Phenolic and flavonoid contents of mandarin (Citrus reticulata Blanco)fruit tissues and their antioxidant capacity as evaluated by DPPH and ABTS methods

ZHANG Hua, YANG Yi-fei ZHOU Zhi-qin

1 College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, P.R.China

2 College of Life Science & Engineering, Chongqing Three Gorges University, Chongqing 404120, P.R.China

3 Key Laboratory of Horticulture for Southern Mountainous Regions, Ministry of Education, Chongqing 400715, P.R.China

4 Laboratory of Quality & Safety Risk Assessment for Citrus Products of Ministry of Agriculture, Citrus Research Institute,Southwest University, Chongqing 400712, P.R.China

RESEARCH ARTICLE

Phenolic and flavonoid contents of mandarin (Citrus reticulata Blanco)fruit tissues and their antioxidant capacity as evaluated by DPPH and ABTS methods

ZHANG Hua1,2, YANG Yi-fei1, ZHOU Zhi-qin1,3,4

1 College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, P.R.China

2 College of Life Science & Engineering, Chongqing Three Gorges University, Chongqing 404120, P.R.China

3 Key Laboratory of Horticulture for Southern Mountainous Regions, Ministry of Education, Chongqing 400715, P.R.China

4 Laboratory of Quality & Safety Risk Assessment for Citrus Products of Ministry of Agriculture, Citrus Research Institute,Southwest University, Chongqing 400712, P.R.China

The total phenolic and flavonoid contents in the fruit tissues (peels, pulp residues, seeds, and juices) of 19 citrus genotypes belonged to Citrus reticulata Blanco were evaluated and their antioxidant capacity was tested by 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) method and 2,2´-azino-bis(3-ethylbenzthiozoline-6)-sulphonic acid (ABTS) method. The total phenolic and flavonoid contents, and their antioxidant capacity varied in different citrus fruit tissues. Generally, the peel had both the highest average of total phenolics (27.18 mg gallic acid equivalent (GAE) g–1DW) and total flavonoids (38.97 mg rutin equivalent (RE) g–1DW). The highest antioxidant capacity was also the average of DPPH value (21.92 mg vitamin C equivalent antioxidant capacity (VCEAC) g–1DW) and average of ABTS value (78.70 mg VCEAC g–1DW) in peel. The correlation coef ficient between the total phenolics and their antioxidant capacity of different citrus fruits tissues ranged from 0.079 to 0.792, and from –0.150 to 0.664 for the total flavonoids. The antioxidant capacity of fruit tissues were correlated with the total phenoilc content and flavonoid content except in case of the peel. In addition, the total phenolic content and antioxidant capacity varied in different citrus genotypes. Manju and Karamandarin were better genotypes with higher antioxidation and the phenolic content, however Shagan was the poorest genotype with lower antioxidation and the phenolic content.

Citrus L., fruit tissues, phenolics/ flavonoids, antioxidant capacity

1. lntroduction

Citrus fruits are rich sources of natural antioxidants, which are now widely accepted as being bene ficial to human health (Zou et al. 2016). The antioxidant capacity of citrus fruits has been the research subjects of many studies in the literatures (Bocco et al. 1998; Zhang et al. 2014; Lee et al. 2015; Zou et al. 2016). From these studies, vitamins,flavonoids and phenolic acids were suggested to have anti-oxidant capacity (Zou et al. 2016). Many different methods have been reported for the antioxidant capacity evaluation of plant samples (Zhang et al. 2014). There are numerous methods to estimate the antioxidant activities such as 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) method(Rivero-Pérez et al. 2007), 2,2´-azino-bis(3-ethylbenzthiozoline-6)-sulphonic acid (ABTS) method (Miller et al. 1993),ferric reducing/antioxidant power (FRAP) method (Benzie et al. 1999), and oxygen radical absorbance capacity assay(ORAC) method (Ou et al. 2001). DPPH and ABTS methods are convenient and independent of expensive equipment,thus they are widely used.

China is one of important center origins for the genus Citrus L., and some important citrus genotypes are originated from there. Over the past few years, many mandarin genotypes in China have been described. For example, the content and composition of phenolic compounds in Chinese wild mandarin genotypes had been determinated and their antioxidant capacity was evaluated (Zhang et al. 2014).Up till now, however, the information about the antioxidant compounds in different mandarin fruit tissues, especially those of the Chinese native Citrus genotypes, and their antioxidant capacity is still limited.

