Determination of Antioxidant Content and Capacity of Four Jordanian Fresh Citrus Fruits

Citrus fruits are considered one of the most grown crops worldwide including Jordan with high nutritive and non-nutritive value. The consumption of foods that contain natural antioxidants is thought to be an efficient way for reducing the risk for oxidative stress diseases. Determination of antioxidant content and capacity allows the screening of plants that are probably involved in the prevention and/or treatment of oxidative stress diseases. This study aimed at comparing four fresh Jordanian citrus fruits (namely: orange, lemon, pomelo, and mandarin) for their antioxidant content (using two methods namely: Folin-Ciocaltaeu method and total flavonoids method) and capacity (using two methods namely: 2,2-diphenyl-picrylhydrazyl (DPPH) and cupric antioxidant reducing capacity (CUPRAC) assays). Three solvents were used for the fruit extraction (ethanol, methanol, and water). There were significant (P < 0.05) differences between the fruits in terms of antioxidant content and capacity. Regardless of the extraction solvent, the fruit content of total polyphenols (M catechin/100 g) was as follows: lemon > mandarin > pomelo > orange. The total flavonoid content (mM rutin/100 g) of the fruits was: orange > mandarin > lemon > pomelo. On the other hand, the antioxidant capacity (M trolox/100 g) of the fruits was: lemon > mandarin > orange > pomelo. The antioxidant capacity (measured as mg vitamin C/ml extract) of the fruits was: mandarin > orange > pomelo > lemon. Different extracts of different fruits showed significantly (P < 0.05) different antioxidant contents and capacities. No correlation between antioxidant content and antioxidant capacity of the studied fruits has been found.


Introduction
Fruits and vegetables are considered of the most important natural sources of antioxidants (Apak et al., 2007).Citrus fruits are considered one of the most grown crops worldwide, including Jordan, with high nutritive and health values.The health benefits of citrus fruits have been attributed mainly to the presence of antioxidants such as phenolics and ascorbic acid (Kumar, Lamers, Singh, Ladaniya, & Sthapit, 2015).Jordan environment is rich and there is diversity in the Jordanian crop production (Qura'n, 2010).The Jordanians' consumption of citrus fruit is estimated to be 7.64 Kg/caput/year (FAO, 2018).
Free radicals are highly reactive chemical species that are able to affect the unstable atoms or molecules leading to a subsequent cascade of free radical oxidative reactions.Oxidative reactions are considered important because they might lead to imbalanced oxidative status of a living cell and to oxidative stress diseases accordingly.Many chronic diseases are thought to have oxidative stress background in their pathogenesis.Diabetes mellitus, cardiovascular diseases, and some types of cancers are considered nowadays as oxidative stress diseases.Antioxidants are chemical substances that are present naturally or synthetically capable for stopping a free radical chain reaction either by scavenging free radicals or by neutralizing the effects of free radicals.The consumption of antioxidants is thought to reduce the risk for oxidative stress diseases (Halliwel & Gutteridge, 1995).There were many attempts to find the antioxidant content, capacity, and specific antioxidant compounds in citrus fruits.Nonetheless, there is no figures regarding the antioxidant content and capacity for fresh citrus fruit produced in Jordan using different extraction solvents.Thus, the purpose of this study is to screen four Jordanian fresh citrus fruits that are consumed regularly in Jordan (namely: orange, lemon, pomelo, and mandarin) for their antioxidant content and capacity.Antioxidant content was determined by two methods namely: Folin-Ciocultae method and total flavonoids method.Antioxidant capacity was determined by two methods namely: 2,2-diphenyl-picrylhydrazyl (DPPH) and cupric antioxidant reducing capacity (CUPRAC) assays.This study aimed also to find (if any) a correlation between the antioxidant content and antioxidant capacity of the selected fruits.It is probably the first study that evaluated the antioxidant content and capacity of locally produced fresh Jordanian citrus fruits extracted by three solvents (i.e. ethanol, methanol, and water).We expect to add a value to the scientific antioxidant database.
Based on the previous research reviewed by the researchers, the null hypotheses (H 0 ) of this research state that: (a) there is a significant difference between the antioxidant content and capacity values of four fresh Jordanian citrus fruits namely: orange, lemon, pomelo, and mandarin extracted by three solvents (ethanol, methanol, and water) and (b) there is a correlation between the antioxidant content and capacity values of the fruits (the antioxidant capacity of the fruits is related to the fruit content of the extracted antioxidants).The alternative hypotheses (H 1 ) of this research are: (a) there is no significant difference between the antioxidant content and capacity values of four fresh Jordanian citrus fruits namely: orange, lemon, pomelo, and mandarin extracted by three solvents (ethanol, methanol, and water) and ( 2) there is no correlation between the antioxidant content and capacity values of the fruits.

