Introduction

Free radicals or reactive oxygen species (ROS) are the outcomes of normal essential metabolic processes. Exposure to air pollutants, cigarette smoking, industrial chemicals, ozone and X-rays also contribute to the production of free radicals. The implication of free radicals and ROS exposure employs a multitude of biological effects covering many chronic diseases.¹ Therefore, food with a high content of antioxidant compounds may lower the chances of chronic disease development because natural antioxidant from plant owns a protective effect against the progress of chronic diseases.² Thus, the antioxidant is essential in protecting the human body from harmful effects of oxidation.³ The Citrus genus belongs to the family of Rutaceae which includes around 40 species of citrus; the citrus trees are widely planted in China, India, Malaysia, Australia and Sri Lanka.⁴

Citrus fruit has been largely cultivated in the tropical and subtropical countries with a total annual production of approximately 102 million tonnes.⁵ In 2014, the production of citrus in Malaysia alone was around 42,212 tonnes. Citrus is planted for domestic and export purposes on 6,208 hectares of land around the country.^6,7 Fruit from Rutaceae plants are typically rich in phytochemicals, and these plants are the good sources of bioactive compounds that responsible for antioxidation and many other biological activities.⁸ Citrus fruit prevents free radical generation and reduces homocysteine level; it also possesses anti-carcinogenic, anti-diabetic, anti-inflammatory and anti-arthritic properties.^9,10

It is important to incorporate citrus fruit into our daily diets as it is rich in antioxidants. It supplies energy and nutrient to our body for combating diseases.¹¹ Among the different parts of citrus fruit, citrus peel contains the highest proportion of natural antioxidants such as natural flavonoid, phenolic, ascorbic acid and carotenoids, as well as reducing sugar. Literature shows that citrus peel extracts had the highest antioxidant activity compared with pulp and seeds.^12-14 The citrus peel that represents around 50-65% of the fruit is commonly treated as a primary citrus by-product and discarded as the massive load into the environment.¹⁵

Antioxidants in a plant are susceptible to degradation, mostly due to the handling practices such as chilling injury, fungal decay, irradiation, incorrect holding temperature, relative humidity and other factors of stress that can contribute to the reduction of antioxidant content and nutritional value.¹⁶ There is an established investigation of postharvest exposure at a temperature above the freezing point that can contribute to a negative effect on nutrient quality and antioxidant.¹⁷ However, there are inconsistencies in the finding on the effect of freezing toward various plant samples, where some studies reported a positive effect of frozen samples and some were contradicted. The current study aims to determine antioxidant content and antioxidant activity of fresh and frozen selected citrus peels. This study used fresh and frozen peel samples of lemon, key lime and musk lime.

Material and Methods

Compound Name

In this study, the chemicals used were 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tri-(2-pyridyl)-s-triazine (TPTZ), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium bicarbonate (Na₂CO₃), sodium nitrate (NaNO₃), sodium acetate trihydrate (C₂H₃NaO₂∙3H₂O), ferric chloride hexahydrate (FeCl₃∙6H₂O), ferrous sulphate heptahydrate (FeSO₄∙7H₂O), aluminium chloride (AlCl₃), glacial acetic acid, Folin-Ciocateu reagent, gallic acid and quercetin. These chemicals and reagents were supplied by Sigma-Aldrich, Co. (USA).

Sample Preparation

Selected citrus species were Citrus limon (lemon) C. aurantifolia (key lime) and C. microcarpa (musk line). All samples were purchased at the Selangor wholesale market, Malaysia. The fruit peel was removed from the pulp, and the peel was divided into two parts. Half of each fresh peel was extracted for antioxidants right after sample preparation; while the other half was the frozen sample. All samples were kept frozen at –80°C for 24 h prior prior to freeze-drying.

Extraction Method

Antioxidants from both fresh and frozen peels of citrus fruit were extracted according to Ferreira et al.,¹⁸ with slight modification. The fresh citrus peels (10.0 g) and freeze-dried (frozen) citrus peel powders (25.0 g) were added to 100 mL of 70% ethanol solution in a conical flask. The extracts were then sonicated for 60 min and filtered using a Whatman No. 1 filter paper. The ethanol was removed using a vacuum rotary evaporator (Buchi Rotavapor R-200, Switzerland) at 50°C. The extract was kept at –20°C before further analysis. The extraction yield was obtained by weighing the extract after the solvent removal; the percentage of the extraction yield was calculated based on the equation as follows:

