Metabolites, Nutritional Quality and Antioxidant Activity of Red Radish Roots Affected by Gamma Rays

: Radish


Introduction
There is a converse relationship between fruit and vegetable consumption and the incidence of various illnesses, including cardiovascular diseases, cancers, and inflammation disorders [1]. Brassica vegetables improve the immune system, protect against allergies, and decrease the hazards of heart disease and various kinds of cancers [2]. The role of The un-irradiated and irradiated seeds were counted and put in Petri dishes to calculate germination percentage. Each germination assay was performed in triplicate. Samples were collected 45 days after sowing to determine the following.

Photosynthetic Pigments Determination
Chlorophylls a and b plus carotenoids of red radish leaves were evaluated spectrophoto metrically using the method of Vernon and Seely [21]. Fresh leaf samples (0.5 g) were homogenized in a mortar with 85% acetone in the presence of washed and dried sand and CaCo 3 (Ca 0.1 g) in order to neutralize organic acids in the homogenate of the fresh leaf. The homogenate was then filtered through a sintered glass funnel. The residue was washed several times with acetone until the filtrate became colorless. The optical density of the obtained extracts was determined using a spectrophotometer (Jasco model V-530, Tokyo, Japan) at 662 and 644 nm for chlorophyll a and chlorophyll b, respectively, as well as 440.5 nm for carotenoids. Pigment contents were calculated in mg/g FW.

Nutritional and Metabolite Components of Roots 2.6.1. Vitamin C Content
Ascorbic acid (vitamin C) in fresh root samples was examined as mg/100 g FW by 2,6-dichlorophenolindophenol for titration [22]. Twenty grams of red radish roots were homogenized using 40 mL of 3.0% oxalic acid solution. After filtration, 10 mL of the filtrate was titrated with 2,6 dichlorophenolindophenol. Ascorbic acid content was calculated as mg/100 g FW.

Anthocyanin Content
The amount of anthocyanin was measured by the colorimetric method [23]. Findings were calculated as grams of cyanidin-3-O-glucoside/100 g FW (%).

Root Extracts
In brief, 5.0 g of fresh root samples were crushed by liquid nitrogen, and after that, 25 mL of 80% methanol was added, and the mixture shaken for 24 h at 25 • C. After filtration, the extraction was carried out two times [24]. The obtained methanolic extract volume was completed and utilized for the evaluation of free amino acids, soluble protein, phenols, and flavonoid, as well as DPPH.

Total Soluble Protein
The Bradford [25] method was used to determine the total soluble protein amount in root extracts.

Total Phenolic Content
In accordance with Shahidi and Naczk [26], root extracts were spectrophotometrically (Jasco model, V-530, Tokyo, Japan) utilized to determine the total phenolic content with the Folin-Ciocalteu reagent method utilizing gallic acid for the standard curve. The total phenolic concentration was measured at 725 nm as milligrams gallic acid (GAE)/g FW.

Flavonoid Content
The aluminum colorimetric procedure was utilized to evaluate the flavonoid contents. Absorbance rate was estimated at 510 nm by a spectrophotometer (Jasco model V-530,Tokyo, Japan), as followed by Marinova et al. [27]. The obtained results were stated in milligrams of quercetin per gram FW.

Antioxidant Activity
According to Gulluce et al. [28], the purple-colored methanol reagent of 2,2-diphenyl-1picrylhydrazyl was bleached to test the corresponding extract's capacity to donate electrons or hydrogen atoms at 517 nm in comparison to a blank.

Nutrient Concentrations
Dried and ground radish root samples (0.5 g) were placed into a burning cup, and 15 mL of pure HNO 3 was added. The samples were incinerated in a microwave oven at 200 • C, and the solution was diluted to the desired volume with deionized water. The minerals were determined as follows: Nitrogen concentration was evaluated with Kjeldahl's procedure [29]. The contents of calcium (Ca), potassium (K), sulfur (S), phosphorus (P), and magnesium (Mg) in digested dry samples were analyzed separately using an inductively coupled plasma (ICP) optical emission spectrometer, model Prodigy Prism High Dispersion (Teledyne Leeman ICP-OES USA). The minerals were expressed as mg/kg dry sample, except for N, K, and S concentrations, which were in percentage.

Organic Acid Contents
Organic acids were separated by HPLC (Knauer, Germany) using Rezex@ columns. The flow rate was set at 0.6 mL/min, the UV detector was set at 214 nm, the oven temperature was kept stable at 65 • C, the Rezex@ column used for the mobile phase was 0.005 M H 2 SO 4 , and data assimilation was conducted utilizing clarity-chrome software.

Profile of Amino Acids
The amino acid profiles of red radish roots were examined by high performance liquid chromatography (HPLC). Defatted samples (100 mg) were put in screw-capped tubes together with 5.0 mL of HCl 6.0 N. The hydrolyzed tubes were linked to a system that permitted the connection of nitrogen with vacuum lines without disturbing the samples. The tubes were placed in an oven for 24 h at 110 • C [22]. Filtered tubes were evaporated till dryness by a rotary evaporator.
The HPLC investigation was performed with an Agilent 1260 series. Amino acid separation was conducted through an Eclipse Plus C18 column (4.6 mm × 250 mm i.d.,  45:45:10 (B), at a flow rate of 1.5 mL/min. The column was maintained at 40 • C. The data were distinguished from those of amino acid standard solutions. Measurements of free amino acids were performed by contrasting retention time in addition to the peak area of the amino acid standards (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine) compared to the contents found in the treatments (Sigma-Aldrich, 3050 Spruce Street, Saint Louis, MO 63103, USA).