The Material Studied
Fresh citrus fruits that were locally produced in Jordan were studied.As reported by the seller, fruits were collected in the same day of purchasing (morning time), neither stored nor treated.

Sample Preparation
The fruits were purchased from local (Jordanian) market and analyzed freshly.Fruits were prepared by peeling, chopping finely by knife or food chopper (Ariete ® , China).Representative samples (1-3 g) were extracted conventionally by 10 ml of each of the three extraction solvents (methanol, ethanol, and water) at 50 °C, 50 °C, and 90 °C respectively for 2 hours with intermittent shaking.The extracts were centrifuged at 3000 rpm for 10-15 minutes (HuMax ® , Germany) and filtered (Wattman filter paper No.4), purged with liquid nitrogen (Apak et al., 2007), and stored at -20 °C (for not more than two months) until analyzed.Deionized water was used for the preparation of all standard solutions and to complete the reactions (Apak et al., 2007).

Analyses
Chemicals were purchased from GCC ® (UK), Fischer ® (China), Labscan ® (Thaihland), LabChem ® (USA) and Sigma ® (China).Standard curves were prepared to have r 2 value of 0.96-0.99.Samples were analyzed in duplicate with an accuracy of not less than 95% (Luterotti, Bicanic, & Pozgaj, 2007) and coefficient of variation not more than 15%.Samples were analyzed in duplicates.Absorbance values were measured using UV-visible spectrophotometer (SCO Tech, Model SPUV ® ) at the specified wavelength values against standard concentrations of certain antioxidants and blank solutions.

Determination of Antioxidant Content
(1) Folin-Ciocultae Method Folin-Ciocaltae method was used for the determination of antioxidant content according to Agbor, Vinson, and Donnelly (2014).Ten to 100 μl sample was completed to the volume of 1000 µl by 10x freshly prepared Folin-Ciocultae reagent.The reaction was completed within 15-minutes.Sample concentration for antioxidants was measured against freshly prepared catechin standard (catechin standard was dissolved in methanol) at 750 nm wavelength.
(2) Total Flavonoid Method Total flavonoids were analyzed by the method of Pękal and Pyrzynka (2014).Half milliliter of the methanolic solution (2% w/v) of AlCl 3 was added to 1 ml sample.Then, 0.5 ml of deionized water and 0.5 ml of 1M HCl were added respectively, the mixture was shaken vigorously, and the reaction was completed within 10 minutes.The absorbance was measured at 400 nm wavelength against different concentrations of rutin standard solutions (rutin was dissolved either in ethanol or in methanol).The absorbance was measured at 400 nm wavelength against different concentrations of rutin standard solutions (rutin was dissolved either in ethanol or in methanol).

Determination of Antioxidant Capacity
(1) CUPRAC Assay Concentrated (36%) hydrochloric acid (10.21 ml) was added to a suitable amount of sample extracts (0.5-5 ml), the volume was then completed to 100 ml by 50% methanol and refluxed at 80 °C for 2 hours and cooled down to room temperature.Sample mixture was then neutralized to pH 7 by 1M NaOH.Then, 1 ml CuCl 2 , 1 ml neucoprine, and 1 ml acetate buffer, and suitable sample volume (500-1100 µl) were added respectively to complete the reaction volume to 4.1 ml.The reaction mixture was then incubated at 50 °C for 20 minutes, cooled to room temperature and centrifuged at 3000 rpm for about 7 minutes.Sample absorbance was measured using a spectrophotometer at 450 nm (Apak et al., 2007) against different concentrations of trolox standard solutions (trolox was dissolved either in ethanol or methanol).
(2) DPPH Assay The DPPH assay procedure was performed according to Molyneux (2003).The free radical 2,2-diphenyl-picrylhydrazyl (DPPH) (2.95 ml of 0.1 mM, prepared in 80% ethanol) was added to 50 μl sample.The mixture was incubated at room temperature for 30 minutes in dark place.The absorbance was measured at 517 nm wavelength against ascorbic acid as a standard to detect DPPH radical scavenging percentage.The scavenging percentage was calculated according to the following equation: Where, A 0 : is the absorbance of the control; A 1 : is the absorbance of the sample.

Statistics and Data Analysis
The statistical analysis of data was performed using the software package for social sciences (SPSS, version 23).
To detect the differences between the 4 different fruits as well as the extraction solvent, data were analyzed by factorial mixed (effect of type of plant and extract type) analysis of variance (ANOVA) (Laerd Statistics, 2018).Significant differences were considered at P < 0.05.Data are expressed in the tables as mean±standard deviation.
Pearson's correlation coefficients were calculated and considered significant at P < 0.05.

