Phytochemical Content, Antioxidant Properties, and Phenolic Profile of Tunisian Raisin Varieties (Vitis Vinifera L.)

Abstract

Four raisin (Vitis vinifera L.) varieties, Chriha, Razeki, Assli, and Meski, were evaluated for total phenolic content, total o-diphenol content, total flavonoid content, total condensed tannin, total carotenoid content, and total anthocyanin content. Antioxidant potential was assessed by three assays: 2,2-Diphenyl-1-Picrylhydrazyl and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid radical scavenging capacity and ferric reducing power. Individual phenolic profiles were determined by high-performance liquid chromatography. The results revealed that the four raisin varieties had considerable phenolic content and antioxidant activity. Chriha had the highest total phenolic content (534.2 mg/g dry weight) while Meski had high total condensed tannin (208.6 mg CEQ/g dry weight), TAC (137 mg/100 g dry weight), total o-diphenol content (115.8 mg/g dry weight), total flavonoid content (93 mg CEQ/g dry weight), and total carotenoid content (33 mg/100 g dry weight). There were significant differences in phenolic content among the four varieties (p < 0.05). Meski had the highest 2,2-Diphenyl-1-Picrylhydrazyl scavenging capacity, while Chriha had adequate reducing power and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid scavenging capacity. The individual phenolic compounds (2.96–6.54 mg/g dry weight) were variety-dependent.

Keywords:

• Raisins
• Phytochemical content
• Antioxidant activity
• Phenolic profile

INTRODUCTION

In biological systems, oxidative damage results from an imbalance in free radicals and antioxidants.^[1,^2] When oxidative damage is cumulative it contributes to oxidative stress,^[3] which leads to cell membrane disintegration, protein damage, and DNA mutation,^[4] which can further initiate or propagate ageing^[5] and several diseases, such as neurodegenerative disorders, cancer, liver cirrhosis, cardiovascular diseases, atherosclerosis, cataracts, diabetes, and inflammation.^[6–8] Phenolic compounds,^[9] such as phenols, phenolic acids, flavonoids, tannins, and anthocyanins, have received considerable attention for their high antioxidant activity.^[9,^10] Phenolic compounds are free radical scavengers because they are nucleophiles that inhibit lipid peroxidation and chelators of metal ions that induce oxidation.^[11]

Antioxidant activity and polyphenol content in dried fruits are high due to their low moisture content.^[12] Dried fruits have been studied by many researchers.^[13–15] Raisins (Vitis vinifera L.) are obtained by dehydrating grapes, usually with sun exposure or tunnel drying.^[16–18] Solar drying is the most ancient and most widely used method in Tunisia. In solar drying, food samples are submerged in hot water (87–93°C) and dried under direct sunlight for 2–3 weeks.^[19]

Even though raisins are widely known for their nutritional properties, only the chemical composition and health benefits of grapes and vines have been studied in Tunisia.^[20–22] The objective of this study was to determine the phenolic profile and antioxidant activities of four varieties of raisins grown in Tunisia.

MATERIALS AND METHODS

Plant Material

Dried raisins used in these experiments consisted of four varieties (Chriha, Raseki, Assli, and Meski). They were procured in August–mid September 2013 season from different regions of Tunisia: Chriha and Meski from Rafraf (located in the region of Bizerte; latitude: 37° 16′N, longitude: 9° 52′E), Raseki from Tunis (latitude: 36° 48′ N, longitude: 10° 11′ E) and Assli from Sfax (latitude: 34° 44′N, longitude: 10° 46′E). Specimens have been deposited in the Herbarium of the Laboratory of Biochemistry and immediately frozen at –80°C until analysis.

Chemicals and Reagents

All chemical, standards including phenolic acids and reagents used were purchased from Sigma–Aldrich Co. Ltd (St. Louis, MO. USA).