Statistical Analysis
Three replicates were used in a randomized full block design, and the results were presented as means plus standard deviations. Statistical analysis was performed via oneway ANOVA, and the differences in means were calculated utilizing Duncan's multiple rate tests [30] at p ≤ 0.05.

Results and Discussion
Similar to other plants, the occurrence of adverse biotic and abiotic circumstances results in morphological, physiological, and biochemical alterations that may have an impact on the chemical constituents of storage parts [31].

The Germination Percentage
The germination percentage of un-irradiated and γ-irradiated red radish seeds is described in Figure 1. The irradiated seeds demonstrated a high germination percentage of ≥96% for the dose levels of ≤20 Gy, and the germination was continued more than 90% in the γ-irradiated seeds at doses up to 80 Gy. These results are in agreement with Aly et al. [32], who found that 50 Gy enhanced the germination percentage of eggplant.  Red radish seeds appeared to have a high resistance to radiation in terms of their ability to germinate. Gamma-ray doses up to 80 Gy may not have affected the germination of red radish seeds. Additionally, Zanzibar and Sudrajat [33] reported that a low dosage (10 Gy) of γ-rays may be employed to enhance seeds germination, storability, and seedlings development of M. champaca. On the other hand, Waje et al. [34] found that high germination (≥97%) was obtained in red radish seeds irradiated at ≤5 kGy. The sensitivity of plants to radiation depends on features such as plant type or cultivar, plant part, and irradiation dose level [35]. Red radish seeds appeared to have a high resistance to radiation in terms of their ability to germinate. Gamma-ray doses up to 80 Gy may not have affected the germination of red radish seeds. Additionally, Zanzibar and Sudrajat [33] reported that a low dosage (10 Gy) of γ-rays may be employed to enhance seeds germination, storability, and seedlings development of M. champaca. On the other hand, Waje et al. [34] found that high germination (≥97%) was obtained in red radish seeds irradiated at ≤5 kGy. The sensitivity of plants Agronomy 2022, 12, 1916 6 of 20 to radiation depends on features such as plant type or cultivar, plant part, and irradiation dose level [35].

Vegetative Growth
Gamma-rays have been broadly utilized to improve different traits of several plant species [36]. The results presented in Table 2 revealed that the evidenced vegetative growth factors were definitely attributed to gamma-rays. The highest recorded results of plant heights and leaf number/plant were 32.65 cm and 8.08, respectively, at doses of 10 and 40 Gy, respectively. While the lowest values were 23.35 cm and 7.80, respectively, in the control, all the irradiation treatments led to an increase in the length and width of leaves, where the dose of 80 Gy gave the highest value of these parameters (16.41 cm and 9.04 cm, respectively) compared to the control (13.59 cm and 7.53 cm, respectively). Data in Table 3 indicated that the maximum values of root parameters of root length, root diameter, root size, and root weight were confirmed when seeds were exposed to 20 Gy (17.51 cm, 5.45 cm, and 85.25 cm 3 as well as 78.12 g, respectively) followed by 40 Gy (15.71 cm, 5.14 cm, and 85.00 cm 3 and 71.55 g, respectively).  However, the lowest results were achieved by control sample that permitted 14.75 cm, 5.01 cm, 67.50 cm 3 , and 62.96 g, respectively. The use of low doses of γ-irradiation at the dose level of 5.0 Gy produced the greatest values in terms of plant height, branch numbers, and weight of fresh and dry blackberry and Jerusalem artichoke [36,37]. Additionally, Aly et al. [32] declared that 50 Gy enhanced the growth of eggplant in all growth parameters studied (plant height, root length, fruit length, fruit diameter, fresh weight, and dry weight). The current results are in accordance with Aly et al. [38], who found that all considered morphological characteristics (leaf number/plant, leaf length, root diameter, as well as root weight) of red radish were gradually enhanced by increasing gamma-ray dosage level, resulting in enhancement for the dosage of 40 Gy and reduction for the dosage level of 80 Gy. Jaipo et al. [39] stated that low doses of gamma rays might enhance the germination level and seedling development, primarily in these edling condition of several vegetable seeds, where a dose of 50 Gy gave the highest germination percentage and seedling lengths of cucumber and okra. Moreover, Zanzibar and Sudrajat [33] studied different gamma doses up to 200 Gy and found that the growth rate represented by seedling length, neck diameter, leaves number and dry weight of M. champacab increased at γ-ray dose levels up to 80 Gy, and the dosage of 10 Gy produced the greatest results for the majority of the traits.
Low doses of irradiation may lead to amplification of enzymatic activation and awakening of the juvenile embryo, thereby motivating the rate of cell division as well as affecting germination and vegetative growth [40].

