Phenolic profile of leaves obtained from autochthonous grapevine cultivars in Isparta Province of Türkiye

Abstract

In the current age, nutrition is a widely discussed topic, with foods rich in antioxidants being advocated to support healthy living. Phenolic compounds, which are used in treating various diseases due to their antimicrobial properties, have health-promoting properties. Consequently, producing extracts from grape cultivars with high phenolic content for use in pharmaceuticals, food supplements, and cosmetics is essential for health and the economy. It is well established that grape leaves are rich in phenolic compounds. However, they are not currently utilized to their full potential and are considered mainly waste. This study was conducted in vineyards in Isparta, located in Türkiye’s Mediterranean Region, in 2018. The study utilized four autochthonous leaf varieties: two red (Vitis vinifera cv. ‘Burdur Dimriti’ and Vitis vinifera cv. ‘Siyah Gemre’) and two white (Vitis vinifera cv. ‘Razaki’ and Vitis vinifera cv. ‘Tilki Kuyruğu’). Total phenolic and flavanol content, as well as the presence of other phenolic compounds, were determined in the grape leaves. The study’s findings indicated significant variations in the phenolic content of different grapevine leaf cultivars. Total phenolic content ranged from 3.25 to 7.38 mg CE·g⁻¹, while total flavanol content ranged from 67.66 to 120.24 mg CE·g⁻¹. Phenolic compounds identified in the leaves included gallic acid, ferulic acid, caffeic acid, chlorogenic acid, catechin, quercetin, epicatechin, rutin, p-Coumaric acid, ο-Coumaric acid, protocatechuic acid, hesperidin, vanillin, kaempferol, luteolin, and tannic acid, which were found to vary among the cultivars. Leaves from white cultivars had higher phenolic content than red cultivars. ‘Razaki’ is the most bioactive cultivar because it has the richest flavonols. ‘Razaki’ and ‘Tilki Kuyruğu’ could be an important resource for the development of functional products.

Introduction

Grapes are among the most extensively cultivated fruits globally. Türkiye is among the foremost grape-cultivating nations worldwide. Türkiye ranks among the top 10 countries in the world in terms of both area and production volume, producing 3.8 million tonnes of grapes on an area of 340.000 hectares¹. Türkiye’s viticultural history spans millennia, and the country is widely regarded as the grape’s ancestral home. It is evident that the country has experienced a considerable degree of cultivation over centuries, and consequently, it is now characterized by a rich genetic diversity. The cultivation of grapes is widespread throughout Türkiye, and the fruit is consumed in various forms. While they are primarily valued for salads, drying, and winemaking, traditional consumption methods are also widely practiced in Türkiye². The consumption of grape leaves has been a practice for many years, both in their fresh state and in the form of pickles. In Türkiye, stuffed grape leaves made with pickled leaves are considered a traditional dish. Gürkan³ demonstrates that analogous culinary preparations are also favored in Greece, Albania, Bulgaria, France, and Spain.

In recent years, there has been an increased focus on healthy living, leading to a greater emphasis on plants rich in phenolic compounds and antioxidants. These secondary metabolites are found in various parts of plants and perform different functions within them. They have been demonstrated to play a role in the plant’s defense mechanisms and to possess protective properties against various stress conditions, such as UV radiation, pathogens, and diseases^4,5. Phenolic compounds have a cultivar of applications in the study of various characteristics of plants, including bitterness, astringency, aroma, the coloration of flowers and fruits^6–8 taxonomic differentiation^9,10 growth and development¹¹ rooting¹²and graft incompatibility mechanisms^13,14. The properties above include antioxidant, antimutagenic, anticarcinogenic, anti-aging, and antimicrobial characteristics^15–17.

Grapes are among the most notable fruit types in terms of their phenolic compound content. The substantial secondary metabolite content of the grape plant is a salient feature that distinguishes it from numerous other plants. Resveratrol, a polyphenol found in grapes, is also available in tablet form. Research has indicated the presence of phenolic compounds in various parts of the grapevine, including leaves, shoots, fruit flesh, peel, and seeds^18,19–21. Consequently, identifying grapevine cultivars with elevated phenolic compound content and the subsequent production of extracts for utilization in pharmaceuticals, food supplements, and cosmetics is significant from both health and economic standpoints. The initial studies on determining the phenolic profile of grapevines focused primarily on wine, seeds, seed husks, and pulp. However, in recent years, there has been an increase in studies on grapevine leaves. Scientific studies have demonstrated that grape leaves are abundant in phenolic acids, tannins, anthocyanins, and flavonoids^5,21–23. The use of grape leaves in traditional medicine to treat various diseases has a long history, as evidenced by numerous studies^24,16. It is well established that grape leaves are endowed with a plethora of pharmacological properties, including but not limited to antioxidant, antidiabetic, liver-protective, cholesterol-lowering, antibacterial, anti-ageing, antiviral, anti-carcinogenic, collagen-preserving, hair-growth-promoting, antifungal, anti-inflammatory, and cell damage prevention properties^16,26,27,27. Over the past two decades, the potential of grape leaves in terms of phenol provision has attracted the attention of numerous pharmaceutical companies, which have invested significant economic resources in research to identify a safe and effective source of phenols.