Extraction Method

Frozen raisins were mixed and homogenized. The resulting powder was freeze-dried and stored protected from light under vacuum. A sample of the ground powder of each raisin variety (5 g) was extracted with 50 mL of pure methanol at room temperature (25°C) for 24 h using an orbital shaker. Extractions were done in triplicate. The extracts were then filtered through a Whatman No. 4 filter paper and concentrated under vacuum at 50°C by using a rotary evaporator. The extracts were stored in darkness at 4°C until used within a maximum period of 1 week.

Phytochemical Composition

Total phenols and o-diphenols

Total phenolic contents (TPCs) and o-diphenols of fractions were determined according to the method of Montedoro et al.^[23] with minor modifications. For total phenols, 0.4 mL of each fraction and 10 mL of diluted Folin–Ciocalteu reagent were mixed. After 1 min of incubation, 8 mL of sodium carbonate (75 g/L) was added and the mixture was incubated for 1 h. The absorbance was measured at 765 nm. The same extract was used to determine total o-diphenols. Then, 1 mL of a solution of HCl (0.5 N), 1 mL of a solution of a mixture of NaNO₂ (10 g) and NaMoO₄·2H₂O (10 g) in 100 ml·H₂O, and finally 1 mL of a solution of NaOH (1 N) were added to 100 µL of the extract. After 30 min, o-diphenols were read at 500 nm. The total phenols and o-diphenols were expressed on a dry weight (DW) basis as mg gallic equivalents/100 g of sample.

Determination of total flavonoids

Total flavonoid contents (TFCs) of the extracts were determined according to the colorimetric assay developed by Zhishen et al.^[24] One milliliter of properly diluted extract was mixed with 4 mL of distilled water. At zero time, 0.3 mL of (5% w/v) NaNO₂ was added. After 5 min, 0.3 mL of (10% w/v) AlCl₃was added. At 6 min, 2 mL of 1 M solution of NaOH were added. Finally, the volume was made up to 10 mL, immediately by the addition of 2.4 mL of distilled water. The mixture was shaken vigorously and the absorbance was read at 510 nm. The results were also expressed on a DW basis as milligram catechin equivalents (CEQ)/g of DW.

Determination of condensed tannins

Condensed tannins were determined according to the method of Julkunen-Titto.^[25] An aliquot (50 µL) of each extract or standard solution was mixed with 1.5 mL of 4% vanillin (prepared with MeOH), and then 750 µL of concentrated HCl was added. The well-mixed solution was incubated at ambient temperature in the dark for 20 min. The absorbance against blank was read at 500 nm. (+)- Catechin was used to make the standard curve (0.05–1 mg/mL; y = 0.801x + 0.0555; R² = 0.9992; y is the absorbance; x is the solution concentration). The results were expressed as mg CEQ/g of DW.

Measurement of total carotenoids

Total carotenoids were extracted according to the method of Talcott and Howard,^[26] with slight modifications. Two grams of the sample was extracted using 25 mL of acetone/ethanol (1:1, v/v) with 200 mg/L butylated hydroxytoluene (BHT) added. All manipulations were carried out under a yellow fluorescent light (Thorn), to avoid light-induced changes. After extraction, sample was centrifuged at 1500 × g for 15 min at 4–5°C. The supernatant was collected, and the remaining residue was re-extracted using the same method until the residue was colorless. Finally, the combined supernatants were brought to 100 mL with the extraction solvent, and the absorbance at 470 nm was measured using a UV-1601 Shimadzu spectrophotometer (Shimadzu). Total carotenoids were calculated according to the method of Gross,^[27] using the following equation, and expressed as mg/100 g of DW.

Ab is the absorbance at 470 nm, V is the total volume of extract, A1% is the extinction coefficient for a 1% mixture of carotenoids at 2500, and G is sample weight (g).