Total Polyphenol Content of the Fruits
Table 1 shows the antioxidant content (M catechin/100 g) of the fruit extracts determined by Folin-Ciocaltae method.The descending order of total polyphenol content of the fruits (regardless of the extraction solvent) is: lemon, mandarin, pomelo, orange.Nonetheless, there were no significant (P > 0.05) differences between the following pairs of fruits in terms of total polyphenol content: lemon-mandarin, mandarin-orange, and pomelo-orange.Within the same context, different extraction solvents exhibited different (P < 0.001 ** ) extraction efficacies.Water had extracted the highest (P < 0.001 ** ) amounts of antioxidants from orange, lemon, mandarin, and orange.Methanol had extracted the highest amounts of antioxidants from pomelo.On the other hand, methanol extracted the lowest (P < 0.001 ** ) amounts of antioxidants from all of the fruits.Note. 1 Values of the tables are average of duplicates ± SD with c.v. of not more than 15%.
2 P values are used to express significant differences between different fruit extracts at P < 0.05.

Total Flavonoid Content of the Fruits
Table 2 shows the antioxidant content (mM rutin/100 g) of the fruit extracts determined by total flavonoid method.The descending order of the total flavonoid content (regardless of the extraction solvent) in the fruits is: orange, mandarin, lemon, pomelo.Nonetheless, there were no significant (P > 0.05) differences among orange, mandarin, and lemon.Within the same context, different extraction solvents exhibited different (P < 0.001 ** ) extraction efficacies.Water had extracted the highest (P < 0.001 ** ) amounts of antioxidants from all of the fruits.Ethanol had extracted more antioxidants than methanol from orange, mandarin, and lemon.On the other hand, methanol had extracted more antioxidants than ethanol from pomelo.

CUPRAC Assay
Table 3 shows the antioxidant capacity (M trolox/100 g) of the fruit extracts determined by CUPRAC assay.The descending order of antioxidant capacity for the fruits (regardless of the extraction solvent) is: lemon, mandarin, orange, pomelo.In the same context, different extraction solvents exhibited different (P < 0.001 ** ) extraction efficacies.The highest value for antioxidant capacity expressed by CUPRAC assay for lemon was for methanol despite the fact that methanol extracts of lemon contained the least (P < 0.05) amounts of antioxidants (Tables 1  and 2).Methanol had the highest (P < 0.001 ** ) extraction efficiency from lemon and mandarin.Water had the highest (P < 0.001 ** ) extraction capacity from orange and pomelo.On the other hand, ethanol had the lowest (P < 0.001 ** ) extraction efficiency from all of the fruits.Note. 1 Values of the tables are average of duplicates ± SD with c.v. of not more than 15%.
2 P values are used to express significant differences between different fruit extracts at P < 0.05.

DPPH Assay
Table 4 shows the antioxidant capacity (expressed as % of DPPH radical scavenging and mg vitamin C/ml) of the fruit extracts determined by DPPH assay.The descending order (P < 0.01 ** ) of antioxidant capacity (as DPPH radical scavenging percentage) of the fruits (regardless of the extraction solvent) is as follows: orange, mandarin, pomelo, lemon.Within the same context, different extraction solvents exhibited different (P < 0.001 ** ) extraction efficacies.The descending (P < 0.01**) order for the fruits in terms of antioxidant capacity (measured as mg vitamin C/ml) (regardless of the extraction solvent) is: mandarin, orange, pomelo, lemon.Nonetheless, there were no significant (P > 0.05) differences between the following pairs of fruits: pomelo-lemon and mandarin-orange.Within the same context, different extraction solvents exhibited different (P < 0.001 ** ) extraction capacities.Water was the most (P < 0.001 ** ) efficient extraction solvent from all of the fruits when the result was expressed as % DPPH scavenging.Methanol was the least (P < 0.001 ** ) efficient extraction solvent from orange and pomelo.Ethanol was the least efficient extraction solvent from lemon and mandarin.When the results were expressed as (mg vitamin C/ml extract), water was the most (P < 0.001 ** ) efficient extraction solvent from orange and pomelo.Methanol was the most (P < 0.001 ** ) efficient extraction solvent from lemon and pomelo.On the other hand, ethanol was the least efficient (P < 0.001 ** ) extraction solvent from all of the studied fruits.No correlation has been found between antioxidant content and capacity of the studied fruits.