Photosynthetic Pigments
Gamma rays are ionizing irradiation and react with atoms or molecules to produce free radicals in cells, and high doses of them can destroy photosynthetic pigments and thus lose the ability of plants to photosynthesize [41]. Figure 2 shows that the concentration of chlorophylls a and b as well as that of carotenoids of red radish plants (0.738, 0.664, and 0.151 mg/g F.W.), respectively, were significantly (p ≤ 0.05) higher at a dose of 20 Gy in contrast with the other treatments followed by the dose of 40 Gy (0.607, 0.484, and 0.145 mg/g FW), respectively, in comparison to the control sample, which had the lowest contents (0.344, 0.212, and 0.062 mg/g FW, respectively). The concentrations of Chl a were greater than Chl b in irradiated and un-irradiated plants. The maximum content of total chlorophylls was obtained by the 40 Gy sample (1.402 mg/g FW). On the other hand, the control had the lowest total chlorophyll levels (0.556 mg/g FW). There was a favorable relationship between the outcomes of growth as well as biochemical indicators. These results were consistent with Aly et al. [37], who reported that low doses of gammarays increased Chl a, Chl b, and carotenoid content in blackberry plantlets. Additionally, Aly et al. [38] showed that γ-irradiation had a stimulating effect on chlorophylls, as the content of chlorophylls a and b was increased by radiation (40 Gy) in red radish. Therefore, the stimulating impact of low doses of γ-rays on the pigments is in turn reflected in the vegetative development. The biological effect of gamma radiation is mainly due to the development of free radicals through the hydrolyses of H 2 O, which can lead to modification of the antioxidative structure, and the gathering of phenols and chlorophyll pigments [42]. Moreover, Jan et al. [43] noted that low doses of gamma-irradiation may lead to activation of the photo synthetic pigments (chlorophyll a, chlorophyll b, and total chlorophylls) in Cullen corylifolium L. Medik by improving photosynthesis capabilities. Similarly, dry lettuce (Lactuca sativa var. capitata) seeds treated by gamma-ray dose levels between 2 and 70 Gy revealed that seeds irradiated at dose levels up 30 Gy had increased photosynthesis pigment (Chl a, Chl b, and carotenoid) concentrations, whereas the highest dose (70 Gy) caused a reduction of the pigments [44].
Agronomy 2022, 12,1916 8 of 21 stimulating impact of low doses of γ-rays on the pigments is in turn reflected in the vegetative development. The biological effect of gamma radiation is mainly due to the development of free radicals through the hydrolyses of H2O, which can lead to modification of the antioxidative structure, and the gathering of phenols and chlorophyll pigments [42]. Moreover, Jan et al. [43] noted that low doses of gamma-irradiation may lead to activation of the photo synthetic pigments (chlorophyll a, chlorophyll b, and total chlorophylls) in Cullen corylifolium L. Medik by improving photosynthesis capabilities. Similarly, dry lettuce (Lactuca sativa var. capitata) seeds treated by gamma-ray dose levels between 2 and 70 Gy revealed that seeds irradiated at dose levels up 30 Gy had increased photosynthesis pigment (Chl a, Chl b, and carotenoid) concentrations, whereas the highest dose (70 Gy) caused a reduction of the pigments [44]. Additionally, plants are able to acclimatize to the environment more broadly by large photon energy utilization effectiveness by enhancing the proportion of Chl b in response centers (complex structures of protein, lipid, cofactors, and metals that are optimized via complicated mechanisms to capture and convert light energy) included in photosynthesis. These results are in a harmony with the current findings, presenting a constructive association with development features of red radish.

Nutritional and Metabolite Components of Roots
Vitamin C is a main compound of the plant defense system that protects plant bio-  Additionally, plants are able to acclimatize to the environment more broadly by large photon energy utilization effectiveness by enhancing the proportion of Chl b in response centers (complex structures of protein, lipid, cofactors, and metals that are optimized via complicated mechanisms to capture and convert light energy) included in photosynthesis. These results are in a harmony with the current findings, presenting a constructive association with development features of red radish.

Nutritional and Metabolite Components of Roots
Vitamin C is a main compound of the plant defense system that protects plant biological systems from reactive oxygen species produced during biotic and abiotic stresses [13]. Humans cannot synthesize vitamin C and thus it should be provided from different sources, such as fruits and vegetables [45].
In addition, adults are advised to consume 75 mg of vitamin C daily for women and 90 mg daily for men [46]. Representative findings of ascorbic acid (mg/100 g FW) generated in red radish roots determined with various doses of gamma irradiation are shown in Figure 3. The results acquired showed that the mean contents of ascorbic acids increased significantly (p ≤ 0.05) with low doses of gamma irradiation treatments, and the highest value (19.62 mg/100 g FW) of ascorbic acid was proven for the irradiation dose of 20 Gy, followed by 40 Gy (18.63 mg/100 g FW), whereas the lowest result (14.30 mg/100 g FW) for ascorbic acid was found for the control sample. Thus, the use of gamma rays can also enhance crop quality; therefore, the content of ascorbic acid is affected by many factors, including variety, temperature, storage conditions, and age [47]. The obtained results of the current study had the same pattern as those of Aly et al. [37,48], who indicated that ascorbic acid content was at its maximum increase at the gamma-ray dose level of 40 Gy in blackberry (Rubus fruticosus L.) plantlets and cilantro (Eryngium foetidum L.), respectively. Hanafy and Akladius [19] reported that plants produced from fenugreek seeds exposed to gamma irradiation up to 25 Gy contained more vitamin C in their leaves compared to untreated plants, in equivalence with an increase in radiation dose, while this content was decreased by the dose level of 400 Gy. Depending on the absorbed dosages, gamma-rays are one of the potent agents that can alter the physiological and biochemical characteristic [49]. Several studies have shown that high dosages of gamma-rays inhibit plant growth, causing the generation of free radicals, which have devastating impacts on physiological, morphological, and anatomical features regarding the irradiation dose levels [19]. After thiamine (vitamin B), ascorbic acid is considered of the main radiationsensitive water-soluble vitamins [50]. The potential cause of the accelerating loss of vitamin C at high radiation dose levels might be attributed to an enhancement in respiration values, which would then lead to an increase in enzyme activity, which quickly degrades ascorbate, or it might be because of a partial conversion of ascorbate to dehydroascorbic, which may clarify the loss of vitamin C level in the plants [51]. The low level of vitamins might be associated with the neutralization of reactive oxygen species (ROS) induced by radiation [52].