The use of by-products generated during the preparation of grapes for the market, both fresh and processed, is quite limited in Türkiye. Vines are pruned during the growing season to obtain higher quality products, and the prunings are either left in the vineyard or used for animal feed. After harvest, the leaves are left on the vines. At the end of the season, the leaves are mixed into the soil as organic fertilizer. It is evident that grapevine leaves are not sufficiently utilized and are largely considered waste. Their use as a natural source of bioactive compounds could increase their economic value. Therefore, determining the phenolic compounds in grapevine leaves during the post-harvest period could open up opportunities for their utilization in the food, pharmaceutical, and cosmetic industries. Additionally, the potential therapeutic applications of grape leaves, along with their use as a functional food, signify the re-evaluation of this product within the framework of sustainable agricultural development²⁸. Grape leaves are rich in minerals, vitamins, and phenolic compounds. Studies^{5,22,23,29,30} on the phenolic content of grape leaves have been conducted; however, the composition and concentration of these compounds are influenced by various factors such as genotype and environmental conditions^31,33,33. Therefore, determining the phenolic profiles of grape leaves using different varieties in different ecologies is important for waste management and human health.

This study aimed to ascertain the phenolic compound content of leaves from four autochthonous grape cultivars cultivated in vineyards in Isparta, which is located in the Mediterranean Region of Türkiye. This was undertaken to identify their potential as a valuable resource for the food and pharmaceutical industries and to facilitate their re-evaluation. Thus, while preserving grapevine genetic resources, the phenolic profiles of leaves collected from four red and white grapevine varieties after harvest were determined as a source of bioactive compounds in grapevine leaves.

Materials and methods

Plant material

The present study was conducted in 2018 in vineyards in the province of Isparta, in the Mediterranean Region of Türkiye. Leaves from four grapevine cultivars were utilized as materials in the study: two colored cultivars (Vitis vinifera cv. Burdur Dimriti, Vitis vinifera cv. Siyah Gemre) and two white cultivars (Vitis vinifera cv. Razaki and Vitis vinifera cv. Tilki Kuyruğu). The vines of the cultivars had been cultivated for 20 years and had grown on their roots. The Goble training system was implemented in the vineyards. As the available climate data demonstrated, the total precipitation in 2018 was 399.8 mm, which is lower than the long-term average of 543.6 mm. Concurrently, the average temperature was recorded as 13.8 °C, higher than the long-term average of 12.2 °C. The monthly precipitation and temperature data are presented in Fig. 1.

Fig. 1.

Monthly precipitation and temperature data for the test area.

The soil in the designated trial area is characterized by a predominantly clay-textured composition, exhibiting a neutral reaction with a pH value of 7.01. Electrical conductivity measurements indicate a low level of conductivity (0.09 dS/m), suggesting conditions that are not saline. The calcium carbonate content was found to be 2.96%, and the organic matter content was found to be 1.01%. Nutrient analysis indicates the presence of phosphorus at a high level (41 ppm), while potassium is present at a low level (68.7 ppm). The analysis revealed that calcium (1960 ppm) and magnesium (195.3 ppm) were found at moderate levels.

A total of 15 grapevine leaf samples were collected in Isparta vineyard area (Central Isparta, 37,81026° N – 30,51397″ E; elevation 1100 m a. s. l). Healthy, green grapevine leaves were collected from five individual plants of each cultivar during the whole ripening stage of the grape. It is imperative to emphasize that meticulous attention was devoted to consistently ensuring that the leaves were positioned and orientated. Mature and healthy, south-facing, sun-exposed leaves from the 6th − 12th node were collected in September 2018. After collection, the leaves were placed in ice boxes to prevent water loss and kept in ice boxes until they reached the laboratory. The leaves transferred to the laboratory were stored in a deep freezer at −18 °C until the analysis period for phenolic compound analysis.

The ‘Burdur Dimriti’ grape cultivar utilized in the present study is a table grape cultivar distinguished by a red-purple coloration, a medium berry size (3.8 g), a round shape, and the presence of seeds. The buds open in the first and second weeks of April, and floering occurs in the second week of June. The ‘Burdur Dimriti’ grape cultivar, which reaches maturity in late July, continues to ripen through late August and September. The ‘Siyah Gemre’ grape cultivar is a table grape characterized by its dark purple coloration, medium size (7.8 g), round shape and seed-bearing nature. The buds open in the second week of April, and flowering occurs in the second week of June. The ‘Siyah Gemre’ grape cultivar, which reaches maturity in the second week of August, continues to ripen until the end of September. The ‘Tilki Kuyruğu’ cultivar is a table grape characterized by its yellow-green color, medium size (4.7 g), cylindrical shape, and the presence of seeds. The buds open in the third week of April, and flowering occurs in the second week of June. The ‘Tilki Kuyruğu’ grape cultivar, which reaches maturity in the third week of August, is ready for harvest in early October. The ‘Razaki’ grape cultivar is a table grape characterized by its light yellow-green color, medium size (5.5 g), elongated elliptical shape, and seeds. The buds open in the third week of April, and flowering occurs in the second week of June. The ‘Razaki’ grape cultivar, which reaches maturity during the last week of August, continues to ripen until the end of September or the beginning of October³⁴.