The total anthocyanin content (TAC) assay

The anthocyanin contents of the extracts from G. paraguayense were analyzed according to the method of Padmavati et al.^[28] modified by Chung et al.^[29] The extracts were mixed with acidified methanol (1% HCl/methanol) for 2 h at room temperature in the dark, and then centrifuged at 1000 ×·g for 15 min. The anthocyanin concentration in the supernatant was measured spectrophotometerically at 530 and 657 nm, respectively. The contents of anthocyanins have been made from the methanol extract as follows A530 – (0.24 × A653).^[30] The anthocyanin levels are expressed in mg cyanidin 3-glucoside, using an extinction coefficient (ε) of 26,900 L mol^–1 cm^–1 at 530 nm and a molar mass (MM) of 449.2 g mol^–1.^[31] The concentration was calculated using the following equation:

High-Performance Liquid Chromatography (HPLC) Analysis of Phenolic Composition

HPLC separation of phenolic extracts was carried out on a Hewlett Packard system (Waldbronn, Germany) comprising a HP-1100 pump, a Rheodyne model 7725 injector (Cotati, CA, USA, loop volume 20 µL), a UV detector (280 nm) and a C18 Technochrom Eurosphere 100 analytical column (250 mm × 8 mm). Three milliliters of each raisin extract was passed through a 0.45 µm filter and 20 µL extracts were directly injected into the HPLC. The flow rate was 0.5 mL min^–1. The mobile phases for chromatographic analysis were: (A) Acetonitrile and (B) sulfuric acid/water (2:98). A linear gradient was run from 15% (A) and 85% (B) to 40% (A) and 60% (B) during 12 min; it changed to 60% (A) and 40% (B) in 2 min; after 4 min it changed to 80% (A) and 20% (B); and then to 90% (A) and 10% (B) after 2 min (20 min, total time). After 4 min (At 24 min), it becomes 100% (A) during 4 min. The data were stored and processed by an HPLC Chemstation (Dos Series; Hewlett Packard). Phenolic compounds were identified on the basis of their retention times and quantified using external standard calibration curves. The results are expressed as µg g^–1 of DW.

Antioxidant Activity

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The DPPH method^[32] was used to determine antioxidant activity of dried raisins extracts. Different dilutions of the phenolic extract were prepared for each variety. Twenty microliters from the stock solution of the sample were dissolved in absolute methanol to a final volume of 1 mL and then added to1 mL DPPH (0.1 mM, in absolute methanol). The reaction mixture was kept at room temperature. The optical density (OD) of the solution was measured at 517 nm, after 20 and 60 min. The ODs of the samples in the absence of DPPH were subtracted from the corresponding OD with DPPH. The percentage reduction values were determined and compared to appropriate standards. Inhibition of the free radical DPPH, in percent (I_DPPH %) was calculated using the following equation:

where A blank is the absorbance of the control reaction (containing all reagents except the tested compound), and A sample is the absorbance of the tested compound. The sample concentration providing 50% inhibition (EC₅₀ DPPH) was calculated from the graph of DPPH inhibition percentage against sample concentration. Tests were carried out in triplicate.

2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS⁺) radical cation scavenging

For the determination of the antiradical activity, a protocol based on the ABTS⁺ free radical decolorization assay was used, as described previously.^[32] In brief, 5.0 mL of a 7.0 mM ABTS⁺ solution was treated overnight in the dark with 88.0 µL of a 140 mM potassium persulfate solution to yield the ABTS⁺ radical cation. Prior to use in the assay, the ABTS⁺ radical cation was diluted with ethanol to an initial absorbance of about 0.700 (ratio of 1:88) at 734 nm, with 30°C. Free radical scavenging activity was assessed by mixing 1.0 mL of diluted ABTS⁺ radical cation with 10 µL of sample. The reaction mixture was allowed to stand at 23°C for 6 min and the absorbance at 734 nm was immediately recorded. Results were expressed as percent of inhibition. All tests were carried out in triplicate. The sample concentration providing 50% scavenging ability on ABTS⁺ (EC₅₀ ABTS⁺) was calculated from the graph of ABTS⁺ scavenging ability against sample concentration. Tests were carried out in triplicate.