Discussion
The first null hypothesis of the research has been accepted as there were significant differences among the studied fruits in terms of antioxidant contents and capacities.However, the second alternative hypothesis has been rejected since no correlation between the antioxidant content and capacity of the studied fruits has been found.Note. 1 Values of the tables are average of duplicates ± SEM with c.v. of not more than 15%.
2 P values are used to express significant differences between different fruit extracts (columns) at P < 0.05.Mandarin corresponds to 103.7213 mg rutin equivalent/g fresh sample.
Assuming that mandarin contains about 87% moisture (Barros, Ferreira, & Genovese 2012); our value correspond to 81.6703 mg rutin equivalent/ g dry mandarin.Table 5 shows a comparison between the values found in this research and the values found in other reports for all of the studied parameters.Some of the values for total polyphenols found in this research are near to those found by other researchers, other are either higher or lower.The highest value for antioxidant capacity expressed by CUPRAC assay for lemon was for methanol despite the fact that methanol extracts of lemon contained the least (P < 0.05) amounts of antioxidants (Tables 1 and 2).This probably implies that there are methanol-soluble antioxidants that can be analyzed by other assay methods that might have contributed to the antioxidant capacity of the methanolic extracts (Table 3).
The highest antioxidant capacity expressed by DPPH% scavenging was for water extract of lemon.A result that is expected since the highest antioxidant content (measured by Folin-Cioculteau and total flavonoid methods) were exhibited for water extracts of lemon.The values of DPPH % scavenging were reflected as mg vitamin C/ml in Table 4.
Most of the antioxidants of mandarin analyzed in this research (total polyphenols and total flavonoids) were extracted by water (P < 0.05).This was reflected by the highest (P < 0.05) antioxidant capacity (measured by DPPH% and vitamin C equivalent) for water extracts among all of the extraction solvents of mandarin.Nonetheless, the antioxidant capacity measured by CUPRAC assay of the methanol extract of mandarin was the highest among the extraction solvents.This probably reflects the presence of certain methanol-soluble/water-insoluble (or having low water solubility) antioxidants that might have contributed to the highest antioxidant capacity (quantified by CUPRAC assay) of mandarin methanol extract.Due to the result that water had extracted the highest (P < 0.05) concentration of polyphenolic compounds from orange, it seems that orange contains water soluble antioxidants higher than those that are soluble in ethanol and/or methanol.Similar to total polyphenols, it seems that the flavonoids that had been extracted from orange were water soluble.This was reflected by the highest values (for orange) of antioxidant content and capacity in water extracts.Table 5 shows a comparison between values of antioxidant content and capacity found in this study and other researches.
The differences between the antioxidant content and capacity values found in this study and values found in the previous literature are probably due to the analyzed fruit variety, fruit growing conditions, and experimental procedures.The difference in extraction solvent polarity allows the extraction and quantification of different antioxidant content and capacity of the fruits (Zefang, Zhao, Hongmei, Zhiqin, & Jie, 2016;Wang, Yang, & Zhou, 2018).No correlation has been found between any of the studied parameters.This probably implies that there are antioxidants other than those analyzed by the researchers that might have contributed to the antioxidant capacity values shown in Tables 3 and 4.
This study is limited by the type of extraction solvent (in terms of polarity), method of extraction (conventional extraction), and the method of analysis (spectrophotometric determination).However, results of this research will probably start a database for the antioxidant content and capacity for Jordanian fruits and vegetables that is recommended to be completed by other researchers.

Conclusions
It has been concluded that the studied citrus fruits contained polyphenols in descending order of: lemon, mandarin, pomelo, orange.On the other hand, the fruits contained total flavonoids in descending order of: orange, mandarin, lemon, pomelo.Water has extracted the highest amounts of antioxidants.In terms of antioxidant capacity, the fruits exhibited the antioxidant capacity (expressed as M trolox/100 g, analyzed by CUPRAC assay) in descending order of: lemon, mandarin, orange, pomelo.On the other hand, the fruits efficiency in scavenging the DPPH radical was in descending order of: mandarin, orange, pomelo, lemon.Different solvents exhibited different efficacies of extraction.No correlation has been found between any of the studied parameters.This probably implies that there are antioxidants other than those analyzed by the researchers that might have contributed to the antioxidant capacity values.

Table 1 .
The antioxidant content (M catechin/100 g) of the methanolic, ethanolic, and water extracts of the fruits determined by Folin-Ciocaltaeu method1,2

Table 2 .
The antioxidant content (mM rutin/100 g) of the methanolic, ethanolic, and water fruit extracts determined by total flavonoid method1,2

Table 3 .
The antioxidant capacity (M trolox/100 g) of the methanolic, ethanolic, and water fruit extracts determined by CUPRAC assay1,2Note.1Values of the tables are average of duplicates ± SD with c.v. of not more than 15%. 2 P values are used to express significant differences between different fruit extracts at P < 0.05.

Table 4 .
The antioxidant capacity of the methanolic, ethanolic, and water fruit extracts determined by DPPH assay1,2

Table 5 .
Comparison between values of antioxidant content and capacity of this study and of other researches