Extraction yield (%) = (Weight of yield obtained)/(Weight of fresh sample) × 100%

Estimation of Total Phenolic Content

Folin-Ciocalteu (FC) reagent assay was used to determine total phenolic content (TPC) of citrus samples. The assay was performed according to the procedure described by Meda et al.,¹⁹ with slight modification. FC solution (0.2 N) was prepared by adding 0.15 mL of FC reagent with 15.0 mL of methanol, whereas Na₂CO₃ solution was prepared by mixing 0.6 g Na₂CO₃ with 10.0 mL of distilled water. Briefly, 1.0 mL of sample was added with 1.0 mL of FC reagent and incubated for 5 min in the dark before the addition of 2.0 mL of Na₂CO₃ solution. The absorbance was measured spectrophotometrically at 725 nm. Gallic acid (1-1000 µg mL^-1) was used for standard calibration. The results were expressed as gallic acid equivalent (GAE) per gramme fresh sample.

Estimation of Total Flavonoid Content

Total flavonoid content (TFC) of citrus samples was estimated based on the aluminium chloride colourimetric method as described by Ghasemzadeh et al.,²⁰ Briefly, 2.0 mL of the diluted sample was mixed with 0.2 mL of 5% NaNO₃ and incubated at room temperature for 5 min before the addition of 0.2 mL of 10% AlCl₃. After 6 min of incubation, the mixture was added with 2.0 mL of 1 M NaOH. The volume was then made up to 5.0 mL by adding 70% ethanol and incubated for another 10 min. Absorbance of the reacting mixture was read at 430 nm. Quercetin was used as a standard at concentrations ranged between 1.0 µg mL^-1 and 1000.0 µg mL^-1. The results were presented as milligramme quercetin equivalent per gramme (mg QE g^-1) fresh sample.

DPPH Radical Scavenging Assay

DPPH radical scavenging activity of citrus samples was determined according to the method described by Thaipong et al.,²¹ with slight modification. DPPH stock solution was prepared by adding 24 mg DPPH with 100 mL of methanol, whereas DPPH working solution was prepared by mixing 10 mL of the DPPH stock solution with 45 mL of methanol. Briefly, 500 μL of the sample was added with 500 µL of DPPH working solution. The mixture was incubated for 2 h at room temperature in the dark. Absorbance of the mixture was determined at 515 nm against a blank. The results were expressed as EC₅₀ value of DPPH assay in mg mL^-1.

Ferric Reducing Antioxidant Power

Ferric reducing antioxidant power (FRAP) of citrus samples was determined based on the method described by Ghasemzadeh et al.,²⁰ FRAP reagent was prepared by adding 2.5 mL of TPTZ (10 mM in 40 mM HCl) with 2.5 mL of FeCl₃ solution (20 mM in distilled water) and 25 mL of acetate buffer (300 mM, pH 3.6). In brief, 3.0 mL of the freshly prepared FRAP reagent was added with 50 µL of the sample, and stand at 37°C for 30 min in the dark. Absorbance was determined at 593 nm after 30 min of incubation. Antioxidant activity was expressed as millimoles ferrous ion per gramme fresh weight (mM Fe²⁺ g^-1 FW).

Statistical Analysis

The data were presented as mean ± standard deviation of three replicates (n = 3). The mean differences were statistically analysed based on one-way analysis of variance (ANOVA) with posthoc test (Tukey HSD). Pearson correlation coefficient was determined between the content of antioxidant compounds and antioxidant activity of the fresh and frozen citrus peels. The p-value of less than 0.05 was considered significant differences between different citrus samples. Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 23.

Results and Discussion

Extraction Yield

Solvent extraction is the most commonly used technique in isolation of antioxidant compound because it is a widely applicable technique and efficient in extracting bioactive compounds. Ethanol is one of the popular solvent used in extraction as it offers higher antioxidant properties compared with water extraction.^22,23 The extraction yields obtained are presented in Table 1. Aqueous ethanol (70%) was chosen as the extraction solvent due to the reason that it is an ideal solvent for extracting polyphenolic compounds; it is also not toxic to the environment and safe for human consumption.^3,24 The results of this study showed that the extraction yields of all frozen citrus peels were significantly higher than the fresh citrus peels (p < 0.05).

Table 1: Extraction yield, total phenolic content (TPC) and total flavonoid content (TFC) of frozen and fresh citrus peels.

The values are presented as mean ± standard deviation (n = 3).

Different superscript lowercase letters denote a significant difference at p < 0.05, Tukey posthoc test.