Reducing power (RP)

In the RP assay, the yellow color of the test solution changes to various shades of green and blue, depending on the RP of each extract. The presence of reducers (i.e., antioxidants) causes the reduction of the Fe³⁺/ferricyanide complex to the ferrous form. Therefore, Fe²⁺ concentration can be monitorized by measuring the formation of Perl’s Prussian blue at 700 nm. The capacity of date extracts to reduce Fe³⁺ was assessed by the method of Oyaizu.^[33] An aliquot of each sample or standard solution prepared with methanol (250 µL) was mixed with 250 µL of sodium phosphate buffer (0.2 M, pH 6.6) and 250 µL of 1% K₃Fe (CN) 6 incubated at 50°C for 20 min. After adding 250 µL of 10% trichloro acetic acid, the mixture was centrifuged at 3750 g for 10 min. The supernatant (100 µL) was then taken out and immediately mixed with 100 µL of methanol and 25 µL of 0.1% ferric chloride. After incubation for 10 min, the absorbance against blank was determined at 700 nm. The EC₅₀ value is the concentration at which the absorbance is 0.5.

Statistical Analysis

All assays were run in triplicate. The results are reported as mean values of three analysis and standard deviation. Data were subjected to statistical analysis using the SPSS program, release 11.0 for Windows (SPSS, Chicago, IL, USA). The one-way analysis of variance (ANOVA) followed by Duncan multiple range test were employed to study the differences between individual means were deemed to be significant at p < 0.05. Principal component analysis (PCA) was carried out using XLSTAT (2014) for Windows (Addinsoft, New York, USA).

RESULTS AND DISCUSSION

Phytochemical Composition

Phenolics, such as flavonoids, phenolic acids, and tannins, are considered to be the main antioxidants in fruits and vegetables. In this study, we measured the TPC, total o-diphenol content (ToPC), TFC, condensed tannin content (CTC), total carotenoid content (TCC), and TAC of four raisin varieties, Chriha, Raseki, Assli, and Meski (Table 1).

TABLE 1 Phytochemical contents of Tunisian raisins varieties

	Chriha	Raseki	Assli	Meski
Total phenols	534.2 ± 24.7^a	401.5 ± 25.2^b	459.7 ± 22.7^ab	405.6 ± 30.1^b
Condensed tannins	189.3 ± 0.5^b	206.5 ± 0.4^a	192.1 ± 2.9^b	208.6 ± 0.4^a
Total flavonoids	86.7 ± 4.9^ab	79.9 ± 3.8^b	66.5 ± 1.6^c	93 ± 4.2^a
Total o-diphenols	76.6 ± 2.2^c	106.5 ± 2.7^b	55.9 ± 0.6^d	115.8 ± 3.1^a
Total carotenoids	16.4 ± 1.2^c	26.3 ± 1.9^b	12.7 ± 1^d	33.0 ± 1^a
Total anthocyanins	107.4 ± 2.7^b	136.1 ± 2.3^a	116.1 ± 4.7^b	137 ± 4.4^a

Results are expressed as means ± standard errors (n = 3); Values in the same row with different letters showed statistically significant differences (p < 0.05) according to Duncan test; Total phenol and total o-diphenols contents were determined as gallic acid equivalents in milligrams per gram of dry weight; Total flavonoid and condensed tannins contents were determined as mg catechin equivalents CEQ/g of dry weight; Total carotenoids and anthocyane contents were expressed in mg/100 g of dry weight.

TPC was expressed as gallic acid equivalents in mg/g DW (Table 1). In this study, there were statistical significant differences in TPC among the raisin varieties (p < 0.05); TPC ranged from 401.5 ± 25.2 to 534.2 ± 24.7 mg/g DW. Chriha had the highest TPC followed by Assli, Meski, and Razeki. Our TPC results were lower than those reported by Mishra et al.^[34] (0.83 g/100 g) and Ouchemoukh et al.^[35] (470–770 g/100 g DW). However, different solvents and measurement methods affect the TPC of raisins.^[36] Additionally, the differences in raisin TPC could be attributed to varietal, seasonal, and agronomical differences, genomics, and moisture content.^[37]

On the other hand, ToPC showed the opposite trend as that of TPC (Table 1): the highest ToPC levels were obtained in Meski and Raseki with 115.8 ± 3.1 mg/g DW and 106.5 ± 2.7 mg/g DW, respectively, followed by Chriha and Assli. Phenolic compounds are mostly present in skin and seeds of grape;^[38] therefore, raisins originating from seed-containing grapes (i.e., Raseki and Meski) had higher phenolic content than those originating from seedless grapes (i.e., Chriha and Assli).

