Bioactive Compounds and Antioxidant Capacity in Dark Green, Old Gold Crimson, Ripening Inhibitor, and Normal Tomatoes

Abstract

Bioactive compounds and antioxidant capacity were determined in one high lycopene normal genotype (Berika), two color mutants [dark green (BCT-115) and old gold crimson (BCT-119)], one ripening mutant [ripening inhibitor (BCT-111)], and six ordinary tomato genotypes (Punjab, Chhuhara, FEB-2, BCT-53, Patharkutchi, CLN-B, and CLN-R). Significant variation (mg/100 g fresh weight) in ascorbic acid (~25–38), lycopene (~3–5), β-carotene (~0.6–1.6), total flavonoids (~0.15–48.0), and total phenols (~23–41) was recorded in the pulp. The peel fraction of the tomatoes was identified as an important reservoir of antioxidant bioactive compounds viz. lycopene (~0.26–28), β-carotene (~1.7–4.7), total flavonoids (~0.6–109), and total phenols (~43–104). The radical scavenging activity was ranged from ~53–82 and ~25–51% in peel and pulp, whereas the metal chelating activity was found to range from ~21–53 and ~12–25% in peel and pulp, respectively, among all genotypes. The tomato genotypes with the highest content of bioactive molecules and antioxidant potential in the edible portion were BCT-115, Berika, and BCT-119. The results show great potential of color mutants (dark green and old gold crimson) for all the parameters. Thus, these genotypes could be used in the future breeding programs to enhance the synthesis of beneficial bioactive molecules in the new cultivars.

Keywords:

• Lycopersicon esculentum
• Color mutant
• Ripening mutant
• Phenolic compounds
• Antioxidant activity
• By-products

Introduction

The favorable effects of fresh fruits and vegetables on human health are associated with various classes of compounds, which are responsible for antioxidant properties.^[1–3] Interest in the function of antioxidants in human health has promoted research to evaluate fruit and vegetable antioxidants and to determine how their content and activity can be maintained or even improved through crop breeding, cultural practices, postharvest storage, and processing.^[3,4] Enhancing the health benefits of fresh produce could, therefore, add value and create new opportunities for growers and processors by enabling them to earn lucrative shares in health-oriented markets. In fact, estimation of antioxidants is becoming an important evaluation parameter for the nutritional quality of food and its quantification allows a real evaluation of this nutritional value.^[3,5,6]

The health benefits of tomato have been ascribed principally to its phenolics (flavonoids, tannins, phenolic acids, coumarins, stilbenes, xanthones, anthraquinones, etc.), vitamin C content, and carotenoid constituents (particularly lycopene and β-carotene) that accumulate in plasma and tissues.^[7,8] The skin, being rich in bioactive compounds, is an important component of waste originates during various stages of tomato processing. Tomato pomace constitutes the major part of the waste that comes from the pulper. Pomace consists of skin that could be utilized for extracting bioactive molecules.^[9] The overall antioxidant potential of tomatoes could vary considerably according to the genetic, variety, ripening stage, growing conditions, and postharvest handling.^[5,8,10,11]

The characterization of genotypes is imperative for their efficient use in plant breeding efforts to improve postharvest qualities comprising storage and processing potential. Genetic profiles of mutant tomatoes are clearly different from those of normal/non-mutant tomatoes.^[12] The composition of fresh tomato can vary between the tissues of a single fruit^[8,13] and between different tomatoes, according to the cultivar, its cultivation conditions (light, temperature, soil, fertilization, and ripeness at harvest), and handling and storage methods.^[7,8] In the tomato, a small number of single gene mutations exist, such as ripening-inhibitor (rin), non-ripening (nor), never-ripe (Nr), and colorless non-ripening (Cnr), which have pleiotropic effects, which result in the reduction or almost complete abolition of ripening. Improving the color and lycopene content of tomatoes can be accomplished using typically defined genes. The genes dark green (dg), old gold crimson (og^c), high pigment-1 (hp-1), and high pigment-2 (hp-2) are examples of genes that have a positive effect on the concentration of lycopene. The mutant og^c has been widely used in breeding programs to improve red-fruited tomatoes.^[14]

However, no study has been carried out so far on ripening (rin) and color mutants (dg and og^c) for quantifying the bioactive molecules of their peel and pulp fractions. Moreover, we are not aware of any published data on metal chelating (MC) activity of these genotypes (in terms of peel and pulp). Thus, the intent of the study was to assess the variation among the different tomato genotypes for bioactive molecules such as ascorbic acid (AsA), lycopene, β-carotene, total phenols (TPs), total flavonoids (TFs), and in vitro antioxidant potential in relation to the importance of tomatoes for functional aspects.