GAE – gallic acid equivalent; QE – quercetin equivalent; FW – fresh weight.

The result showed that the extraction yield for fresh lemon peel (11.0%) was significantly lower than frozen lemon peel (33.9%). Similarly, extraction yields for fresh key lime peel (6.7%) and fresh musk lime (7.9%) were also significantly lower than frozen key lime peel (31.7%) and frozen musk lime peel (33.0%), respectively. Based on the result obtained, frozen lemon peel had 207.3% total extractable yield higher than fresh lemon peel. Similarly, frozen key lime peel and frozen musk lime peel had 373.6% and 315.9% total extraction yield higher than fresh key lime peel and fresh musk lime peel, respectively. Freezing technique serves as a better method to preserve the antioxidant activity of polysaccharide from plant samples as the bioactive compound in the sample can be maximally protected under the freezing condition.²⁵ Literature also showed that frozen carrot samples had higher extraction yield of carotenoids than the non-frozen samples.²⁶

Total Phenolic Content

In this study, all frozen citrus samples had significantly higher TPC than the fresh citrus samples (Table 1). The results showed that TPC of fresh lemon peel (72.0 ± 0.67 mg GAE g^-1 FW) was significantly lowered than frozen lemon peel (100.0 ± 0.96 mg GAE g^-1 FW). The TPC was also significantly different between fresh musk lime peel (108.6 ± 1.34 mg GAE g^-1 FW) and frozen musk lime peel (136.5 ± 0.58 mg GAE g^-1 FW). Moreover, TPC of fresh key lime peel (75.7 ±1.34 mg GAE g^-1 FW) and frozen key lime peel (118.6 ± 0.77 mg GAE g^-1 FW) had the same trend, where the frozen samples possess significantly higher TPC than the fresh sample. Furthermore, the finding of this study shows that frozen lemon peel had 38.9% increment in TPC compared with the fresh lemon peel. Similarly, the frozen key lime peel and frozen musk lime peel showed 56.7% and 25.7% increment in TPC, respectively.

Fruit possesses a high concentration of phenolic in the outer compartment. The phenolic compounds are known to be accumulated in the vacuoles. Food processing such as freezing helps to accelerate a more release of the bound phenolic compound from the breakdown of the cellular part.²⁷ Besides, the enzymes that can destroy antioxidant such as oxidative and hydrolytic enzymes are deactivated during freezing.^28,29 Our finding is supported by a previous report that the TPC of frozen Ajwain leaf sample was significantly higher than the fresh sample.³⁰ On the contrary, freezing the freshly cut peach at −20 °C for 24 h did not significantly affect TPC of the peach compared with the freshly cut peach.³¹ The content of total phenolics of frozen raspberry and blackberry of different cultivars was also significantly lower than the fresh samples.³² The freshness of fruit samples reported in these studies could be varied. Postharvest storage of different fresh fruit samples should be further investigated. As the studied frozen fruit samples showed a better TPC than the non-frozen fresh samples; therefore, cold storing or freezing of freshly harvested fruit retains the bioactive antioxidants of the fruit.

Total Flavonoid Content

Fruit contains naturally occurring compounds. These compounds are hydroxybenzoic acid, anthocyanins, hydroxycinnamic acids, proanthocyanidins, flavonoids, lignans and stilbenes.³³ As shown in Table 1, TFC of the frozen citrus peels was significantly higher than the fresh citrus peels. Based on the results obtained, TFC of fresh lemon peel (50.51 mg QE g^-1 FW) and frozen lemon peel (78.15 mg QE g^-1 FW) was significantly different. TFC of the fresh key lime peel (50.75 mg QE g^-1 FW) was also significantly lower than the frozen key lime peel (82.22 mg QE g^-1 FW). Similar with frozen lemon and key lime peels, TFC of frozen musk lime peel (178.32 mg QE g^-1 FW) was significantly higher than the TFC of fresh musk lime peel (120.92 QE g^-1 FW). The finding also showed that frozen lemon peel had 54.7% increment in TFC compared with the fresh lemon peel. Similarly, frozen key lime peel and frozen musk lime peel had 62.0% and 47.5% increment of TFC, respectively. Based on a previous study, frozen red raspberries had higher TFC than the fresh and refrigerated red raspberries.³⁴ The reason might be due to the deactivation of oxidative and hydrolytic enzymes during the freezing process.²⁸