The aims of this study are to evaluate the phenolic composition and antioxidant capacity of different mandarin fruit tissues (peels, pulp residues, seeds and juices), especially those of 10 Chinese native genotypes; and to identify the relationship between the antioxidant capacity evaluated by DPPH and ABTS free radical methods and the total phenolic and flavonoid contents. The results will provide important information for the future study and use Citrus genetic resources fully.

2. Materials and methods

2.1. Chemicals

ABTS and DPPH reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rutin, gallic acid, and L-ascorbic acid (vitamin C) were supplied by J & K Scienti fic Ltd.(Beijing, China). Ultrapure water was prepared using a Millipore System (Millipore, Bedford, MA, USA). Standards were stored in dark at –20°C. All the other reagents of analytical grade were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Citrus materials

Nineteen citrus genotypes belonged to Citrus reticulata Blanco were grown at the National Citrus Germplasm Repository, Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing, China and were used in this study (Table 1). Fruit samples were harvested at a commercial maturity stage. The fruits were separated into peels (containing flavedo and albedo), pulp and seeds.The juice was manually squeezed from the pulp and was dried (Chen et al. 2010). The pulp residues were obtained after the juice was squeezed from pulp. The peels, pulp residues and seeds of the fruits were separated and dried in a draught drying cabinet (Shanghai Yiheng Instruments Co., Ltd., Shanghai, China) at 50°C for 3 days (Chen et al.2011). Then they were shredded into homogeneous pow-der (0.280 mm) by a shredder (Star Infuluence, Beijing,China). The powders of peels, pulp residues, juices and seeds were stored at –80°C for phenolic composition and antioxidant analyses.

Table 1 The Citrus genotypes used in the present study

2.3. Preparation of standard and stock solutions

Individual stock solutions of gallic acid and rutin were dissolved in 80% methanol into a concentration of 1.00 mg mL–1. Nine concentrations of gallic acid solutions (0, 0.001,0.006, 0.008, 0.01, 0.05, 0.10, 0.20, and 0.50 mg mL–1gallic acid solution) were prepared for the total phenolic content determination. Nine concentrations of rutin solutions (0,0.008, 0.02, 0.05, 0.08, 0.10, 0.20, 0.30 and 0.40 mg mL–1rutin solution) were prepared for total flavonoid content measurement.

L-Ascorbic acid (vitamin C, as a standard for antioxidant analysis) was dissolved in HPLC-grade water to a concentration of 1.00 mg mL–1. L-Ascorbic acid solution was freshly prepared.

2.4. Preparation of DPPH and ABTS radical working solutions

DPPH radical solutionDPPH powder was dissolved in 500 mL methanol to prepare the 0.10 mmol L–1DPPH solution for DPPH method. The prepared solution was stored at 4°C in dark.

ABTS radical working solution (ABTS radical cation solution, ABTS•+ solution)Potassium persulfate solution was mixed with ABTS solution (1:1, v/v) to react overnight at the room temperature in dark to obtain ABTS radical working solution. When the absorbance value of working solution was 0.70±0.02 at 400 nm, the concentration of working solution was used for ABTS method. The ABTS radical working solution was freshly prepared.

2.5. Preparation of citrus extracts

The method of Ramful et al. (2011) with some modi fications was applied for the citrus extracts preparation. Dried samples of peels (0.250 g), pulp residues (0.250 g), seeds(0.625 g), and juices (0.050 g) were transferred into the 50 mL falcon tubes (BD Biosciences, Franklin Lakes, NJ,USA). The tested samples were dissolved and reached to 25 mL with 80% methanol (Siddhuraju and Becker 2003).The samples were mixed well and put into a shaker (Thermo Scienti fic, Hudson, NH, USA) overnight at 25°C. Next day,the extracts were centrifuged at 5 000×g for 10 min. Then supernatant was separated into 10 mL falcon tubes, which were stored at –80°C for the total phenolic/ flavonoid content and antioxidant capacity analyses.

2.6. Determination of total phenolic content and total flavonoid content

The Folin-Cio calteu method was used to determine total phenolic content (Xu et al. 2008). The aluminum chloride method of Wang et al. (2008) was used to test the total flavonoid content.

2.7. Determination of antioxidant capacity

The total antioxidant capacities of citrus extracts from different tissues of fruits were analyzed by DPPH and ABTS radical scavenging assays (DPPH and ABTS methods).Radical scavenging activity of citrus extracts against stable DPPH radical was performed as described by Brand-Williams et al. (1995). Radical scavenging activity of citrus extracts against stable ABTS radical was performed as method described by Arnao et al. (2001).