MATERIALS AND METHODS

Plant Materials and Growing

A total of ten diverse tomato genotypes consisting one high lycopene normal genotype (Berika), two color mutants [dg (BCT-115) and og^c (BCT-119)], one ripening mutant [rin (BCT-11)], and six ordinary tomato genotypes (Punjab, Chhuhara, FEB-2, BCT-53, Patharkutchi, CLN-B, CLN-R) were selected for the study (Table 1). The field experiments were carried out in a field in the Central Research Farm, Gayeshpur, Nadia, West Bengal, India, in the October–March growing season. The seedlings (21 day old) were transplanted on raised beds. Before sowing, seeds were treated with Thiram (2–3%) of seed and sown 1–2 cm deep in rows spaced 5 cm apart. To facilitate protection against the risk of frost injury the crop was protected with polythene sheets aligned in E–W direction on the northern side of each row until mid-February. All the genotypes were grown simultaneously in the same field and subjected to identical cultural practices (irrigation, nutrient, and pesticide application, etc.) and, of course, environmental conditions in order to minimize the influence of pre- and postharvest factors on genotype-related variability of field-grown tomatoes.^[11]

TABLE 1 Description of the genotypes used in the experiment

Genotypes	Specific features	Source
Berika	High lycopene containing variety	Institute of Genetics, Bulgarian Academy of Science, Sofia, Bulgaria
BCT-115	Dark green and high-pigmented line carrying dark green (dg) gene	United States Department of Agriculture (USDA), USA
BCT-119	High-pigmented line containing old gold crimson (og^c) gene	United States Department of Agriculture (USDA), USA
BCT-111	Slow-ripening line having ripening inhibitor (rin) gene, devoid of lycopene	Department of Vegetable Science, HAU, Hisar, Haryana, India
Punjab Chhuhara	Popular variety having good processing characteristics	AICRP on Vegetable Crops, BCKV, Mohanpur, West Bengal, India
FEB-2	Early blight resistant variety	Division of Vegetable Science, IARI, New Delhi, India
BCT-53	A high-yielding line with appreciable fruit quality	Department of Vegetable Crops, BCKV, Mohanpur, West Bengal, India
Patharkutchi	Local adaptable cultivar, low in pigment with stay-green shoulder	Department of Vegetable Crops, BCKV, Mohanpur, West Bengal, India
CLN B	Heat-tolerant line, low in pigment	Asian Vegetable Research and Development Center (AVRDC), Taiwan
CLN R	Heat-tolerant line, low in pigment	Asian Vegetable Research and Development Center (AVRDC), Taiwan

USA refers to United States of America; AICRP refers to All India Coordinated Research Project; BCKV refers to Bidhan Chandra Krishi Viswavidyalaya; IARI refers to Indian Agricultural Research Institute; HAU refers to Hisar Agricultural University.

Determination of Bioactive Molecules and Antioxidant Assays

Different bioactive molecules of the tomatoes were quantified. AsA was measured with whole fruits, whereas other molecules and antioxidant assays were analyzed for both peel and pulp separately. To obtain the peel, the skins of the fruits were removed manually using a sterile blade. Generally, 18–25 fruits yielded 10–12 g of peels.