DPPH Radical Scavenging Activity

Antioxidants have the capability to slow down or prevent the oxidation process of other molecules.³⁵ DPPH radical scavenging assay is a procedure for estimation of antioxidant capacity by measuring the decrease in absorbance after oxidation processes. The scavenging activity was determined based on the capability of the antioxidants to retard colour loss. The results of this study showed that no significant difference was found between the frozen and fresh citrus samples (Table 2). Among the samples, frozen lemon peel had the lowest EC₅₀ value (0.82 mg mL^-1), followed by fresh lemon peel (1.30 mg mL^-1), frozen key lime peel (1.57 mg mL^-1), fresh key lime peel (1.83 mg mL^-1), frozen musk lime peel (2.70 mg mL^-1) and fresh musk lime peel (3.16 mg mL^-1). The lowest EC₅₀ value represents the highest scavenging activity of a sample. Although no statistically significant difference was found between EC₅₀ values of the frozen and fresh citrus peels, the frozen peels had better efficiency in scavenging DPPH radicals than the fresh citrus peels.

Ferric Reducing Antioxidant Power

Ferric reducing antioxidant power (FRAP) assay is another method for determination of the antioxidant activity of a plant extract. The assay signifies the power of antioxidants as reductants in the redox-linked colourimetric reaction. This assay is also used to quantify the capability of antioxidants in a sample to reduce ferric (III) ions to ferrous (II) ions at a low pH.³⁵ As shown in Table 2, FRAP values of the fresh and frozen citrus peels ranged from 0.38 mM Fe²⁺ g^-1 FW to 0.52 mM Fe²⁺ g^-1 FW. FRAP value of frozen musk lime peel (0.53 mM Fe²⁺ g^-1 FW) was significantly higher than the value of fresh musk lime peel (0.46 mM Fe²⁺ g^-1 FW). Similarly, frozen peels of lemon and key lime had significantly higher FRAP values than the fresh peels (Table 2).

Table 2: Antioxidant activities of frozen and fresh citrus peels.

The values are presented as mean ± standard deviation.

Different superscript lowercase letters denote a significant difference at p < 0.05, Turkey posthoc test.

FW – fresh weight.

There are limited published data on FRAP values of fresh and frozen peels of lemon, key lime and musk lime. Only data on FRAP values of fresh and frozen guava samples are available for reference. Although no significant difference between the FRAP values of fresh and frozen samples was observed, the FRAP value of the frozen guava sample was slightly higher than the FRAP value of fresh guava sample.³⁶ It could be due to the low storage temperature destructed the cell walls of guava, and also the release slightly higher bioactive compounds such as polyphenol and carotenoids from the frozen guava tissue. The higher free phenolics extracted from the frozen fruit sample contributed to the significant increase in antioxidant activity.³⁰

Correlation between Antioxidant Content and Antioxidant Activity

As shown in Table 3, EC₅₀ values of DPPH radical scavenging assay were not significantly correlated with both TPC or TFC. Therefore, an increase in the TPC does not increase the DPPH scavenging activity. It might be due to the fact that the increased value of TPC is attributed to ascorbic acid content.

Table 3: Correlation coefficient of total antioxidant content and antioxidant activity.

NC – no correlation; r – Pearson coefficient

**significant at p < 0.05.

A previous study revealed that ascorbic acid is a strong free radical scavenger.³⁷ Literature also shows that citrus peels exhibited a high vitamin C content.³⁸ As citrus peel contains a high amount of ascorbic acid, the compound might affect the DPPH radical scavenging activity of the peel. The finding of this study is supported by a study which determined vitamin C and antioxidant capacities of fresh and frozen guava samples.³⁶ Based on the study, no correlation was determined between the phenolic compound and antioxidant activity of the guava samples. However, there was a high correlation between vitamin C content and antioxidant capacities. Thaipong et al.,²¹ also showed that there was a strong correlation between ascorbic acid content and antioxidant capacity of the citrus and guava samples. On the contrary, Sir Elkhatim et al.,³⁸ reported a low correlation between vitamin C content and DPPH radical scavenging assay. Another study also revealed that no significant correlation was found between the antioxidant content and scavenging activity of the peels of 13 citrus species.³⁹

FRAP values of the fresh and frozen citrus peels reported in this study (Table 3) were positively correlated with TPC (r= 0.783) and TFC (r= 0.681). The higher the FRAP values of the frozen and fresh citrus peels, the higher the total antioxidants. Therefore, total phenolics and flavonoids were highly correlated with FRAP values. The finding suggests that phenolic compounds in the citrus samples highly contributed to the reduction of ferric ions to ferrous ions. A preceding study also supports our finding that the peels of C. sinensis and C. aurantium had a strong correlation between phenolic content and reducing power.⁴⁰

Conclusion

In brief, frozen citrus peels had higher antioxidant properties than the fresh peels. The frozen peels also possessed higher phenolics content and antioxidant activity than the fresh peels. Based on the results obtained, frozen citrus peels have high dietary antioxidants. Due to the high antioxidant capacity, frozen citrus peels can be used as raw ingredients for production of dietary supplements and in value-added products. Food industries are encouraged to fully utilise the fruit peels resulted from juice processing since these by-products contain many potentials in terms of their health benefits.