Anthocyanin Content
Cultivars of red radishes are a prospective resource of natural colorant attributed to the presence of anthocyanin, which has excellent stability and is extremely similar to the synthetic pigments (Food Red No. 40) [53,54]. Additionally, anthocyanin is well-known for its health advantages, including the capacity to remove free radicals, control cancers and diabetes, avoid nerve cell and cardiovascular disorders, and reduce inflammation. The content of anthocyanin of red radish roots affected by gamma rays is illustrated in Figure 4. The results show that the anthocyanin content ranged from 3.30 to 3.74%. All irradiation doses enhanced the anthocyanin content, and its highest value was at the dose of 40 Gy (3.74%), followed by the dose of 20 Gy (3.66%), while it had the lowest value in the control treatment (3.30%).
sitive water-soluble vitamins [50]. The potential cause of the accelerating loss of vitamin C at high radiation dose levels might be attributed to an enhancement in respiration values, which would then lead to an increase in enzyme activity, which quickly degrades ascorbate, or it might be because of a partial conversion of ascorbate to dehydroascorbic, which may clarify the loss of vitamin C level in the plants [51]. The low level of vitamins might be associated with the neutralization of reactive oxygen species (ROS) induced by radiation [52].

Anthocyanin Content
Cultivars of red radishes are a prospective resource of natural colorant attributed to the presence of anthocyanin, which has excellent stability and is extremely similar to the synthetic pigments (Food Red No. 40) [53,54]. Additionally, anthocyanin is well-known for its health advantages, including the capacity to remove free radicals, control cancers and diabetes, avoid nerve cell and cardiovascular disorders, and reduce inflammation. The content of anthocyanin of red radish roots affected by gamma rays is illustrated in Figure 4. The results show that the anthocyanin content ranged from 3.30 to 3.74%. All irradiation doses enhanced the anthocyanin content, and its highest value was at the dose of 40 Gy (3.74%), followed by the dose of 20 Gy (3.66%), while it had the lowest value in the control treatment (3.30%).  Findings of the recent study are in contrast to Mohajer et al. [55], who investigated the results of γ-rays at 30, 60, 90, and 120 Gy on in vitro development issues and phytochemical and nutritional contents of Sainfoin leaves and found that γ-irradiation significantly reduced the anthocyanin content in inverse proportion to irradiation intensity.

Total Soluble Protein Content
Brassicaceae vegetables, including radish, are attracting interest as functional plants due to their advantageous nutritional compounds, such as amino acids, fibers, minerals, carbohydrates, and proteins [56]. In actuality, plants provide the majority of the proteins needed by human. Changes of total soluble protein content in roots of red radish plants were induced by various dose levels of gamma-rays ( Figure 5). The findings provided a significant increase in total soluble protein with increasing doses up to 40 Gy and then decreased by increasing the gamma-ray dose levels. Accordingly, the 40 Gy dose gave the greatest value (4.90 mg/g FW), followed by the 80 Gy dose (4.75 mg/g FW), compared to the control, which gave the lowest value (3.84 mg/g FW). These results are consistent with Hanafy and Akladius [19], who showed that low doses of γ-rays led to a gradual increase in leaf soluble proteins, especially at 100 Gy in fenugreek plants. In the same context, Aly et al. [32] showed that the total soluble protein concentration of various parts of eggplant was increased by the 50 Gy radiation dose level. One of the plant's protective mechanisms Findings of the recent study are in contrast to Mohajer et al. [55], who investigated the results of γ-rays at 30, 60, 90, and 120 Gy on in vitro development issues and phytochemical and nutritional contents of Sainfoin leaves and found that γ-irradiation significantly reduced the anthocyanin content in inverse proportion to irradiation intensity.

Total Soluble Protein Content
Brassicaceae vegetables, including radish, are attracting interest as functional plants due to their advantageous nutritional compounds, such as amino acids, fibers, minerals, carbohydrates, and proteins [56]. In actuality, plants provide the majority of the proteins needed by human. Changes of total soluble protein content in roots of red radish plants were induced by various dose levels of gamma-rays ( Figure 5). The findings provided a significant increase in total soluble protein with increasing doses up to 40 Gy and then decreased by increasing the gamma-ray dose levels. Accordingly, the 40 Gy dose gave the greatest value (4.90 mg/g FW), followed by the 80 Gy dose (4.75 mg/g FW), compared to the control, which gave the lowest value (3.84 mg/g FW). These results are consistent with Hanafy and Akladius [19], who showed that low doses of γ-rays led to a gradual increase in leaf soluble proteins, especially at 100 Gy in fenugreek plants. In the same context, Aly et al. [32] showed that the total soluble protein concentration of various parts of eggplant was increased by the 50 Gy radiation dose level. One of the plant's protective mechanisms against gamma ray damage is to increase the levels of soluble proteins [57]. In current research, red radish seeds exposed to low dose levels of gammarays elevated the content of total soluble proteins in the generated plants in comparison to un-irradiated plants. Other studies also demonstrated that total protein decreases with elevating gamma-ray doses induced by higher metabolites and hydrolase enzyme activity in germinating seeds [58].