AsA and Carotenoids (Lycopene and β-Carotene)

AsA was quantitatively determined by the 2, 6-dichlorophenol indophenol method.^[15] Lycopene and β-carotene from tomato fractions (peel and pulp) were extracted with hexane: methanol: acetone (2:1:1), containing 2.5% butylated hydroxy toluene (BHT). During the extraction process, some precautions were taken, such as working in a less luminous room and wrapping glass materials in aluminium foil to avoid lycopene loss by photo-oxidation. The optical density of the hexane extract was measured spectrophotometrically at 502 nm against a hexane blank. Concentration of lycopene was calculated using the extinction coefficient (E%) of 3150. The β-carotene contents in the hexane extract was monitored at 452 nm and calculated using a calibration curve against a higher purity β-carotene. The results are expressed as mg/100 g fresh weight (FW) for both lycopene and β-carotene.^[2]

Preparation of Extracts for TP, Flavonoid, and Antioxidant Assays

Extracts were prepared following the method described in our previous article.^[3] The pulp and peel of fresh fruits were homogenized separately in a grinder for 3 min at 15 s intervals to avoid excessive heating. Five grams of paste samples were extracted in 50 mL of absolute ethanol at 20°C for 2 h using an incubator cum shaker. The extract was filtered through Whatman No. 41 filter paper to obtain a particle free extract. The residue was extracted twice and filtered. Solvents of higher polarity (ethanol or ethanol–water mixtures) additionally can extract flavonoid glycosides and higher molecular weight phenols, resulting in higher yields of total extracted polyphenols. The extract was stored at –20°C until used.

TP and TFs

The concentration of TPs in the extracts (peel and pulp) was determined by using Folin–Ciocalteu reagent and external calibration with catechol.^[16] The concentration of the TP contents was expressed as mg/100 g FW catechol equivalent. The estimation of phenolic compounds in the fractions was carried out in triplicate and the results were averaged. TF content was determined using aluminium chloride method.^[17] TF was expressed as milligram of rutin equivalents per 100 g on FW.

Radical Scavenging Activity (RSA)

RSA was estimated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. It is based on the measurement of the scavenging ability of antioxidants toward the stable DPPH radical.^[18] A 3.9 mL aliquot of a 0.001 M of DPPH solution in ethanol (80%) was added to 0.1 mL of extract and shaken vigorously. Change in the absorbance of the sample extract was measured at 517 nm for 30 min. The percentage of RSA was calculated as

where, Abs₀ was the beginning absorbance at 517 nm, obtained by measuring the same volume of solvent, and Abs₁ was the final absorbance of the sample extract after 30 min.

MC Activity

The chelating of ferrous ions by extracts was estimated by the method inspired by Siddiqui et al.^[3] with a few adaptations. Briefly, the extract (0.1 mg/mL) was added to a solution of 2 mM FeCl₂ (0.05 mL). The reaction was initiated by the addition of 5 mM Ferrozine (0.2 mL), the mixture was shaken vigorously, and left standing at room temperature for 10 min. The absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage chelating of metal (ferrozine-Fe²⁺ complex formation) was calculated according to formula as given in RSA.

Statistical Analysis

Each assay was repeated four times for all the 10 genotypes and the results are represented as the means of four replicates. Data were analyzed using a one-way analysis of variance with the SAS statistical system 9.2 (SAS Institute, Cary, NC, USA), and all means of total bioactive compounds and antioxidant assays were compared using Duncan’s Multiple Range test. Significant differences were assessed at the p ≤ 0.05 probability level.

RESULTS AND DISCUSSION

Bioactive Molecules

AsA

A significant (p < 0.05) variation was persisted in AsA contents in pulps of fruits belonging to different genotypes (Table 2). The content was ranged from 26.12 to 38.14 and 24.98 to 37.54 mg/100 g FW in mutants [BCT-115 (dg), BCT (og^c), and BCT-111 (rin)] and normal genotypes, respectively. In case of the mutants, the highest amount was recorded in BCT-115 followed by BCT-119, whereas among normal ones it was the highest in Berika followed by Punjab Chhuhara and Patharkutchi. Considering all genotypes, the lowest value of AsA contents was in FEB-2. The values were, more or less, similar to those reported by Singh et al.,^[19] Gonzalez-Cebrino et al.,^[20] and Ilahy et al.^[11] This inconsistency in AsA may be due to a number of factors such as genotypic make-up, maturity stage, and the conditions during production and after harvest (Favati et al.^[21] and Gonzalez-Cebrino et al.)^[20] The high pigment genotypes concentrated higher AsA content than other genotypes.