L-Ascorbic acid solutions as standards were also analyzed by DPPH and ABTS methods. The total antioxidant values of citrus samples were expressed as mg g–1DW L-ascorbic acid equivalent antioxidant capacity (VCEAC).

2.8. Statistical analysis

Data were presented as means±standard deviation (SD).One-way analysis of variance (ANOVA, SPSS 17.0 software, SPSS Inc., Chicago, IL, USA) was used to assess the difference of the means among different samples (P<0.05).Pearson’s correlation analysis (SPSS 17.0 software, SPSS Inc.) was used for evaluating the correlation between the total phenolic/ flavonoid contents and the total antioxidant capacity values of different citrus tissues (P<0.05 or P<0.01).

3. Results and discussion

3.1. The total phenolic and flavonoid contents in different fruit tissues

For different citrus fruit tissues (Table 2), the total phenolic contents ranged from 22.80 to 32.76, 8.25 to 15.15, 1.09 to 2.81 and 7.28 to 34.03 mg (gallic acid equivalent (GAE))g–1DW for peels, pulp residues, seeds and juices, respectively. Zhang et al. (2014) showed that the total phenolic contents in the peels of C. reticulata Blanco ranged from 29.38 to 51.14 mg GAE g–1DW, which were similar to present results. However, it was much lower than those (66.5 to 396.8 mg GAE g–1DW) of reported by Ghasemi et al. (2009),but was higher (about 0.6 mg GAE g–1DW) than those reported by Chen et al. (2010). These differences could be caused by the difference of citrus species or different growing conditions. In addition, Moulehi et al. (2012) reported that the phenolic composition of the seeds of C. reticulata Blanco and Citrus aurantium L. ranged from 0.68 to 2.11 mg g–1DW, which was similar to current results.For the four tested tissues, the average of the phenolic content(Table 2) was ordered form high to low as the following order:peels (27.18 mg GAE g–1)>juices(24.98 mg GAE g–1DW)>pulp residues (12.33 mg GAE g–1DW)>seeds (1.71 mg GAE DW g–1). This trend was similar to a previous report by Chen et al.(2010). In this study, Manju(MJ, genotype in China, its peel,pulp residue and seed except the juice), Karamandarin (KM,genotype in America, its juice,pulp residue, and seed except the peel), and Parson special mandarin (PSMA, genotype in America, especially in its peel and juice) were generally the genotypes having higher phenolic contents in tissue of fruits.On the contrary, the fruit tissues of Shagan (SG, China) had a lower phenolic content, in which the phenolic content of juice was only 7.28 mg GAE g–1DW.

Flavonoids are the main components of plant polyphenols(Bocco et al. 1998). For the different fruit tissues of the 19 genotypes studied, the total flavonoid contents ranged from 23.29 to 56.52, from 6.38 to 16.74, from 5.96 to 18.23 and from 9.88 to 48.13 mg rutin equivalent (RE) g–1DW in peels,pulp residues, seeds and juices,respectively (Table 2). The average flavonoid content for the different fruit tissue was ordered from high to low as follows:peels (38.97 mg RE g–1DW)>juices (31.32 mg RE g–1DW)>pulp residues (10.34 mg RE g–1DW) and seeds (10.48 mg RE g–1DW). These results were similar to those reported by Lu et al. (2006). In such study, the peel was the highest tissue with flavonoid content compared with that of edible tissue. Zhang et al. (2014) also found that the flavonoid content in peel was higher than that in juice for Citrus grandis L. Osbeck. However,some genotypes showed different trends. For example, the juice of KM (48.13 mg RE g–1DW) showed a relatively higher flavonoid content than in its peel did (23.49 mg RE g–1DW). The different flavonoid patterns in citrus fruit tissues might be in fluenced by different Citrus genotypes (Zhang et al. 2001). Generally, the KM had higher flavonoid contents of fruit tissues (especially for its juice, 48.13 mg RE g–1DW)than other genotypes did. On the other hand, SG (only 9.88 mg RE g–1DW in juice) and Avanaapireno(AA, genotype in Italy, especially for its seed and pulp residue) were the ones with a relatively lower flavonoid content in fruit tissues.