Chemicals

Standards (HPLC-grade, ≥ 98%) of quercetin (QUE), kaempferol (Ka), rutin (RUT), catechin (C), epicatechin (EC), vanillin (VA), Lutein (L), Hesperidin (HD) and phenolic acids (gallic (GA), protocatechuic (PrA), chlorogenic acid (CGA), p-Coumaric (PCA), o-Coumaric acid (OCA), Ferulic acid (FA), Caffeic acid (CA), Tannic acid (TA)) were purchased from Sigma-Aldrich (Steinheim, Germany). Dimethylamino cinnamaldehyde (DMAC) was obtained Merck (Darmstadt, Germany). Folin-Ciocâlteu reagent, acetic acid (MS grade), methanol (HPLC grade), sodium carbonate, were obtained from Merck (Darmstadt, Germany). Standard solutions and dilutions were prepared using ultrapure water (TKA Germany MicroPure water purification system, 0.055 µS/cm). Syringe filters (13 mm, PTFE membrane 0.45 μm) were purchased from Supelco (Bellefonte, PA, USA).

Preparation of grapevine leaf extracts for phenolic analyses

Fresh leaves were utilized for the extraction of phenolic compounds. To achieve this objective, 1 g of leaf samples were collected and homogenized with 70% methanol containing 0.1% HCl for one minute. The samples were then extracted in an ultrasonic water bath for 30 min on each occasion (at room temperature, in shade). After filtration, the extracts were employed in the analysis of phenolic compounds³⁵.

Determination of total phenolic content (TPC)

The total phenolic compound content was determined using the Folin- Ciocâlteu colorimetric method as described by Singleton and Rossi³⁶. The reduction of the Folin– Ciocâlteu reagent by phenolic compounds under alkaline conditions, which resulted in the development of a blue color. After adding distilled water and Folin- Ciocâlteu to the leaf samples extracted in methanol, the mixture was thoroughly stirred, and saturated Na₂CO₃ was added after 30 s. The samples were mixed well again and kept in the dark for 2 h under room conditions, then readings were made on a spectrophotometer at a wavelength of 765 nm (UV–vis model 1601, Shimadzu, Kyoto, Japan). The amount of total phenolic was calculated with the use of a calibration curve made from catechin standard and expressed as mg catechin g⁻¹ FW equivalents.

Determination of total flavanol content (TFC)

The total flavanol content was determined by the DMAC (dimethylamino cinnamaldehyde) method, as described by Arnous et al.³⁷. Spectrophotometric readings were obtained at a wavelength of 640 nm. For this experiment, a DMAC solution was prepared and subsequently added to the leaf extracts. Subsequently, the mixture was subjected to rigorous mixing, after which readings were taken following a period of 10 min. The total flavanol content was determined as catechin equivalents in milligrams per gram (mg CE·g⁻¹) of FW.

HPLC determination of phenolic compounds

The phenolic compound analysis was conducted using HPLC DAD (Agilent Tech. Inc., CA, USA), as regards the methodology established by Caponio.³⁸ The reversed phase (RP)-HPLC analysis was done using a SCL-10Avp system controller, a SIL-10AD vp autosampler, a LC-10AD vp pump, a DGU-14a degasser, a CTO-10 A vp column heater, and a Diode Array Detector (DAD) with wavelengths of 278 nm. The 250 mm × 4.6 mm i.d. 5 μm column which is filled with Agilent Eclipse XDB-C18 was used for the analyses in the HPLC system. The flow rate was 0.8 ml/min, the injection volume was 20 µL, and the column temperature was set at 30 °C. For gradient elution, as a mobile phase, solvent A contained 3% acetic acid in water and solvent B contained methanol (99%) (analytical grade). The data were integrated and analyzed using the Shimadzu Class-VP Chromatography Laboratory Automated Software system. Extract samples, standard solutions and mobile phases were filtered using a 0.45 μm pore size membrane filter (Vivascience AG, Hannover, Germany). The following gradient programme was used: 0–3 min, from 100% A to 93% A, 7% B; 3–20 min, from 93% A, 7% B to 72% A, 28% B; 20–28 min, from 72% A, 28% B to 75% A, 25% B; 28–35 min, from 75% A, 25% B to 70% A, 30% B; 35–60 min 70% A, 30% B to 67% A, 33% B; 60–62 min, 67% A, 33% B to 58% A, 42% B; 62–70 min, 58% A, 42% B to 50% A, 50% B; 70–75 min, 50% A, 50% B to 20% A, 80% B; 75–80 min, 100% B. The amount of phenolic compounds in the leaf samples was calculated as µg·g⁻¹ (fresh weight), using external calibration curves obtained for each phenolic standard. Ferulic acid, gallic acid, caffeic acid, chlorogenic acid, epicatechin, rutin, p-Coumaric acid, o-Coumaric acid, protocatechuic acid, catechin, quercetin, hesperidin, vanillin, kaempferol, luteolin, and tannic acid were determined in the samples. Quantitative analyses of the detected phenolic compounds were performed in triplicate and the results were average. UV chromatogram of 4 grape cultivar is shown in Fig. 2.