Conflict of Interest

The authors declare no conflict of interest with any person or Organization in publishing this article.

Acknowledgements

The authors are grateful to officers and staffs of the Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia for providing the instrumentation and other resources to carry out the study.

References

1. Datta, K., Sinha, S., Chattopadhyay, P. Reactive oxygen species in health and disease. Natl Med J India. 2000;13(6):304-310.
2. Wootton-Beard, P. C., Ryan, L. Improving public health?: The role of antioxidant-rich fruit and vegetable beverages. Food Res. Int.2011;44(10):3135-3148.
   CrossRef
3. Shafekh, S. E., Khalili, R., Catherine, C. C. W., Syakiroh, Z. A., Habibah, A. U., Norhayati, A. H., Farhanah, M.Y., Husna, Z.N., Nafizah, M.B., Azlina, M., Sazura, A. R. Total phenolic content and in vitro antioxidant activity of Vigna sinensis. Food Res. J. 2012;19(4):1393-1400.
4. Ghafar, M. F., Prasad, K. N., Weng, K. K., Ismail, A. Flavonoid, hesperidine, total phenolic contents and antioxidant activities from Citrus Afr. J. Biotechnol. 2010;9(3):326-330.
5. Mehl, F., Marti, G., Boccard, J., Debrus, B., Merle, P., Delort, E., Baroux, L., Raymo, V., Velazco, M. I., Sommer, H., Wolfender, J. L. Differentiation of lemon essential oil based on volatile and non-volatile fractions with various analytical techniques: a metabolomic approach. Food Chem. 2014;143:325-335.
   CrossRef
6. (2016). Malaysia – Citrus fruit production quantity. Retrieved March 20, 2018, from https://knoema.com/atlas/Malaysia/topics/Agriculture/Crops-Production-Quantity-tonnes/Citrus-fruit-production
7. Salleh, M. M., Yusof, R. M. Tropical fruits and vegetables in Malaysia: Production and impact on health. FAO Corporate Document Repository, 2006. http://www.fao.org/fileadmin/templates/agphome/documents/horticulture/WHO/seoul/Tropical_Fruits_Vegetables_Malaysia.pdf. Accessed April 13, 2018.
8. Fejzić, A., Ćavar, S. Phenolic compounds and antioxidant activity of some citruses.  Chem. Technol. B&H. 2014;42:1-4.
9. Milind, P., Dev, C. Orange: Range of benefits. Res. J. Pharm. 2012;3(7):59-63.
10. Sandhya, S., Sai Kumar, P., Vinod, K. R., Banji, D., Kumar, K. Plants as potent anti-diabetic and wound healing agents: A review. HYGEIA – J. Drugs Med. 2011;3(1):11-19.
11. Liu, Y. Q., Heying, E., Tanumihardjo, S. A. History, global distribution, and nutritional importance of citrus fruits. Rev. Food Sci. Food Saf. 2012;11(6):530-545.
    CrossRef
12. Guimarães, R., Barros, L., Barreira, J. C., Sousa, M. J., Carvalho, A. M., Ferreira, I. C. Targeting excessive free radicals with peels and juices of citrus fruits: grapefruit, lemon, lime and orange. Food Chem. Toxicol. 2010;48(1):99-106.
    CrossRef
13. O’Shea, N., Arendt, E. K., Gallagher, E. Dietary fibre and phytochemical characteristics of fruit and vegetable by-products and their recent applications as novel ingredients in food products.  Food Sci. Emerg. Technol. 2012;16:1-10.
    CrossRef
14. Sawalha, S. M., Arráez-Román, D., Segura-Carretero, A., Fernández-Gutiérrez, A. Quantification of main phenolic compounds in sweet and bitter orange peel using CE–MS/MS. Food Chem. 2009;116(2):567-574.
    CrossRef
15. Ramful, D., Tarnus, E., Aruoma, O. I., Bourdon, E., Bahorun, T. Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps. Food Res. Int. 2011;44(7):2088-2099.
    CrossRef
16. Chan, Z., Tian, S. Induction of H₂O₂-metabolizing enzymes and total protein synthesis by antagonistic yeast and salicylic acid in harvested sweet cherry fruit. Postharvest Biol. Tec. 2006;39(3):314-320.