3.2. Antioxidant capacity variation in different fruit tissues

Based on the antioxidant values obtained by two free radical antioxidant methods (DPPH and ABTS),an analysis of the antioxidant capacity of different fruit tissues was obtained. From Table 3, it could be seen that the antioxidant capacity detected by the ABTS method was higher than the results detected by DPPH method. The phenomenon is caused by different reaction mechanisms (Layina-Pathirana et al. 2006; Zhang et al. 2015).The antioxidant capacity among the different genotypes varied, which could be explained by different kinds or contents of antioxidants insides.

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Antioxidant capacity variation of the peels among 19 citrus genotypesThe DPPH values varied from 11.19 to 44.40 mg VCEAC g–1DW with Chinese Daxianggan (DXG) genotype having the highest and SG the lowest DPPH values; the ABTS values varied from 64.24 to 97.31 mg VCEAC g–1DW with MJ genotype having the highest, whereas Cleopatramandarin (CM,India) and KM genotypes having the lowest ABTS values.

Antioxidant capacity variation of the pulp residues among 19 citrus genotypesThe DPPH values varied from 2.13 to 4.47 mg VCEAC g–1DW with Shinamanatsu(Japan-No. 1) (SA, Japan), KM, Hayaka (HA, Japan), Tankantangor (TT, China), Yuanhongxianggan (YHXG, China)having the highest (P>0.05), whereas Baiju (BJ, China)genotype having the lowest. The ABTS values varied from 6.75 to 16.95 mg VCEAC g–1DW with TT, YHXG, HA and DXG having the highest (P>0.05), whereas Jinju (JJ, China)and CM having the lowest. Although the DPPH value of pulp residues was much lower than that of peel, pulp residues still had antioxidant capacity. Abeysinghe et al. (2007) also showed that the pulp residues had antioxidant capacity.

Antioxidant capacity variation of the seeds among 19 citrus genotypesThe DPPH values varied from 0.95 to 2.45 mg VCEAC g–1DW with KM, Miho-core (MC, Japan),MJ having the highest (P>0.05), whereas SG genotype being the lowest; the ABTS values varied from 3.93 to 7.03 mg VCEAC g–1DW in seeds of MJ and KM being the highest(P>0.05), whereas the seed of SG being the lowest. It could see that the seeds of some citrus genotypes were a good source for natural antioxidants (Bocco et al. 1998).

Antioxidant capacity variation of the juice among 19 citrus genotypesThe DPPH values varied from 5.84 to 28.84 mg VCEAC g–1DW with Parson’s special mandarin(PSM, Mexico) and PSMA having the highest (P>0.05),whereas SG genotype being the lowest; the ABTS values varied from 12.95 to 47.84 mg VCEAC g–1DW with PSM and PSMA being the highest (P>0.05), but the juice of SG being the lowest.

Comparation of the antioxidant capacity of four tissues showed that average DPPH value (Table 3) in peel was the highest (21.92 mg VCEAC g–1DW), followed by the DPPH value in juice (16.53 mg VCEAC g–1DW), in pulp residues(3.34 mg VCEAC g–1DW), and in seed (1.73 mg VCEAC g–1DW). For ABTS values, the order of antioxidant (from high to low) was: peel value (78.70 mg VCEAC g–1DW)>juice value (28.84 mg VCEAC g–1DW)>pulp residue value(12.48 mg VCEAC g–1DW)>seed value (4.83 mg VCEAC g–1DW). The trend was similar to that reports by Xu et al.(2009). In their report, the peel normally has higher antioxidant capacity than flesh. The variation of the antioxidant capacity between different fruit tissues might be caused by the different phenolic composition in the different tissues of a citrus fruit (Xu et al. 2009). Based on the above information,the peels of DXG and MJ, seed of KM, pulp residues of TT,YHXG and HA, and the juices of PSM and PSMA were good fruit tissues with a higher antixoidant. However, SG was the poor source for scavenging the free radical.

3.3. The correlation between the total phenolic contents/ flavonoid content in fruit tissues and their antioxidant capacity

To identify the potential chemical compounds which contribute to the antioxidant capacity of the citrus fruit tissues,Pearson’s correlation coef ficients between the total phenolic contents and their antioxidant capacity were analyzed for citrus different tissues (Table 4). For peels, the DPPH and ABTS values were not correlated with the peholic content(r=0.079 and 0.157, respectively). The DPPH method did not show correlation with the total phenolic content in peel,which was similar to the reports of Ghasemi et al. (2009) and Chen et al. (2010). However, there are studies that have found high correlation between DPPH and ABTS values with the total phenolic (TP) and total flavonoid (TF) contents of other Citrus species peels (Barreca et al. 2014; Papoutsis et al. 2016). The different might be caused by different species or different solvents used. Other compounds could be involved in the antioxidant capacity of mandarin apart from phenols, such as vitamin C. For pulp restudies and seeds, the phenolic content was correlated with results of two free radical antioxidant methods (r-value ranged from0.564 to 0.792, P<0.01). For juices, the phenolic content was correlated with results detected by DPPH method(r=0.705, P<0.01) except the ABTS method (r=0.339), which was similar to the reports of Chen et al. (2010).