Meski, Chriha, and Raseki had the highest TFC values ranging from 93.0 ± 4.2 to 79.9 ± 3.9 mg CEQ/g DW (Table 1). The lowest TFC value was obtained in Assli (66.5 ± 1.6 mg CEQ/100 g DW). Significant differences in TFC were observed among the four raisin varieties (p < 0.05). These results were different than those reported by Ouchemoukh et al.,^[35] who obtained low TFC values in raisins (20.8 ± 1.1 mg CEQ/100 g DW).

In this study, CTC, which ranged from 189.3 ± 0.5 to 208.6 ± 0.4 mg CEQ/g DW, was mainly predominant in Meski and Razeki. The lowest CTC values were obtained in Assli and Chriha. There were no significant differences (p > 0.05) in TFC between Meski and Razeki or between Assli and Chriha. On the other hand, there were significant differences (p < 0.05) in TCC among the four raisin varieties. Meski and Raseki had the highest TCC values with 33.0 ± 1 and 26.3 ± 1.9 mg/100 g DW, respectively, followed by Chriha (16.4 ± 1.2 mg/100 g DW) and Assli (12.7 ± 1 mg/100 g DW).

In this study, TAC ranged from 137 ± 4.4 to 107.4 ± 2.7 mg/100 g DW (p < 0.05). Meski and Razeki had the highest TAC values. TPC, ToPC, TFC, CTC, TCC, and TAC can be used as indicators of the antioxidant activity of foods. The differences observed in phenolic compounds among the raisin varieties may be attributed to differences in cultivation methods, growth location, ripeness, harvesting time, climatic conditions, storage time, and environmental factors.^[37]

Identification and Quantification of Phenolic Compounds

The HPLC analysis of raisin methanol extracts revealed the presence of 19 phenolic compounds (Table 2): six hydroxybenzoic acids (HBA), including gallic, hydroxytyrosol, protocatechuic, vanillic, isovanillic, and 3-HBA; four hydroxycinnamic acids (HCA), including, ferulic, p-coumaric, and rosameric acids; seven flavonoids, including catechin, luteolin, luteolin-7-glucoside, apigenin, apigenin-7-glucoside, naringenin, and malvine; one stilbene (resveratrol); and one lignane (pinoresinol). It has been reported that the most predominant phenolic compounds in raisins are quercetin, kaempferol, catechin, epicatechin, rutin, transresveratrol, gallic acid, vanillic acid, syringic acid, ferulic acid, chlorogenic acid, 3,4-dihydroxybenzoic acid, cinnamic acid, protocatechuic acid, phloretic acid caftaric, and coumaric acids.^[13,¹⁷,³⁶,³⁹,^40]

TABLE 2 Phenolic profiles of Tunisian raisins varieties (in mg g^–1 D.W)