TABLE 2 Bioactive contents (mean ± standard deviation) of the tomato genotypes

Genotypes	Ascorbic acid (mg/100 g FW)	Lycopene (mg/100 g FW)		β-Carotene (mg/100 g FW)		Total phenol (mg CE/100 g FW)		Total flavonoid (mg RE/100 g FW)		Lycopene: carotene
		Peel	Pulp	Peel	Pulp	Peel	Pulp	Peel	Pulp	Peel	Pulp
Berika	37.54 ± 1.06^a	22.33 ± 1.06^b	5.03 ± 0.17^a	4.53 ± 0.03^a	1.45 ± 0.04^ab	92.67 ± 4.04^b	32.67 ± 4.04^bc	104.33 ± 2.08^a	41.33 ± 1.52^b	4.93 ± 0.19^cd	3.47 ± 0.21^abc
BCT-115	38.14 ± 0.72^a	27.95 ± 0.75^a	5.16 ± 0.05^a	4.77 ± 0.04^a	1.56 ± 0.08^a	104.0 ± 5.56^a	41.33 ± 2.51^a	108.67 ± 1.52^a	48.0 ± 3.0^a	5.86 ± 0.12^a-e	3.30 ± 0.20^bc
BCT-119	28.43 ± 0.79^e	21.33 ± 3.09^c	4.03 ± 0.05^c	4.16 ± 0.21^b	1.44 ± 0.04^a-c	81.75 ± 5.03^cd	32.08 ± 3.0^b-d	92.17 ± 2.51^b	38.0 ± 4.04^c	5.13 ± 1.02^d	2.81 ± 0.09^c
BCT-111	26.12 ± 0.19^e	0.26 ± 0.04^g	N D	1.72 ± 0.02^f	0.61 ± 0.07^h	46.0 ± 5.56^f	24.67 ± 1.52^de	0.61 ± 0.10^i	0.15 ± 0.03^h	0.15 ± 0.02^e	N D
Punjab Chhuhara	37.48 ± 1.05^a	16.04 ± 0.46^de	3.95 ± 0.21^b	3.06 ± 0.14^cd	0.95 ± 0.16^fg	60.67 ± 7.50^e	23.00 ± 2.0^d-e	70.0 ± 3.0^c	15.67 ± 2.08^d	5.24 ± 0.12^cd	4.17 ± 0.86^a
FEB-2	24.98 ± 0.31^e	11.35 ± 0.67^f	3.39 ± 0.06^cd	2.33 ± 0.10^e	0.92 ± 0.16^fg	69.33 ± 4.04^c-e	28.67 ± 2.51^b-d	48.33 ± 2.52^g	12.0 ± 2.0^ef	4.86 ± 0.08^d	3.70 ± 0.69^ab
BCT-53	29.77 ± 0.44^cd	14.02 ± 1.52^e	3.39 ± 0.13^cd	2.91 ± 0.25^d	0.98 ± 0.06^e-g	75.0 ± 5.56^c	25.67 ± 3.51^de	61.0 ± 3.0^e	12.33 ± 2.51^d-f	4.82 ± 0.43^d	3.47 ± 0.32^abc
Patharkutchi	30.26 ± 0.91^b-d	19.89 ± 0.59^c	4.14 ± 0.11^b	3.41 ± 0.39^c	1.19 ± 0.04^c-e	67.0 ± 6.0^c-e	34.33 ± 3.05^b	67.0 ± 4.0^cd	9.0 ± 2.64^fg	5.83 ± 0.52^a-c	3.48 ± 0.22^abc
CLN-B	28.65 ± 0.54^d	9.85 ± 0.53^f	2.82 ± 0.27^e	1.68 ± 0.10^f	0.78 ± 0.07^gh	42.67 ± 7.67^f	24.00 ± 4.58^de	38.67 ± 4.50^h	9.33 ± 1.52^e-g	5.87 ± 0.33^a-c	3.60 ± 0.49^ab
CLN-R	25.34 ± 0.79^e	10.72 ± 0.56^f	3.19 ± 0.20^d	1.94 ± 0.15^f	1.25 ± 0.26^b-d	63.67 ± 5.68^de	27.00 ± 4.0^c-e	46.0 ± 4.0^g	7.0 ± 2.64^g	5.53 ± 0.73^b-e	2.55 ± 0.83^c

Values within column followed by the same superscript letter were not significantly different (p < 0.05) by the Duncan’s Multiple Range Test; CE: catechol equivalents; RE: rutin equivalents; ND: not detected.