Total Phenolic and Flavonoid Contents
The high total phenolic contents of plants are associated with the antimicrobial and antioxidant capacity of plants [61,62]. As a result, several research teams have concentrated on identifying the bioactive substances from the radish plant that could be effective chemo preventive drugs.
Total phenolic content identified through the Folin assay showed that gamma-rays increased the total phenolic content (Figure 6), and the highest content (0.840 mg/g FW) was obtained by a dose of 40 Gy followed by a dose of 20 Gy (0.722 mg/g FW) compared with the control, which gave the lowest content (0.399 mg/g FW). Comparable results were achieved for the total flavonoid concentration ( Figure 6). The maximum increase of flavonoid (0.029 mg/g FW) was recorded at a dose of 40 Gy followed by a dose of 20 Gy (0.0196 mg/g FW) compared with the control, which gave the lowest content (0.0121 mg/g FW). These results are in agreement with Hanafy and Akladius [19], who found that exposing fenugreek seeds to low dose levels of gamma-irradiation (25, 50, 100, and 200 Gy) resulted in a critical incrementin phenolic and flavonoid contents of the generated plants in comparison with the un-irradiated sample, and the level of stimulation was increased the most at 100 Gy. On the other hand, the total phenol and flavonoid content in plants generated from irradiated seeds was decreased by the highest dose (400 Gy) of irradiation. Additionally, Aly et al. [32] established that the 50 Gy dosage levels of γ-rays increased the phenol and flavonoid content in eggplant fruits. Additionally, the current findings are in line with Mohajer et al. [55], who discovered their remarkable incrementin phenolic and flavonoid contents of in vitro grown Sainfoin leaves, particularly at a dose of 90 Gy. Phytonutrient substances, such as flavonoids and phenols, have an indirectly effect on the quality of red radish. Phytochemical substances demonstrated variations in the state of phenol and flavonoid contents in red radish roots as a result of γ-ray treatments. Gamma- Protein degradation and recycling are essential responses of plants to stresses, since the breakdown of protein generates free amino acids needed for the de novo synthesis of new proteins [59]. Utilizing high doses of gamma-rays may also lead to high component extractability. Furthermore, gamma-rays produce a disulfide bridge linking the polypeptide chain, which can affect the accumulation and conformation of the small molecular weight protein [19,60].

Total Phenolic and Flavonoid Contents
The high total phenolic contents of plants are associated with the antimicrobial and antioxidant capacity of plants [61,62]. As a result, several research teams have concentrated on identifying the bioactive substances from the radish plant that could be effective chemo preventive drugs.
Total phenolic content identified through the Folin assay showed that gamma-rays increased the total phenolic content (Figure 6), and the highest content (0.840 mg/g FW) was obtained by a dose of 40 Gy followed by a dose of 20 Gy (0.722 mg/g FW) compared with the control, which gave the lowest content (0.399 mg/g FW). Comparable results were achieved for the total flavonoid concentration ( Figure 6). The maximum increase of flavonoid (0.029 mg/g FW) was recorded at a dose of 40 Gy followed by a dose of 20 Gy (0.0196 mg/g FW) compared with the control, which gave the lowest content (0.0121 mg/g FW). These results are in agreement with Hanafy and Akladius [19], who found that exposing fenugreek seeds to low dose levels of gamma-irradiation (25, 50, 100, and 200 Gy) resulted in a critical incrementin phenolic and flavonoid contents of the generated plants in comparison with the un-irradiated sample, and the level of stimulation was increased the most at 100 Gy. On the other hand, the total phenol and flavonoid content in plants generated from irradiated seeds was decreased by the highest dose (400 Gy) of irradiation. Additionally, Aly et al. [32] established that the 50 Gy dosage levels of γ-rays increased the phenol and flavonoid content in eggplant fruits. Additionally, the current findings are in line with Mohajer et al. [55], who discovered their remarkable incrementin phenolic and flavonoid contents of in vitro grown Sainfoin leaves, particularly at a dose of 90 Gy. Phytonutrient substances, such as flavonoids and phenols, have an indirectly effect on the quality of red radish. Phytochemical substances demonstrated variations in the state of phenol and flavonoid contents in red radish roots as a result of γ-ray treatments. Gamma-rays might be the reason for oxidative damage and weakened taste in plants. Nevertheless, the effective action and radio-stable nature of antioxidants can protect the chemical oxidation of biomolecules in irradiated plants. By donating hydrogen atoms or electrons, phenol compounds gain antioxidant defense characteristics and may also stabilize intermediate radicals. The obtained data (Figure 6) indicated that the phenol concentrations increased with γ-ray dosage levels, while the control sample resulted in the lowest phenol content. In corn callus tissues and soybean plants exposed to gamma radiation at doses between 50 and 150 Gy, an increase in phenol content was observed [63,64].
Depending on the radiation dosage, gamma-rays reacted with various atoms and particles in the cell, particularly water molecules, and generated free radical that may alter crucial plant cell contents, such as proteins, lipids, etc. [65]. In fact, the formation of free radicals can act as a stress signal, activating stress reactions and increasing phenolic acid contents that provide noteworthy antioxidant activities [66].The increase in total phenols can be attributed to phenylalanine ammonia-lyase (PAL) activity, which is one of the synthesis enzymes of phenolic compounds [67]. In this regard, it was established that radiation may increase PAL activities [68], leading to the result of phenol accumulation in plant tissue. In contrast, the lowest concentration of phenols at high levels of irradiation might be attributed to the degradation or insolubilization of phenol components. Ahn et al. [69] reported that when Chinese cabbage was irradiated with high doses of gamma radiation, the phenol content considerably decreased. Flavonoid is one form of secondary metabolites widely found in plants and repairs the damage caused byradiation stress. The outcomes of are cent study confirmed that a noteworthy increment in flavonoid content re- By donating hydrogen atoms or electrons, phenol compounds gain antioxidant defense characteristics and may also stabilize intermediate radicals. The obtained data ( Figure 6) indicated that the phenol concentrations increased with γ-ray dosage levels, while the control sample resulted in the lowest phenol content. In corn callus tissues and soybean plants exposed to gamma radiation at doses between 50 and 150 Gy, an increase in phenol content was observed [63,64].
Depending on the radiation dosage, gamma-rays reacted with various atoms and particles in the cell, particularly water molecules, and generated free radical that may alter crucial plant cell contents, such as proteins, lipids, etc. [65]. In fact, the formation of free radicals can act as a stress signal, activating stress reactions and increasing phenolic acid contents that provide noteworthy antioxidant activities [66].The increase in total phenols can be attributed to phenylalanine ammonia-lyase (PAL) activity, which is one of the synthesis enzymes of phenolic compounds [67]. In this regard, it was established that radiation may increase PAL activities [68], leading to the result of phenol accumulation in plant tissue. In contrast, the lowest concentration of phenols at high levels of irradiation might be attributed to the degradation or insolubilization of phenol components. Ahn et al. [69] reported that when Chinese cabbage was irradiated with high doses of gamma radiation, the phenol content considerably decreased. Flavonoid is one form of secondary metabolites widely found in plants and repairs the damage caused byradiation stress. The outcomes of are cent study confirmed that a noteworthy increment in flavonoid content resulted due to irradiation. Similar findings were made when soybean seedlings were exposed to UV-B irradiation; the flavonoid contents were elevated [70]. The reduction in flavonoid contents is due to their neutralizing function of the oxidative stress systems provoked with γ-rays. According to Taheri et al. [71], Curcuma alismatifolia leaf may become more effective in scavenging free radicals by accumulating bioactive substances, such as phenol and flavonoid components, when treated by radiation doses up to 20 Gy. Moreover, Said et al. [72] demonstrated that utilizing gamma-rays significantly increased total flavonoid contents of the dill herb.