	Chriha	Razeki	Assli	Meski
Gallic	2.52 ± 0.32^a	1.20 ± 0.1^b	0.58 ± 0.02^c	1.32 ± 0.05^b
Hydroxytyrosol	0.12 ± 0.01^b	0.15 ± 0.01^a	0.08 ± 0.01^c	0.05 ± 0.01^d
Protocatechuic	0.15 ± 0.01^a	0.06 ± 0.01^b	0.07 ± 0.01^b	0.03 ± 0.01^c
Vanillic	0.05 ± 0.01^b	0.05 ± 0.01^b	0.11 ± 0.02^a	0.08 ± 0.01^a
Isovanillic	0.16 ± 0.02^a	0.18 ± 0.03^a	0.15 ± 0.01^a	0.16 ± 0;03^a
3hydroxybenzoic	0.16 ± 0.05^a	0.04 ± 0.01b^c	0.02 ± 0;01^c	0.06 ± 0.01^b
Chlorogenic	0.23 ± 0.03^a	0.06 ± 0.01^b	0.08 ± 0.02^b	0.02 ± 0.01^c
P. Coumaric	0.05 ± 0.01^c	0.05 ± 0.01^c	0.43 ± 0.03^a	0.25 ± 0.03^b
Fereulic	—	—	—	0.03 ± 0.01^a
Rosameric	0.15 ± 0.01^a	0.05 ± 0.01^b	0.10 ± 0.01^a	0.06 ± 0.02^b
Catechin	0.32 ± 0.02^c	0.30 ± 0.03^c	0.55 ± 0.04^b	0.71 ± 0.07^a
Luteolin	0.3 ± 0.05^b	0.06 ± 0.01^c	1.32 ± 0.04^a	0.07 ± 0.01^c
Luteolin 7glucoside	1.17 ± 0.03^a	0.27 ± 0.04^b	0.30 ± 0.03^b	0.27 ± 0.02^b
Apigenin	0.47 ± 0.06^a	0.09 ± 0.01^d	0.29 ± 0.02^b	0.21 ± 0.04^c
Apigenin 7 glucoside	0.08 ± 0.02^ab	0.09 ± 0.01^a	0.05 ± 0.01^bc	0.03 ± 0.01^c
Naringenin	0.20 ± 0.03^a	0.05 ± 0.01^b	—	0.06 ± 0.01^b
Malvine	0.18 ± 0.03^b	0.10 ± 0.02^c	0.26 ± 0.02^a	0.21 ± 0.03^ab
Resveratrol	0.12 ± 0.02^a	0.1 ± 0.05^a	0.02 ± 0.01^c	0.06 ± 0.01^b
Pinoresinol	0.08 ± 0.01^a	0.06 ± 0.01^ab	0.04 ± 0.01^b	—
Σ HBA	3.17 ± 0.21^a	1.68 ± 0.08^b	1.01 ± 0.01^c	1.70 ± 0.04^b
Σ HCA	0.44 ± 0.03^b	0.16 ± 0.01^c	0.61 ± 0.03^a	0.37 ± 0.05^b
Σ Flavonoids	2.74 ± 0.03^a	0.97 ± 0.05^c	2.78 ± 0.02^a	1.57 ± 0.08^b
Σ Phenolic	6.54 ± 0.17^a	2.96 ± 0.17^d	4.47 ± 0.01^b	3.69 ± 0.17^c

Results are expressed as means ± standard errors (n = 3); Values in the same row with different letters showed statistically significant differences (p < 0.05) according to Duncan test; ΣHBA: benzoic acids, ΣHCA: cinnamic acids; —: not detected.

Our results revealed that HBA (1.01–3.17 mg/g DW) was the most predominant phenolic compound in Chriha, Razeki, and Meski followed by flavonoids (0.97–2.74 mg/g DW) and HCA (0.16–0.61 mg/g DW). In Assli, the most predominant phenolic acids were flavonoids followed by HBA and HCA. These results differed from those reported in other fruits, which mainly contain HCA.^[41,^42] HCA was present at low concentrations in raisins probably because esters of HCA are good substrates in enzyme-catalyzed (polyphenol oxidase) browning reactions, which take place during drying.^[43,^44] Quantitatively, the highest and lowest HBA levels were obtained in Chriha and Assli, respectively. HCA and flavonoids levels were higher in Assli than in Razeki (Table 2). Differences observed among the four raisin varieties were statistically significant (p < 0.05).