AsA is a crucial micronutrient required for normal metabolic functioning of the body. The current recommended dietary allowance (RDA) for AsA is set at 60 mg/day to offer an enough margin of safety, as this amount would prevent the development of scurvy for about one month in a diet lacking AsA.^[22] AsA readily scavenges reactive oxygen and nitrogen species, thereby effectively protecting other biomolecules from oxidative damage. It may protect against cancer through several mechanisms, in addition to inhibiting DNA oxidation.^[2]

Lycopene

Lycopene contents showed significant (p < 0.05) variation among all genotypes (Table 2). In general, the high pigment genotypes [Berika, BCT-115 (dg), and BCT-119 (og^c)] were found to contain higher amount of lycopene than ordinary tomato genotypes in both peel and pulp. Regarding BCT-111 having rin gene did not develop lycopene in pulp but only a negligible amount (0.26 mg/100 g FW) was observed in the peel. In case of high lycopene containing genotypes, the highest value in pulp and peel was recorded in BCT-115 followed by Berika and BCT-119. The lycopene contents of peel and pulp of normal genotypes ranged from ~10 to ~20 and ~3 to ~4 mg/100 g FW, respectively, Patharkutchi being the highest followed by Punjab Chhuhara. Variety is an important factor affecting both the composition and content of plant pigments.^[20,23] The reported values for pulp are comparable with those previously published for field grown normal and high pigment tomatoes by others.^[5,11,20,23] These results are also in harmony and confirm those of Ilahy et al.^[5,11] and Lenucci et al.^[6] who recently reported that high-pigment tomatoes are characterized by a very high lycopene content. The variations in lycopene content between high lycopene and normal tomatoes are mainly due to genotypic factors.^[11] The considerable lycopene accumulation in high-lycopene tomato genotypes can be, in fact, due to the reduced cycling rate of this molecule to synthesize carotenes and/or to an enhanced enzymatic activity of phytoene synthase-I that causes a massive production of lycopene precursors in ripening tomato fruits.^[8] Our result revealed that the peel contained about 3 to 5.5 times more lycopene than pulp irrespective of genotype. The results confirm the findings of Singh et al.^[19] who stated that peel contained about 3–5 folds higher lycopene in relation to pulp having a range of 9.78–26.75 and 1.47–5.28 mg/100 g in red and yellow cultivars, respectively. This is the first report characterizing the lycopene contents of peel and pulp specifically to genotypes having diverse profiles, particularly high pigment, and rin mutants.

β-carotene

Like lycopene contents, the β-carotene levels (expressed as mg/100 g FW) showed significant (p < 0.05) differences between peel and pulp as well as among different genotypes (Table 2). In the pulp of high pigment mutant, the β-carotene content ranged from 1.44 to 1.56 with BCT-119 (og^c) being the lowest and BCT-115 (dg) registering the highest. Among normal and high lycopene genotypes, the β-carotene content was the lowest (0.78) in CLN-B and the highest in Berika (1.45). The variation in β-carotene contents as a function of genotype has also been demonstrated in different research studies. β-carotene content (mg/100 g FW) of tomato fruit at full ripe stage ranges from 0.08 to 1.31 (23), and 2.35 to 6.15 (24) in different tomato genotypes without mentioning peel and pulp fraction. On average, the peel of high pigment and normal tomatoes had about 2.5–3 and 1.5–2.5 folds higher β-carotene as compared to pulp. Among high pigment and normal genotypes, the β-carotene contents in the peel ranged between 4.16 (BCT-119) to 4.77 (BCT-115) and 1.68 (CLN-B) to 4.53 (Berika), respectively. The concentration of β-carotene in both peel and pulp of rin mutant (BCT-111) was found to be the lowest (1.72 and 0.61, respectively) in relation to other genotypes. These results indicate that varietal factors can affect (to a considerable extent) the overall biosynthesis of carotenoids in both peel and pulp, particularly β-carotene formation via cyclization of lycopene.^[24] It was found that high pigment genotypes have 1–1.5 times more β-carotene than the normal ones. These results are in accordance with Ilahy et al.^[5] and Ilahy et al.^[11] but not what was reported by Sacks and Francis,^[14] that high lycopene tomato cultivars compensate the increase of lycopene by reducing other antioxidants such as β-carotene.