Antioxidant Activity by DPPH Assay
The concentrations of phenol, flavonoid, and anthocyanin components in plant extracts lead to their antioxidant activities [73]. Flavonoid molecules have many hydroxyl groups, which often have significant antioxidant properties [74]. Red radish is high in antioxidants [53]. In this study, the antioxidant activity was examined using the DPPH radical, which is one of the most effective methods utilized for antioxidant evaluation as well as to determine the free radical scavenging activities of plant extracts. The DPPH free radical scavenging properties of red radish roots are shown in Figure 7. All treatments revealed higher DPPH radical scavenging activity compared to the control, and the 40 Gy dose level had the best scavenging activity of the DPPH radical (87.25%), while the lowest scavenging activity was in the control sample (81.15%). The current findings are in agreement with Aly et al. [32], who studied the effect of gamma rays on eggplant fruits and found that they led to an enhancement in the antioxidant properties, particularly at the dosage of 50 Gy.
Agronomy 2022, 12,1916 13 of 21 as to determine the free radical scavenging activities of plant extracts. The DPPH free radical scavenging properties of red radish roots are shown in Figure 7. All treatments revealed higher DPPH radical scavenging activity compared to the control, and the 40 Gy dose level had the best scavenging activity of the DPPH radical (87.25%), while the lowest scavenging activity was in the control sample (81.15%). The current findings are in agreement with Aly et al. [32], who studied the effect of gamma rays on eggplant fruits and found that they led to an enhancement in the antioxidant properties, particularly at the dosage of 50 Gy. El-Beltagi et al. [17] and Baltrusaityte et al. [75] obtained good correlations between antioxidant activity and phenol creation as well as flavonoid content, demonstrating that phenol and flavonoid components are considered to be main factors influencing the antioxidant capacity of red radish roots. In order to repair the damage initiated by reactive oxygen species (ROS), plants have developed extremely antioxidant protection properties, which include enzymatic and non-enzymatic scavengers that constitute available part within the cellular defense system to overcome irradiation stress depending on the absorbed dose level [44]. Non-enzymatic antioxidants provide imperative functions in metabolism and in scavenging ROS in biological systems [71].
The correlation of total phenolic and flavonoid content with antioxidant activity is shown in Figure 8. High correlations between antioxidant activity and total phenols (R 2 = 0.939) and total flavonoids (R 2 = 0.733) were observed. By comparing the correlation coefficients (R-values), it is possible to suggest that phenolic groups are highly responsible for El-Beltagi et al. [17] and Baltrusaityte et al. [75] obtained good correlations between antioxidant activity and phenol creation as well as flavonoid content, demonstrating that phenol and flavonoid components are considered to be main factors influencing the antioxidant capacity of red radish roots. In order to repair the damage initiated by reactive oxygen species (ROS), plants have developed extremely antioxidant protection properties, which include enzymatic and non-enzymatic scavengers that constitute available part within the cellular defense system to overcome irradiation stress depending on the absorbed dose level [44]. Non-enzymatic antioxidants provide imperative functions in metabolism and in scavenging ROS in biological systems [71].
The correlation of total phenolic and flavonoid content with antioxidant activity is shown in Figure 8. High correlations between antioxidant activity and total phenols (R 2 = 0.939) and total flavonoids (R 2 = 0.733) were observed. By comparing the correlation coefficients (R-values), it is possible to suggest that phenolic groups are highly responsible for the antioxidant activity of red radish roots at a 95 % coincidence level. Phenolic and flavonoid molecules are important antioxidant components which are responsible for deactivating free radicals based on their ability to donate hydrogen atoms to free radicals. They also have ideal structural characteristics for free radical scavenging and there is a linear correlation of total phenolic and flavonoid content with antioxidant capacity [37,76].