Fig. 2.

Chromatograms of four grapevine cultivars (1:gallic acid, 2:catechin, 3:protocatechuic acid, 4:caffeic acid, 5:epicatechin, 6:vanillin, 7:o-coumaric acid, 8:p-coumaric acid, 9:ferulic acid, 10:rutin, 11:chlorogenic acid, 12:hesperidin, 13:tannic acid, 14:quercetin, 15:kaempferol, 16:luteolin).

Statistical analysis

The data obtained in the study were then subjected to variance analysis by the randomised block design. The statistical test known as Duncan’s was utilized to ascertain the disparities in mean values. Pearson correlation analysis, principal component analysis (PCA) and heatmap analysis were performed using the RStudio V4.3.1 program (RStudio Team, 2023; R Core Team, 2023, Vienna, Austria). The ‘devtools’, ‘RColorBrewer’ and ‘pheatmap’ packages were used for the heatmap analysis; the ‘metan’ package was used for the correlation analysis; and the ‘FactoMineR’, ‘factoextra’ and ‘pca3d’ packages were used for the principal component analysis.

Results and discussion

In this study conducted to determine the phenolic compounds of grape leaves from certain grape cultivars grown in Isparta, Türkiye, total phenolic, total flavanol, and phenolic profiles were determined in grape leaves collected during the post-harvest period.

Total phenolic content and total flavanol content

Significant differences were identified between cultivars in total phenolic content in the study (p <.05). The results of the analyses demonstrated that the total phenolic compound content ranged from 3.25 to 7.38 mg CE·g⁻¹FW (Fig. 3). An analysis of the data concerning the total phenolic compound content of the leaves of the two colored and two white grape cultivars employed in the study demonstrated that the highest total phenolic compound content was obtained from the white grape cultivars ‘Tilki Kuyruğu’ and ‘Razaki’. The present study found that the concentration of total phenolic compounds in the sample was consistent with the data previously reported on whole leaves^5,22,39. Lima et al.²² reported that white grape cultivars exhibited higher total phenolic content in their leaves. This finding emerged from a comprehensive study that examined the antioxidant levels and phytochemical composition of leaves from 10 distinct grape cultivars, of which six were white and four were red. White and red grapevine cultivars showed values between 112 and 150 mg GAE·g⁻¹ of total phenols. The total phenol content of white cultivars is marginally higher than that of red cultivars (6.03 mg GAE·g⁻¹ and 7.38 mg GAE·g⁻¹, respectively). This outcome has been corroborated in other studies that have compared white and red grapevine leaf cultivars in terms of their phenolic content^28,30. When looking at total phenolic concentrations, it can be seen that researchers have detected varying concentrations of total phenolics, unlike our study. Indeed, Hallaç Türk³⁹ found the total phenolic content of 6 different grape varieties to be 5.88–10.57 mg CE·g⁻¹ DW, while Dani et al.²⁵ reported 20.2 and 19 mg GAE·ml⁻¹ in Brazilian grape varieties. Additionally, Pantelic et al.⁴⁰ determined that the total phenolic content of grape leaves ranged from 27.5 to 76.0 g GAE·kg⁻¹ DW. Yıldız et al.²³ found that the total phenolic content of 17 Vitis vinifera L. leaves obtained from vineyards in the northeastern region of Türkiye ranged from 172.66 to 483.33 µg GAE·mg⁻¹ DW. When the results are compared with literature values, it is possible that the observed differences are due to cultivar, cultivation conditions and climatic factors. Researchers reported that polyphenol accumulation in grapevine leaves depends on genotype, sunlight, geographical location of the vineyard and seasonal climate^31,32,41.

Fig. 3.

Total phenolic content (TPC) determined in the leaves of four grapevine cultivars.