β-carotene is one of the better-known carotenes because of its high vitamin A activity and its wide distribution in nature.^[22] The protective effects of β-carotene occur through one or several actions, which include singlet oxygen quenching (photo protection), antioxidant protection, and enhancement of the immune response.^[8] β-carotene may also function as a redox reagent, an immunological regulator, or by increasing cell-to-cell communications.^[25] There is substantial evidence that β-carotene is capable of influencing all of these mechanisms, at least in vitro.

Lycopene: β-Carotene Ratio

Along with the concentration of carotenoids, the ratio of lycopene to β-carotene content varied significantly (p < 0.05) between pulp and peel as well as among the genotypes (Table 2). Among high pigment genotypes, the ratio differed between ~3 and ~3.5 in pulp, whereas the ratio ~2.5 to ~4.0 was observed in case of normal genotypes. The lycopene contents in pulp recorded to be 2.5 to 4 times higher (roughly) than β-carotene contents. Considering the peel, this ratio ranged from 4.93 to 5.86 and 4.82 to 5.87 for mutants and normal genotypes, respectively. However, the lycopene contents of peel recorded were 4.5 to 6 folds higher (roughly) than the β-carotene contents.

TP

The TP contents of tomatoes found to vary significantly (p < 0.05) among all genotypes as well as between both peel and pulp (Table 2). The TP contents of pulps of high pigment genotypes (Berika, BCT-115, and BCT-119) were approximately ~32 to ~41 mg CE/100 g FW in which BCT-115 (dg) had the highest amount followed by Berika. In normal genotypes, Patharkutchi and FEB-2 showed the highest TP contents (~34 and ~29 mg CE/100 g FW, respectively) followed by CLN-R, and the lowest value was recorded in Punjab Chhuhara. The values of content for pulp were close to the range reported by Singh et al.^[19] ranging from 7.41 to 57.60 mg gallic acid equivalent (GAE)/100 g FW and confirmed that genotype significantly affects TP content in tomato as stated by other researchers.^[7,26] The high lycopene genotypes had higher amount of TP contents in the peel and pulp. Various authors^[2,5,11] have also reported considerable varying and increased amounts of total phenolic content in high lycopene genotypes. This comprehensive overproduction of phytonutrients is associated with increased plastid biogenesis and, therefore, overproduction of other plastid-accumulating metabolites may be expected in these high pigment mutants.^[27] Generally, the peel contained 2–4 times more TP contents than pulp in all the genotypes. As evident from the Table 2, the TP contents of peels of high-pigment and normal genotypes was ~82 (BCT-119) and 104 (BCT-115) and ~61 (Punjab Chhuhara) and 75 mg CE/100 g FW (BCT-53), respectively. It was found that the peels of high-pigment genotypes had significantly (p < 0.05) higher amount of TP contents than those of normal genotypes. On the other hand, the rin mutant (BCT-111) had lower TP contents in peel (46.0) than other genotypes; however, an appreciable amount was recorded in pulp (~25 mg CE/100 g FW). Excluding high pigment tomatoes, these results are comparable with those previously published for normal ones by Singh et al.,^[19] which roughly ranged from 30–120 mg GAE/100 g FW.