Based on the results, the levels of the main phenolic compounds were variety-dependent. Among HBA, gallic acid was the most abundant compound in the four varieties (0.58–2.52 mg/g DW), especially in Chriha (p < 0.05). Among HCA, chlorogenic acid was the most predominant compound in Razeki and Chriha (0.06 and 0.23 mg/g DW, respectively) and p-coumaric acid was the main compound in Meski and Assli (0.25 and 0.43 mg/g DW, respectively). Moreover, the dominant flavonoids were catechin in Razeki and Meski (0.30 and 0.71 mg/g DW, respectively), luteolin-7-glucoside in Chriha (1.17 mg/g DW), and luteolin in Assli (1.32 mg/g DW). Tsai et al.^[45] reported that catechin is positively correlated with DPPH scavenging capacity. In our study, catechin was concentrated in Meski, which may explain the high DPPH scavenging capacity of this variety. As a result of the great interest that has recently been devoted to resveratrol and its derivatives, special attention was given to the stilbene compounds.^[46] In this study, the four raisin samples had low resveratrol contents (0.02–0.12 mg/g DW); the highest levels were obtained in Chriha (p < 0.05). Karadeniz et al.^[47] reported that fully ripened grapes used for raisin production lose their capacity to synthesize resveratrol during ripening, which could explain the low resveratrol levels obtained in this study.

The low levels of some phenolic compounds and the absence of others could be attributed to the participation of these compounds in various reactions during the drying process, including non-enzymatic browning, auto-oxidation, and especially, oxidation by polyphenol oxidase and peroxidase.

Antioxidant Activity

For a more comprehensive and accurate evaluation of the antioxidant capacity of raisins extracts, three different in vitro assays were carried out: DPPH radical scavenging capacity assay, ferric RP assay, and ABTS⁺ scavenging capacity assay. The antioxidant activity of extracts cannot be reasonably validated by one single method due to the complex nature and interactions of phytochemicals. Therefore, it is crucial to use multi assay systems with different indices.^[48]

In the DPPH radical scavenging capacity assay, a reduction in DPPH absorbance is caused by a reaction between antioxidant molecules and the radical. The degree of discoloration is related to the antioxidant activity of the sample. The extracts displayed DPPH scavenging capacity in a dose-response manner (Fig. 1). Meski and Chriha had better antioxidant activity than Assli and Razeki. Based on EC₅₀ values (Table 3), Meski and Chriha had higher DPPH radical scavenging capacity than Raseki and Assli (2.71 ± 0.03 and 3.01 ± 0.05 versus 3.44 ± 0.06 and 3.50 ± 0.07, respectively; p < 0.05). In order of decreasing DPPH radical scavenging capacity, the raisin varieties were Chriha > Meski > Assli > Razeki. The antioxidant activity against DPPH has been correlated with the concentration, chemical structure, polymerization, and degree of antioxidants.^[49,^50]

TABLE 3 EC₅₀ values of Tunisian raisins varieties

	Chriha	Raseki	Assli	Meski
DPPH	3.01 ± 0.05^b	3.44 ± 0.06^a	3.5 ± 0.07^a	2.71 ± 0.03^c
Reducing Power	3.60 ± 0.03^d	5.31 ± 0.04^b	4.72 ± 0.05^c	5.51 ± 0.03^a
ABTS+	3.64 ± 0.03^d	4.55 ± 0.04^b	3.73 ± 0.02^c	4.94 ± 0.02^a

Results are expressed as means ± standard errors (n = 3); Values in the same row with different letters showed statistically significant differences (p < 0.05) according to Duncan test.

RP is a measure of the conversion of a ferric cyanide complex into the ferrous form. At 10 mg/mL, Chriba extracts had the highest RP value (64%; Fig. 2), whereas Assli, Razeki, and Meski extracts had an RP value of 39%. These results were confirmed by EC₅₀ RP values (Table 3; p < 0.05). Ouchemoukh et al.^[35] reported higher RP values for raisins extracts, while Mishra et al.^[34] reported lower RP values in raisins. The reducing properties in foods are associated with reductones.^[51,52] The phenolic compounds present in raisins may act in a similar fashion as reductones by donating electrons and quenching free radicals.

FIGURE 1 Antioxidant activities of Tunisian raisins varieties at different concentrations as determined by DPPH radical scavenging activity. Results are expressed as means ± standard deviation (n = 3). Different small letters within histogram are significantly different (p < 0.05) with respect to the concentration of the extract according to Duncan test.

FIGURE 2 Antioxidant activities of Tunisian raisins varieties at different concentrations as determined by ABTS⁺ radical scavenging activity. Results are expressed as means ± standard deviation (n = 3). Different small letters within histogram are significantly different (p < 0.05) with respect to the concentration of the extract according to Duncan test.