Table 4 Correlation between the total phenolics or total flavonoids content of different fruit tissues and their antioxidant activities1)

Because of flavonoids are reported the main phenolic compounds in citrus fruits. Pearson’s correlation coef ficients between the total flavoniod contents and their antioxidant capacity were analyzed (Table 4). For peels, the DPPH and ABTS values were not correlated with the flavonoid content(r=–0.150 and 0.216, respectively). For pulp restudies and seeds, the correlation coef ficients (r) between the antioxidant capacity and the phenolic content ranged from 0.456 to 0.664 (P<0.01). The flavonoid content was correlated with results of two free radical antioxidant methods (P<0.01).For juices, the flavonoid content was correlated with results detected by DPPH method (r=0.543, P<0.01) except the ABTS method (r=0.354). Chen et al. (2010) reported the antioxidant capacity of citrus flesh detected by DPPH method was highly related with the total flavonoid concentrations,suggesting that flavonoids ( flavones, in particular) acted as radical scavengers in juices (Bellocco et al. 2009).

Taken together, statistical analysis revealed that the phenolic composition was related to antioxidant capacity in different citrus fruit tissues expect the peel. The different r-values could be explained by the different sample matrices and genetic background (Gardner et al. 2000).

4. Conclusion

The phenolic composition and antioxidant capacity of different mandarin fruit tissues of 19 citrus genotypes belonged to C. reticulata Blanco are reported in this study. It was found that peel, pulp residues, seeds and juices contain phenolic composition and have antioxidant capacity. The peel and juice are the main tissues with higher phenolic content and stronger scavenging free radical ability compared to the other tissues. Phenolic content and DPPH or ABTS values also showed higher correlation in different fruit tissues except in peel. Among 19 mandarin genotypes, Manju (MJ, China,especially for its peel) and Karamandarin (KM, America,especially for its juice) were good genotypes with higher phenolic content. The peel of Daxianggan (DXG, China) and MJ, seed of KM, pulp residues of Tankantangor (TT, China),Yuanhongxianggan (YHXG, China) and Hayaka (HA, Japan)and the juices of Parson’s special mandarin (PSM, Mexico)and Parson special mandarin (PSMA, America) were good sources with high antixoidant. However, Shagan (SG,China) was the poorest genotype for scavenging the free radical ability or phenolic content. Our study provided new information about the variation of the phenolic contents in different fruit tissues and their relationship to the antioxidant capacity of citrus fruits tissues, and for the future utilization of citrus germplasm especially those of C. reticulata Blanco in China.

Acknowledgements

This work was supported by the Identi fication of the Common Nutrients of Edible Agricultural Products and the Character Nutrients of Special Agricultural Products and Their Key Control Points of Quality, China (GJFP201701501), the Chongqing Program for Production of Late Maturing Citrus Fruits, China (20174-4), the Program for Talent Introduction of Chongqing Three Gorges University, China (14RC05), the Program for Chongqing Municipal Education Commission,China (KJ1501015), and the Program for Chongqing Science& Technology Commission, China (cstc2016jcyjA0555). We are also grateful to Dr. Han Jixiang (Conagen, Inc., USA)and Dr. Eun Young Han (Department of Radiation Oncology,University of Arkansas for Medical Sciences, USA) for their suggestions in the preparation of this manuscript. We are grateful to the help of Ms. Zhou Xingyu, Ms. Liu Xin, Ms. Yin Shanshan, and Ms. Zhang Jinmei (Southwest University,China) in this work.

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21 December, 2016 Accepted 1 March, 2017

ZHANG Hua, Tel: +86-23-58102522, E-mail: zhanghua03129@163.com; Correspondence ZHOU Zhi-qin, Tel: +86-23-68250229,Fax: +86-23-68251274, E-mail: zhouzhiqin@swu.edu.cn

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10.1016/S2095-3119(17)61664-2

Managing editor SHI Hong-liang