When the data were evaluated in terms of total flavanol content, statistically significant differences were found between cultivars (p <.05). The total flavanol content in the leaves of the cultivars utilized in the study ranged from 67.66 to 120.24 mg CE·g⁻¹ (Fig. 4). The highest total flavanol content was determined in the leaves of the ‘Razaki’ grape cultivar. In contrast, the lowest total flavanol content was found in the leaves of the ‘Burdur Dimriti’ grape cultivar. Similarly, Katalinic et al.⁴² reported that the total flavanol content in the leaves of six cultivars collected in September ranged from 1100 to 2746 mg·l⁻¹ as epicatechin equivalents. In the study, the total flavanol content of leaves from white grapes was found to be high, similar to the total phenolic content, in leaves from red grapes.

Fig. 4.

Total flavanol content (TFC) determined in the leaves of four grapevine cultivars.

Phenolic profile

The total amount of 16 polyphenols was determined using existing standards for grape leaf extracts. The phenolic acids and their derivatives, flavonols and flavanol-3 contents of the 4 autochthonous grapevine leaves are reported in Table 1. Other studies have reported a similar distribution of these phenolic classes in grapevine^25,43. The results showed some variations depending on grape cultivars. There were observed statistically significant differences among the amounts of phenolic compounds depending on grape varieties (p <.05).

Table 1.

Phenolic compounds identified in the leaves from four grape cultivars (µg·g⁻¹).

Cultivars	FA	GA	CA	CGA	C	Q	EC	RUT
Burdur Dimriti	0.31 ± 0.01 c	0.77 ± 0.02 d	8.96 ± 0.19 c	40.67 ± 0.87 b	405.8 ± 8.67 b	27.93 ± 0.60 b	121.2 ± 2.58 a	203.9 ± 4.37 b
Siyah Gemre	0.33 ± 0.01 c	6.10 ± 0.23 a	8.35 ± 0.32 c	80.47 ± 3.08 a	549.2 ± 21.00 a	24.61 ± 0.47 c	90.00 ± 2.93 b	103.9 ± 4.00 c
Tilki Kuyruğu	1.60 ± 0.02 a	2.37 ± 0.03 b	44.93 ± 0.65 a	30.54 ± 0.44 b	102.9 ± 1.48 c	27.73 ± 0.40 c	75.80 ± 1.07 c	192.6 ± 2.89 b
Razaki	0.47 ± 0.01 b	1.87 ± 0.06 c	13.33 ± 0.41 b	35.13 ± 1.08 b	99.80 ± 3.08 c	30.57 ± 0.97 a	74.27 ± 1.89 c	386.7 ± 11.86 a

Cultivars	PCA	OCA	PrCA	HD	VA	Ka	L	TA
Burdur Dimriti	1.10 ± 0.02 b	965.00 ± 20.62 d	3.21 ± 0.07 c	5.20 ± 0.11 d	0.98 ± 0.02 c	12.30 ± 0.27 c	3.20 ± 0.07 d	0.91 ± 0.02 b
Siyah Gemre	0.74 ± 0.03 c	1017.0 ± 39.47 c	3.96 ± 0.15 c	6.10 ± 0.23 c	2.25 ± 0.09 a	11.26 ± 0.43 d	4.30 ± 0.13 c	0.97 ± 0.03 b
Tilki Kuyruğu	1.35 ± 0.02 a	1215.0 ± 17.50 a	5.13 ± 0.07 b	8.90 ± 0.13 a	1.18 ± 0.02 c	13.05 ± 0.19 b	6.90 ± 0.10 a	1.18 ± 0.02 a
Razaki	0.73 ± 0.02 c	1167.0 ± 35.92 b	6.21 ± 0.19 a	7.10 ± 0.22 b	1.76 ± 0.05 b	14.88 ± 0.46 a	5.76 ± 0.18 b	0.94 ± 0.03 b

FA: ferulic acid, GA: gallic acid, CA: caffeic acid, CGA: chlorogenic acid, C: catechin, Q: quercetin, EC: epicatechin, RUT: rutin, PCA: p-Coumaric acid, OCA: o-Coumaric acid, PrCA: protocatechuic acid, HD: hesperidin, VA: vanillin, Ka: kaempferol, L: luteolin, TA: tannic acid. Means with different letters in the same column were significantly different (p <.05).