The antioxidant activity of raisin extracts was also evaluated by the ABTS radical scavenging assay. As shown in Fig. 3, raisin extracts scavenged ABTS⁺ radical cations. At 10 mg/mL, Chriha and Assli had higher antioxidant activity (~58%) than Razeki and Meski (~43%). The EC₅₀ values are shown in Table 3; in order of decreasing antioxidant activity of raisins based on the ABTS⁺ assay, the varieties were Chriha > Assli > Razeki > Meski (p < 0.05). These results were not similar to those obtained with the DPPH scavenging capacity assay, probably because the phenolic compounds in raisins can react with DPPH better than with ABTS⁺. Differences in growth location and cultivation practices, ripening stage, harvesting conditions, and seasonality could affect the antioxidant activity of raisins.^[53] Moreover, the extraction solvent polarity, extraction method, and drying temperature could affect the antioxidant compounds present in raisins.^[54,^55] Finally, possible interferences from non-antioxidant compounds may also affect antioxidant capacity.^[56]

FIGURE 3 Reducing power activities of Tunisian raisins varieties at different concentrations. Results are expressed as means ± standard deviation (n = 3). Different small letters within histogram are significantly different (p < 0.05) with respect to the concentration of the extract according to Duncan test.

FIGURE 4 Principal component analysis (scores and loading plots, Biplot) applied to the dataset of phytochemical content, phenolic group identified and quantified by HPLC and antioxidant activity (DPPH, reducing power (RP) and ABTS⁺) of raisins varieties. TPC: Total phenols content; TOPC: Total o-diphenols content; TFC: Total flavonoids content; CTC: Condensed tannins content; TCC: Total carotenoids content; TAC: Total anthocyanins content; PH: Phenolic compounds; HBA: Hydroxybenzoic acids; HCA: Hydroxycinnamic acids and FL: Flavonoids.

PCA

A multivariate statistical analysis of the data was performed using PCA. Figure 4 was plotted according to the correlation between phytochemical content (TPC, TOPC, TFC, CTC, TCC, and TAC), phenolic groups (phenolic compounds [PH], HBA, HCA, and flavonoids [FL]), and antioxidant activity (DPPH, RP, and ABTS⁺). PC1 accounted for 67.8% of the total variance (91.9%), and PC2 accounted for 24.11%. The position of each variable in the loading plot describes its relationship with the other variables. Variables that are close to each other have high correlations. Variables on the same side of the origin (0.0) are positively correlated and those on the opposite side of the origin are negatively correlated. Raisin varieties could be discriminated on the PCA plane. PC1 was positively related to TFC, DPPH, TOPC, TCC, CTC, and TAC. PC2 was related to HCA, ABTS, FL, TPC, PH, RP, and HBA. Razeki and Meski were located on the positive side of PC1. On the other hand, Chriha and Assli were located on the negative side of PC1. Therefore, TAC, CTC, TCC, TOPC, and TFC were mainly responsible for the discrimination between Meski and Razeki. These compounds exhibit potent antioxidant activities based on the DPPH assay. Chriha and Assli were mainly discriminated by HBA, PH, TPC, FL, and HCA, which contributed to the ABTS⁺ scavenging capacity and RP.

CONCLUSION

The results of this study demonstrate that phytochemical content, phenolic compound levels, and antioxidant capacities of Chriha, Razeki, Assli, and Meski were variety-dependent. The raisin varieties had high phenolic compound levels, which were very active as antioxidants. These findings confirm the antioxidant potential and health-promoting properties of Tunisian raisin varieties.

ACKNOWLEDGMENTS

The authors express their sincere thanks to the members of LR-NAFS/LR12ES05, Nutrition-Functional Food et. Vascular Health, at Faculty of Medicine—University of Monastir, Tunisia.

FUNDING

This study was supported by the Ministry of Higher Education and Scientific Research in Tunisia.

Additional information

Funding

This study was supported by the Ministry of Higher Education and Scientific Research in Tunisia.