Elemental Composition
The qualities of plants and their parts may be greatly influenced by their mineral nutritional state [77]. The composition of elements (N, K, S, P, Ca, and Mg) in red radish roots affected by gamma rays is displayed in Table 4. The exposure to gamma-rays had a beneficial effect on enhancing the content of the studied nutrients. The effectiveness of gamma-ray exposure could also be seen in the quantity of nutrients, as it increased with the use of gamma-rays, whereas the contents of N and Mg were increased by irradiation up to 20 Gy (1.78% and 2414.2 ppm, respectively) compared to the control (1.44% and 2249.9 ppm, respectively) and then decreased with increasing irradiation dosage levels. The greatest increase in the concentration of both P and Ca was obtained at the dose level of 80 Gy (1942.5 and 2346.8 ppm, respectively). Regarding potassium and sulfur content, it was observed that the 40 Gy dose level gave the highest values (2.88% and 0.652%, respectively) compared with the control (2.18% and 0.253%, respectively). It is clear from these results that potassium was the most abundant in roots of red radish. These results are consistent with Mohajer et al. [55], who found that gamma irradiation increased the amount of P and N of Sainfoin seeds, especially at a dose of 30 Gy. El-Beltagi et al. [17] showed that the γ-irradiation dose level of 50 Gy was more successful for enhancing the ion balances in roots and shoots of cowpea plants. Nutrients are

Elemental Composition
The qualities of plants and their parts may be greatly influenced by their mineral nutritional state [77]. The composition of elements (N, K, S, P, Ca, and Mg) in red radish roots affected by gamma rays is displayed in Table 4. The exposure to gamma-rays had a beneficial effect on enhancing the content of the studied nutrients. The effectiveness of gamma-ray exposure could also be seen in the quantity of nutrients, as it increased with the use of gamma-rays, whereas the contents of N and Mg were increased by irradiation up to 20 Gy (1.78% and 2414.2 ppm, respectively) compared to the control (1.44% and 2249.9 ppm, respectively) and then decreased with increasing irradiation dosage levels. The greatest increase in the concentration of both P and Ca was obtained at the dose level of 80 Gy (1942.5 and 2346.8 ppm, respectively). Regarding potassium and sulfur content, it was observed that the 40 Gy dose level gave the highest values (2.88% and 0.652%, respectively) compared with the control (2.18% and 0.253%, respectively). It is clear from these results that potassium was the most abundant in roots of red radish. These results are consistent with Mohajer et al. [55], who found that gamma irradiation increased the amount of P and N of Sainfoin seeds, especially at a dose of 30 Gy.
El-Beltagi et al. [17] showed that the γ-irradiation dose level of 50 Gy was more successful for enhancing the ion balances in roots and shoots of cowpea plants. Nutrients are important for plants; for instance, sulfur is the fourth most important macro element for phytonutrients after nitrogen, phosphorus, and potassium. Sulfates may be absorbed by plants that can transform them into essential amino acids and are needed for metabolism. Higher plants' development and growth depend heavily on sulfur [78]. Additionally, potassium is one of the necessary elements of the plant and has an important part in the biosynthesis of vitamins and proteins as well as in the metabolism of nucleic acids [13]. Additionally, potassium is one of the vital elements involved in stomatal-opening, osmoregulation, photosynthesis and enzyme activation, and cell wall enlargement [79].

Organic Acid Content
Organic acids are important plant components for biological processes and human health because they promote mild antibacterial activity [80]. Oxalic, malic, malonic, and erythorbic acid are the main organic acids found in radish root [81]. Additionally, organic acid content varies by climate, cultivation condition, and cultivar [61]. In addition, they are essential for the quality and sensory attractiveness of fruits and vegetables [65]. In this study, four organic acids, namely, oxalic, malic, succinic and glutaric acids were obtained from red radish roots affected by gamma rays (Figure 9). The results indicated that the content of organic acids was increased using radiation doses, and the highest content was provided by the dose level of 20 Gy (108.08, 21.59, and 5.05 mg/mg DW, respectively) for oxalic, succinic, and glutaric acids, respectively. Furthermore, malic acid gained the highest value (40.18 mg/mg DW) at the 80 Gy dose level.
The preservation of organic acids is of most importance, as they are related to organoleptic properties of plant-based foods, such as their taste, fragrance, and texture [82]. Additionally, potassium is one of the vital elements involved in stomatal-opening, osmoregulation, photosynthesis and enzyme activation, and cell wall enlargement [79].

Organic Acid Content
Organic acids are important plant components for biological processes and human health because they promote mild antibacterial activity [80]. Oxalic, malic, malonic, and erythorbic acid are the main organic acids found in radish root [81]. Additionally, organic acid content varies by climate, cultivation condition, and cultivar [61]. In addition, they are essential for the quality and sensory attractiveness of fruits and vegetables [65]. In this study, four organic acids, namely, oxalic, malic, succinic and glutaric acids were obtained from red radish roots affected by gamma rays (Figure 9). The results indicated that the content of organic acids was increased using radiation doses, and the highest content was provided by the dose level of 20 Gy (108.08, 21.59, and 5.05 mg/mg DW, respectively) for oxalic, succinic, and glutaric acids, respectively. Furthermore, malic acid gained the highest value (40.18 mg/mg DW) at the 80 Gy dose level.
The preservation of organic acids is of most importance, as they are related to organoleptic properties of plant-based foods, such as their taste, fragrance, and texture [82].

