31)		22.95
(±0.41)		22.14
(±0.32)		28.63
(±0.44)		35.55
(±0.52)		26.45
(±0.43)		24.75
(±0.41)		29.03
(±0.42)		2963.32 ***
DPPH %78.45
(±1.12)		52.725
(±0.76)		36.74
(±0.52)		39.255
(±0.56)		22.515
(±0.32)		32.135
(±0.46)		50.64
(±0.72)		52.195
(±0.74)		1225.19 ***
(Mean values ± standard deviation). Ns not significant (p > 0.05), *** p < 0.001.
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Crăciun, A.L.; Gutt, G. Study on Kinetics of Trans-Resveratrol, Total Phenolic Content, and Antioxidant Activity Increase in Vine Waste during Post-Pruning Storage. Appl. Sci. 2022, 12, 1450. https://doi.org/10.3390/app12031450

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Crăciun AL, Gutt G. Study on Kinetics of Trans-Resveratrol, Total Phenolic Content, and Antioxidant Activity Increase in Vine Waste during Post-Pruning Storage. Applied Sciences. 2022; 12(3):1450. https://doi.org/10.3390/app12031450

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Crăciun, Alina Lenuța, and Gheorghe Gutt. 2022. "Study on Kinetics of Trans-Resveratrol, Total Phenolic Content, and Antioxidant Activity Increase in Vine Waste during Post-Pruning Storage" Applied Sciences 12, no. 3: 1450. https://doi.org/10.3390/app12031450

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Crăciun, A. L., & Gutt, G. (2022). Study on Kinetics of Trans-Resveratrol, Total Phenolic Content, and Antioxidant Activity Increase in Vine Waste during Post-Pruning Storage. Applied Sciences, 12(3), 1450. https://doi.org/10.3390/app12031450

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