Phenolic contents are imperative contributors to functional excellence. Most antioxidant activities from plant sources are derived from phenolic compounds.^[13,28] TP contents promote optimum health partly via their antioxidant and free radical scavenging effects thereby minimizing molecular and cellular damage against free radical induced damage.^[8] However, due to their varied chemical structures, they are likely to possess different antioxidant potential.^[29]

TFs

The TF contents varied significantly (p < 0.05) among the genotypes in terms of both pulp and peel (Table 2). However, the peel of fruits of all genotypes recorded higher (2.5 to 7 folds roughly) TF contents than pulp. The TF contents of peel and pulp were varied from ~92 to ~109 and 38 to 48 mg RE/100 g FW in high lycopene genotypes, respectively, with BCT-115 (dg) being the highest. In case of ordinary tomatoes, the TF contents ranged from ~39 (CLN-B) to 70 (Punjab Chhuhara) and 7.0 (CLN-R) to ~16 (Punjab Chhuhara) mg RE/100 g FW in both peel and pulp, respectively. The rin mutant (BCT-111) failed to develop appreciable amount of flavonoid contents in both peel (0.61) and pulp (0.15).

These values, particularly for pulp, were similar to what was reported by Lenucci et al.^[6] and Ilahy et al.^[5,11] for different normal and high lycopene tomato cultivars ranging from 16.8–47 and 10.56–51.19 mg RE/100 g FW, respectively. Variations can be ascribed to the high-lycopene traits mainly due to genotypic differences.^[11] In fact, it has been reported that in high-pigment mutants the increase in carotenoid content, is accompanied by a dramatic rise in plastid biogenesis and the synthesis of other compounds such as flavonoids and AsA.^[11,30] The darker fruits of color mutants are due to elevated levels of both flavonoids and carotenoids.^[2] Stewart et al.^[31] reported that the majority of the flavonoids in tomatoes are present in the skin. Bovy et al.^[32] opined that in tomato fruit, flavonoids accumulate mainly in the peel, whereas in the flesh, which comprises 95% of the total fruit weight, only traces of flavonoids can be found. However, this is not true in our case, the pulp of all genotypes showed appreciable TF concentration except BCT-111 possessing rin gene.

As a dietary element, the flavonoids do have health-promoting properties, probably due to their high antioxidant capacity. This function/activity is performed by their competence, in vitro, to induce human protective enzyme systems and by a number of epidemiological studies that advocate a protective effect against cardiovascular disease in particular, cancer, and other age-related diseases such as dementia.^[2,8,33]

Antioxidant Assays

RSA and Fe⁺⁺ chelating activity

The DPPH RSA of all genotypes is presented in Table 3. The RSA of tomatoes, in terms of both peel and pulp, differed significantly (p < 0.05). Disregarding the genotypes, peel of the fruits showed higher RSA than the pulp. In case of high pigment genotypes, the highest RSA of peel was recorded in BCT-115 (~75%) followed by Berika, whereas among the ordinary tomato, RSA of the peel was ~58 to ~71%, in BCT-53 being the highest followed by Patharkutchi. The RSA of pulp in both high pigment and normal genotypes was ranged between ~46–52% (the highest in BCT-115) and ~33–46% (the highest in FEB-2), respectively. The rin (BCT-111) had the lowest RSA of both peel (~53%) and pulp (~25%) compared to other genotypes (Table 3). Guil-Guerrero and Rebolloso-Fuentes^[11] also observed similar variation in DPPH scavenging activity among eight tomato varieties without considering the peel and pulp fractions. Odriozola-Serrano et al.^[34] reported the radical (DPPH) scavenging capacity of the six cultivars of tomato ranging from 9.8 to 26.3% of DPPH inhibition in which cv. Bola showed the significantly highest antioxidant capacity. Mansour et al.^[35] pointed out that DPPH radical scavenging capacity ranged from 55–80% among 11 tomato cultivars with significant differences.

TABLE 3 Radical scavenging and metal chelating power (mean ± standard deviation) of tomato genotypes