Phenolic acids commonly found in grapes and wine generally describe the phenolic compounds having one carboxylic acid group. Phenolic acids are one of the main classes of plant phenolic compounds⁴⁴. Phenolic acids are the most prominent group of bioactive compounds present in various plant sources. Hydroxybenzoic acids and hydroxycinnamic acids, the aromatic secondary metabolites imparting typical organoleptic characteristics to food are the major phenolic acids, and they are linked to several health benefits⁴⁵. Phenolic acids (hydroxycinnamic and hydroxybenzoic acid derivatives) were found ferulic acid, gallic acid, caffeic acid, p-Coumaric acid, o-Coumaric acid, chlorogenic acid, protocatechuic acid and tannic acid in this study. The study revealed the following phenolic acids concentration: ferulic acid (0.31–1.60 µg·g⁻¹), gallic acid (0.77–6.1 µg·g⁻¹), caffeic acid (8.35–44.93 µg·g⁻¹), (30.54–80.47 µg·g⁻¹), p-Coumaric acid (0.73–1.35 µg·g⁻¹), o-Coumaric acid (965–1215 µg·g⁻¹), protocatechuic acid (3.21–6.21 µg·g⁻¹) and tannic acid (0.98–1.18 µg·g⁻¹) (Table 1). There were observed significant fluctuations among the amounts of phenolic acids of grapevine leaves depending on cultivars (p <.05). Gallic acid was found in high amounts (6.10 µg·g⁻¹) in the leaves of the red cultivar ‘Siyah Gemre’ compared to other varieties (p <.05). The leaves of the white cultivar ‘Tilki Kuyruğu’ contain caffeic acid (44.93 µg·g⁻¹), ferulic acid (1.60 µg·g⁻¹), p- Coumaric acid (1.35 µg·g⁻¹), o- Coumaric acid (1215 µg·g⁻¹) and tannic acid (1.18 µg·g⁻¹) were found to be higher than all other varieties (p <.05).

The white cultivar ‘Razaki’ was superior to all other cultivars only in terms of protocatechuic acid content (p <.05). In general, leaves belonging to white cultivars were found to be richer in phenolic acids except gallic acid than red leaves. Similarly, Lima et al.²² reported that hydroxycinnamic derivatives (caffeic acid, ferulic acid, p-Coumaric acid, o-Coumaric acid) in leaves belonging to white cultivars ranged from 39 mg CAE·g⁻¹ to 54 mg CAE·g⁻¹ and were higher than those in red-colored ones. Pantelic et al.⁴⁰ reported that the levels of gallic and protocatechuic acid were low in the leaves of 22 red and white grape cultivars, while ferulic acid (89.8 mg·kg⁻¹ DW in ‘Petra’) was the most abundant hydroxycinnamic acid in a white cultivar. Similar studies support our findings.

Significant differences were found in the flavonols contents of grapevine cultivars (p <.05). Grapevine leaves contained quercetin (24.61–30.57 µg·g⁻¹), kaempferol (11.26–14.88 µg·g⁻¹), hesperidin (21 µg·g⁻¹), rutin (103.9–386. 7 µg·g⁻¹), and luteolin (3.21–5.76 µg·g⁻¹) (Table 1). In previous studies, quercetin and kaempferol are the main leaf flavonoids found in Vitis vinifera species^46,47 while Dani et al.²⁵ reported that rutin showed the highest concentration in grapevine leaves, followed by quercetin and kaempferol. Andelkovic et al.⁴³ reported that the most abundant flavonol in grapevine leaf was quercetin glucoside, followed by rutin, kaempferol glucoside, quercetin, luteolin-glucoside, quercetin galactoside, and myricetin glucoside. So, our research also supports this knowledge. The white grape varieties ‘Razaki’ and ‘Tilki Kuyruğu’ have high concentrations of flavanols. The highest levels of quercetin (30.57 µg·g⁻¹), kaempferol (14.88 µg·g⁻¹), and rutin (386.7 µg·g⁻¹) were detected in the leaves of the ‘Razaki’ cultivar, followed by the ‘Tilki Kuyruğu’ cultivar.

Among the other flavonols detected, hesperidin and rutin were found in the highest concentrations in the leaves of the ‘Tilki Kuyruğu’ grapevine cultivar. All cultivars were rich in rutin (content was up to 386.7 µg·g⁻¹in ‘Razaki’), which is in accordance with the data previously reported^40,48.

The presence of flavan-3-ols (catechin, epicatechin) was detected in the leaves of four different grape varieties, and their amounts were found to vary among varieties (p <.05). The flavan-3-ols content was higher in red grapevine leaves than in white grapevine leaves of ‘Burdur Dimriti’ and ‘Siyah Gemre’. The most abundant flavan-3-ol was catechin, followed by epicatechin (Table 1), consistent with the results of another study⁴⁷. Catechin varied between 99.8 and 549.2 µg·g⁻¹ and epicatechin between 74.27 and 121.2 µg·g⁻¹ in grape leaves. Leaves from red-colored grapes were found to be richer in both flavan-3-ols than those from white-colored grapes. The highest catechin levels were found in leaves of the “Siyah Gemre” cultivar, while the highest epicatechin levels were detected in leaves of the “Burdur Dimriti” cultivar.

The results of this study, which determined the total phenolic, total flavanol, and phenolic profiles of leaves belonging to four different autochthonous grapevine cultivars, are consistent with previous studies. As demonstrated in previous studies^22,40,49,50the present study’s findings are from preceding research. The results indicate that the grapevine cultivars exhibit varying richness levels in different leaf phenolic compounds.