Amino Acid Profiles
The amino acid pool represents a minor portion of the total dissolved organic nitrogen pool, which generally includes less than ten percent of free amino acids in moderate ecosystems, and free amino acids may react as membrane-defensive agents that protect cells from biotic and abiotic stress before and after harvest [19]. The main significant purpose of amino acids is their function as a component of protein, and they are required in secondary metabolites, and the biosynthesis of components like glucosinolates and phenols in plants, which are imperative tasks, both directly and indirectly, for plant environment reactions and human health [56].

Amino Acid Profiles
The amino acid pool represents a minor portion of the total dissolved organic nitrogen pool, which generally includes less than ten percent of free amino acids in moderate ecosystems, and free amino acids may react as membrane-defensive agents that protect cells from biotic and abiotic stress before and after harvest [19]. The main significant purpose of amino acids is their function as a component of protein, and they are required in secondary metabolites, and the biosynthesis of components like glucosinolates and phenols in plants, which are imperative tasks, both directly and indirectly, for plant environment reactions and human health [56].
The content of amino acids of red radish roots as affected by different doses of gammarays is presented in Table 5. Seventeen amino acid were obtained in the treatments: aspartic acid (Asp), glutamic acid (Glu), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), Cystine, methionine (Met), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys), and proline (Pro). It was observed that the gamma irradiation at various dosage levels significantly increased the values of the amino acid pool, more than the control. Thr, Arg, Val, Met, Phe, Ile, Leu, and Lys were found to be essential amino acids, and histidine was classified as a non-essential amino acid [83]. Radiation was used to increase the majority of these significant amino acids, with the exception of Lys, which was reduced. In the amino acid profile of red radish roots, Pro was predominant, and the remaining amino acids behind it were found in low percentages, and the current findings were in accordance with the results of Schiavon et al. [84]. The amount of amino acids that proceed as originators of glucosinolate (GLS) (phenylalanine, tyrosine, and methionine) increased after exposure to gamma irradiation, especially at doses 40 and 80 Gy. It was obvious that the amino acid pool was enhanced with irradiation in comparison to the un-irradiated, and the 40 Gy dose level gave the highest total amino acids content (95.10 µg/g DW) followed by 80 Gy (89.04 µg/g DW) compared to the un-irradiated (86.18 µg/g DW). These results are in line with Moemen [85], who found that gamma rays (20,40, and 80 Gy) significantly increased amino acid pool levels in Ambrosia maritima plants, and the 40 Gy or 80 Gy dose was more effective. In the same context, Aly et al. [86] observed that the used gamma-ray dose levels (100-300) increased the content of the amino acid pool. Methionine is a sulfur-including amino acid that is important for humans and therefore has to be provided entirely from dietary sources, and its content was increased using irradiation, with a dose of 80 Gy giving the highest content (2.16 µg/g DW) followed by 40 Gy (2.13 µg/g DW), while the control gave the lowest content (1.77 µg/g DW). A slight enhancement in the value of aromatic amino acids, including phenylalanine and tyrosine, was found with increasing irradiation dose, and the highest content was at the dose 40 Gy (3.24 µg/g DW) compared to the un-irradiated treatment (3.15 µg/g DW). Findings are provided as means ± SD (n = 3), and different letters within the same column are statistically different (p ≤ 0.05).
The human body requires the intake of essential amino acid through the consumption of food to perform its metabolic processes [87][88][89][90], and the recent results indicated that the amino acids were increased with the use of γ-radiation dose levels of 40 Gy followed by 80 Gy.

Conclusions
The consumption of radish roots due to the presence of phytochemicals with diverse beneficial properties confers health promotion with natural medicinal value. The current results indicated that lower doses of gamma-ray irradiation increased the development traits characteristics and several biochemical components of red radish roots, whereby doses of 20 and 40 Gy of γ-radiation were superior in enhancing the quality characteristics associated with biochemical constituents of red radish roots. Additionally, there was an increase in the content of vitamin C using gamma rays, and the highest content (19.62 mg/100 g FW) was obtained at the dose of 20 Gy. The content of some metabolites such as anthocyanin, phenols, and flavonoids was affected by gamma rays, which increased the antioxidant activity to a maximum at a dose of 40 Gy. In addition, exposure to γ-irradiation had encouraging impacts on enhancing the concentrations of the elements (N, K, S, P, Ca, and Mg). The efficiency of exposure to gamma-rays was also observed in the content of elements, which increased with the use of gamma-rays. Similarly, the amino acids pool was significantly increased by radiation, as were the contents of amino acids that act as precursors to glucosinolate GLS (phenylalanine, tyrosine, as well as methionine), which increased after exposure to gamma irradiation, especially at doses of 40 and 80 Gy. Therefore, gamma-rays are one of the tools utilized to enhance the growth plus quality of crops, particularly in low doses. The future prospects of this study is to extract some important compounds that were affected by radiation such as phenols, flavonoids and anthocyanins, which are natural antioxidants, and separate them to identify them, in addition to studying the enzymatic activity of some enzymes related to the synthesis of these compounds and demonstrating the extent of the radiation effect on them.