Genotypes	DPPH scavenging activity (%)		Fe^++ chelating activity (%)
	Peel	Pulp	Peel	Pulp
Berika	75.46 ± 1.43^b	50.93 ± 1.75^ab	46.44 ± 4.86^b	19.33 ± 0.85^b
BCT-115	81.71 ± 1.45^a	52.78 ± 0.69^a	53.12 ± 2.64^a	24.87 ± 2.01^a
BCT-119	71.99 ± 1.51^c	46.53 ± 2.78^a-c	40.55 ± 2.49^c	18.72 ± 1.38^bc
BCT-111	53.01 ± 3.13^h	25.93 ± 5.12^f	20.71 ± 2.39^g	12.44 ± 2.86^f
Punjab Chhuhara	65.05 ± 2.12^f	43.98 ± 1.75^b-d	25.59 ± 2.69^e-g	15.82 ± 2.30^c-e
FEB-2	67.82 ± 3.43^d-f	46.30 ± 1.06^a-c	22.08 ± 2.92^fg	14.46 ± 0.77^d-f
BCT-53	71.30 ± 1.75^cd	44.44 ± 1.84^b-d	26.54 ± 2.04^ef	13.35 ± 1.08^ef
Patharkutchi	68.29 ± 2.23^d-f	47.92 ± 1.39^a-c	33.45 ± 3.07^d	16.89 ± 1.50^b-d
CLN-B	58.56 ± 2.21^g	33.56 ± 1.42^e	29.89 ± 1.04^de	13.10 ± 2.90^ef
CLN-R	67.13 ± 1.06^ef	40.51 ± 2.44^c-e	31.87 ± 3.04^d	14.75 ± 1.84^d-f

Values within column followed by the same superscript letter were not significantly different (p < 0.05) by the Duncan’s Multiple Range Test.

Evaluation of antioxidant potential is becoming increasingly relevant in the field of nutrition and food science as it provides useful information with regard to health promoting and functional quality of raw material without the analysis of each antioxidant compound.^[33,36] Odriozola-Serrano et al.^[34] indicated that most of the antioxidant capacity comes from the natural combination of different phytochemicals. The reduction capability of DPPH radical was determined by the decrease in absorbance by plant antioxidants.

The MC activity of the tomatoes differed significantly (p < 0.05) among genotypes, as well as between peel and pulp of the fruits (Table 3). In case of high lycopene containing genotypes, the highest MC activity of peel was recorded in BCT-115 (~53%) followed by Berika and BCT-119. Among normal genotypes, the MC activity of the peel was varied between ~22 and 33%, being the highest in Patharkutchi followed by CLN-R. In general, the fruit peel of all genotypes had higher MC activity than that of the pulp. The MC activity of pulp was ~18–25% (BCT-115 was the highest and BCT-119 was the lowest) and ~13–17% (Patharkutchi was the highest and CLN-B was the lowest) in high pigment and normal genotypes, respectively. The BCT-111 (this genotype had the rin gene) had the lowest MC activity of peel and pulp, i.e., only ~20 and ~12.5%, respectively (Table 3).

Our results are in accordance with Toor and Savage^[37] who reported the same variation in antioxidant potential of different fractions (peel, pulp, and seed) of three tomato varieties. Varying degrees of antioxidant capacities of the peel and pulp of studied genotypes in all the antioxidant assays could be due to differences in reaction kinetics and steady-state antioxidant potentials of various reductive substrates in the genotypes.^[38,39] In addition, the peel fraction of the studied tomatoes was identified as an important reservoir of antioxidant bioactive compounds. The antioxidant capacity of tomatoes depends on a large number of phytochemical compounds and the interactions that occur between them.^[20,34] Higher antioxidant potential recorded in color mutants (dg and og^c) could be attributed to their high content of lycopene and phenolic compounds.^[2] In contrast, the lowest antioxidant potential was recorded in BCT-111 carrying rin gene, this observation is due to the fact that the BCT-111 (rin mutant) lacks lycopene, which partly contributes to the antioxidant potential of the produce. This also confirms the fact that TPs are important constituents in giving antioxidant power to the BCT-111 genotype.

CONCLUSIONS

Significant differences in bioactive molecules and antioxidant capacities were found between peel and pulp of the fruits, irrespective of the genotypes. The high lycopene containing normal genotypes (Berika) and mutants like BCT-115 and BCT-119 possessing dg and og^c genes were the best among the assessed genotypes. In spite lacking all the desirable characteristics and being devoid of lycopene making it unsuitable for fresh consumption as well as processing, BCT-111 (rin) possessed good antioxidant attributes. The patterns of variation observed in this work would give impetus for planning breeding strategies to develop and improve the new tomato cultivars with good antioxidant properties. Additionally, utilizing tomato peel as a source of phytochemicals could offer diverse opportunities for nutraceutical and functional food applications.