Maia et al.⁵⁰ mentioned the presence of bioactive compounds, including caffeic acid, quercetin, resveratrol, kaempferol, and catechin, in ‘Pinot Noir’ grape leaves. The authors further reported that such leaves could be used as a source of bioactive compounds for both human and animal nutrition. Pintac et al.⁴⁹ identified 28 phenolic compounds in four red and four white grape cultivars collected post-harvest. The quantity of these phenolic compounds was found to vary depending on the grapevine cultivar. Furthermore, it was reported that the phenolic compounds present in the highest amounts in the leaves were flavonoids, ellagic acid, and chlorogenic acid. In a separate study undertaken by Pantelic et al.⁴⁰it was ascertained that the leaves of 22 distinct grape cultivars cultivated in Serbian vineyards exhibited a high concentration of phenolic acids, flavonols, and flavan-3-ols. Fernandes et al.²⁸ reported that leaves from 20 different grape cultivars, including 10 white and 10 red cultivars, grown in Portugal contained trans-caftaric, trans-Coumaryl tartaric acid, myricetin-3-O-glucoside, quercetin-3-O-galactoside, and kaempferol-3-O-glucoside, with the last two compounds being present in the highest amounts.

The observed differences in the studies are hypothesized to be attributable to diversity, geographical location, climatic conditions, location, harvest time, climate, ecological factors, soil structure cultural activities and stress conditions^30,31,41.

Heat map analysis

Another aim of this study is to investigate the relationship between individual polyphenols using heat maps, PCA values, and Pearson correlation analysis. The heat map, created using the phenolic compound data obtained in the study, revealed significant phenolic differences between the four grapevine cultivars (Fig. 5). ‘Tilki Kuyruğu’ exhibited a distinctive profile, characterized by elevated concentrations of numerous phenolic compounds, including ferulic acid, caffeic acid, p-Coumaric acid, tannic acid hesperidin and luteolin. Conversely, ‘Razaki’ has garnered attention due to its high concentrations of flavonoid compounds, including rutin, quercetin, kaempferol, and protocatechuic acid. Furthermore, both white grape cultivars exhibited elevated levels of total phenolic and flavanol content, a finding corroborated by visual analysis⁵¹. In contrast, ‘Siyah Gemre’ exhibited moderate levels of certain phenolic acids (gallic acid, chlorogenic acid) and flavan-3 ols (catechin), while ‘Burdur Dimriti’ generally showed low content, except for epicatechin, which was present at high levels. The results of the vertical clustering indicate that the phenolic profiles of ‘Tilki Kuyruğu’ and ‘Razaki’ are similar to each other, while ‘Burdur Dimriti’ is distinctly different. In the horizontal dendrogram, compounds such as chlorogenic acid, gallic acid, and vanillin were found to be concentrated in similar cultivars, indicating that these compounds act together. This approach is consistent with studies suggesting that phenolic compounds should be evaluated through clustering⁵². The application of a heat map facilitates comparative evaluation of the cultivars in terms of their industrial use potential, with the results highlighting that the unprocessed leaves of ‘Tilki Kuyruğu’ and ‘Razaki’ could be an important resource for the development of functional products.

Fig. 5.

Heat map showing the concentrations of phenolic compounds in leaves from four different grape cultivars. VA: vanillin, GA: gallic acid, CGA: chlorogenic acid, C: catechin, EC: epicatechin, Q: quercetin, RUT: rutin, Ka: kaempferol, TF: Total flavanol, PrCA: protocatechuic acid, TPC: Total phenolic content, HD: hesperidin, OCA: o-Coumaric acid, L: luteolin, PCA: p-Coumaric acid, TA: tannic acid, FA: ferulic acid, CA: caffeic acid.

Principal component analysis (PCA)

In this study, PCA (Principal Component Analysis) was employed to elucidate the variations of phenolic compounds that were the subject of evaluation (Fig. 6). The application of PCA also enabled the visualization of the relationships between the various cultivars. The initial two components (Dim1 and Dim2) account for approximately 80% of the total variance, suggesting that the phenolic variables employed in the analysis are adequate for differentiating between cultivars. The direction and distance relationships between cultivars and phenolic compounds are clearly visible in the biplot graph. ‘Tilki Kuyruğu’ is located in the upper right quadrant and shows strong positive relationships with ferulic acid, caffeic acid, p-Coumaric acid, tannic acid, hesperidin, and total phenolic content. This finding substantiates the hypothesis that this cultivar possesses a high degree of phenolic content and that the related compounds within this cultivar undergo aggregation. ‘Razaki’ is located in the lower right region and is distinguished by its high flavonoid content, which is significantly correlated with flavonoid-characterized compounds, including quercetin, rutin, kaempferol, protocatechuic acid, and total flavanol. PCA has heretofore been utilized to characterize phenolic compounds in grape varieties, with demonstrable efficacy documented^40,41. Conversely, the ‘Siyah Gemre’ and ‘Burdur Dimriti’ cultivars have been observed to be located in areas exhibiting a negative charge and have been found to demonstrate weak or inverse relationships with compounds such as chlorogenic acid gallic acid and catechin. This finding suggests that these two cultivars possess reduced overall phenolic levels and divergent profiles in terms of compounds. When PCA results are evaluated in conjunction with heat maps and chemical analyses, ‘Tilki Kuyruğu’ and ‘Razaki’ are found to have a higher potential for functional product development.

Fig. 6.

PCA (Principal Component Analysis) biplot graph of varieties according to phenolic compounds. VA: vanillin, GA: gallic acid, CGA: chlorogenic acid, C: catechin, EC: epicatechin, Q: quercetin, RUT: rutin, Ka: kaempferol, TF: Total flavanol, PrCA: protocatechuic acid, TPC: Total phenolic content, HD: hesperidin, OCA: o-Coumaric acid, L: luteolin, PCA: p-Coumaric acid, TA: tannic acid, FA: ferulic acid, CA: caffeic acid.

Pearson’s correlation analysis

Pearson’s correlation analysis of the phenolic compounds identified in the leaf samples revealed that some compounds exhibited strong and significant correlations with each other (Fig. 7). Notably, a very high positive correlation was observed between ferulic acid and tannic acid (r =.98; p o-Coumaric < 0.001). Similarly, high positive correlations were observed between ferulic acid, caffeic acid, p-Coumaric acid, luteolin, hesperidin and total phenolic content. These findings support the presence of these compounds at high concentrations in the ‘Tilki Kuyruğu’ grapevine cultivar. This is consistent with literature investigating the structural and functional relationships of phenolic compounds^40,41,43. Conversely, negative correlations were found between total phenolic content and certain compounds, such as epicatechin, catechin, gallic acid, and chlorogenic acid. These compounds were found to be concentrated in cultivars with lower total phenolic content. Negative correlations indicate that certain compounds dominate different cultivars and can influence total content in different directions. Positive correlations between flavonoid compounds and total flavanols, for example, are consistent with the literature on flavonoid-rich cultivars such as ‘Razaki’⁵². Furthermore, positive correlations between quercetin, rutin, kaempferol, and total flavanols corroborate the flavonoid richness of the ‘Razaki’ grapevine cultivar. Overall, correlation analysis has provided important insights into phenolic compound patterns among cultivars and into the identification of compounds that exhibit common variation.

Fig. 7.

Pearson correlation matrix showing the relationships between phenolic compounds detected in grape leaves. The color scale represents the correlation coefficient, with red tones indicating a positive correlation and green tones indicating a negative correlation. ***, ** and * indicate p <.001, p <.01, and p <.05 levels, respectively.

Conclusion

Grapevines produce a large amount of waste every year. The wine industry considers the reuse of leaves left to dry on the vine after pruning and harvesting to be valuable for adding value to by-products. The potential use of grape leaves in supplements supporting healthy living necessitates their identification across different regions and grape cultivars. In this study, the total phenolic, total flavanol, and phenolic profile content and composition of leaves from four different autochthonous grape cultivars (2 white, 2 red) collected from a vineyard in Isparta, Türkiye, were determined for the first time. The results showed that the leaves of 4 different autochthonous grape cultivars are rich in phenolic compounds. The evaluation of the study’s findings indicated that the levels of total phenolic, total flavanol, and phenolic compounds varied according to the cultivars. Leaves belonging to white cultivars had higher antioxidant activity than those belonging to red cultivars due to their phenolic content. The ‘Razaki’ cultivar, rich in flavonoids, and the ‘Tilki Kuyruğu’ cultivar are varieties rich in phenolic acids and total phenolic compounds.

Grape leaves have shown high potential as a source of bioactive compounds. The finding that grape leaves are particularly rich in phenolic compounds indicates their potential as a natural antioxidant source in the pharmaceutical and food industries such as nutraceuticals. In this regard, the study is significant for identifying the differences between cultivars and evaluating autochthonous grapevine cultivars that have lost their original value due to urbanization, which has led to their disappearance. Moreover, the study has emphasized the potential for utilizing leaves devoid of any post-harvest value. The findings of the study indicated that the leaves of the grape cultivars ‘Tilki Kuyruğu’ and ‘Razaki’ possess considerable potential for utilization as functional components within the food industry, pharmaceuticals, and cosmetics.

Author contributions

F.H.T. designed and planned the experiments. F.H.T. and A.A.U. collected the data, F.H.T. and A.A.U. performed the laboratory analyses, F.H.T. performed the statistical analysis, and wrote the manuscript. F.H.T. edited the manuscript. All authors reviewed and approved the final version of the manuscript.

This article was not supported by any funding.

Data availability

Data will be made available on request. Contacts: [filizhallac@isparta.edu.tr](mailto: filizhallac@isparta.edu.tr) (Filiz HALLAC TURK).

Declarations

Competing interests

The authors declare no competing interests.