    CrossRef
17. Villa-Rodriguez, J. A., Palafox-Carlos, H., Yahia, E. M., Ayala-Zavala, J. F., Gonzalez-Aguilar, G. A. Maintaining antioxidant potential of fresh fruits and vegetables after harvest.  Rev. Food Sci. Nutr. 2015;55(6):806-822.
    CrossRef
18. Ferreira, S. S., Silva, A. M., Nunes, F. M. Citrus reticulata Blanco peels as a source of antioxidant and anti-proliferative phenolic compounds. Ind Crops Prod. 2018;111:141-148.
    CrossRef
19. Meda, A., Lamien, C. E., Romito, M., Millogo, J., Nacoulma, O. G. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 2005;91(3):571-577.
    CrossRef
20. Ghasemzadeh, A., Jaafar, H. Z., Rahmat, A. Antioxidant activities, total phenolics and flavonoids content in two varieties of Malaysia young ginger (Zingiber officinale Roscoe). Molecules 2010;15(6):4324-4333.
    CrossRef
21. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., Byrne, D. H. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts.  Food Compos. Anal. 2006;19(6-7):669-675.
    CrossRef
22. Kim, J. S. Preliminary evaluation for comparative antioxidant activity in the water and ethanol extracts of dried citrus fruit (Citrus unshiu) peel using chemical and biochemical in vitro assays. Food Nutr. Sci. 2013;4(2):177-188.
    CrossRef
23. Mao, L. C., Pan, X., Que, F., Fang, X. H. Antioxidant properties of water and ethanol extracts from hot air-dried and freeze-dried daylily flowers.  Food Res Technol. 2006;222(3-4):236-241.
    CrossRef
24. Dai, J., Mumper, R. J. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 2010;15(10):7313-7352.
    CrossRef
25. Li, X., Wang, L., Wang, Y., Xiong, Z. Effect of drying method on physicochemical properties and antioxidant activities of Hohenbuehelia serotinaProcess Biochem. 2016;51(8):1100-1108.
    CrossRef
26. Fikselová, M., Šilhár, S., Mareček, J., Frančáková, H. Extraction of carrot (Daucus carota) carotenes under different conditions. Czech J. Food Sci. 2008;26(4):268-274.
    CrossRef
27. Minatel, I. O., Borges, C. V., Ferreira, M. I., Gomez, H. A. G., Chen, C. Y. O., Lima, G. P. P. Phenolic compounds: Functional properties, impact of processing and bioavailability. London, United Kingdom: InTech; 2017.
    CrossRef
28. Chang, C. H., Lin, H. Y., Chang, C. Y., Liu, Y. C. Comparisons on the antioxidant properties of fresh, freeze-dried and hot-air-dried tomatoes.  Food Eng. 2006;77(3):478-485.
    CrossRef
29. Pérez-Gregorio, M. R., Regueiro, J., González-Barreiro, C., Rial-Otero, R., Simal-Gándara, J. Changes in antioxidant flavonoids during freeze-drying of red onions and subsequent storage. Food Control, 2011;22(7):1108-1113.
    CrossRef
30. Raza, M., Shukla, A. K., Fatima, T., Ali, S. Comparative study of antioxidant activity of polyphenols isolated from frozen and fresh leaves of Trachyspermum ammi (Ajwain).  Pharmacogn. Phytochem. 2015;3(6):122-124.
31. Oliveira, A., Alexandre, E. M., Coelho, M., Barros, R. M., Almeida, D. P., Pintado, M. Peach polyphenol and carotenoid content as affected by frozen storage and pasteurization. LWT-Food Sci. Technol. 2016;66:361-368.
    CrossRef
32. Türkben, C., Sarıburun, E., Demir, C., Uylaşer, V. Effect of freezing and frozen storage on phenolic compounds of raspberry and blackberry cultivars. Food Anal. Method. 2010;3(3):144-153.
    CrossRef
33. Cheong, M. W., Chong, Z. S., Liu, S. Q., Zhou, W., Curran, P., Yu, B. Characterisation of calamansi (Citrus microcarpa). Part I: Volatiles, aromatic profiles and phenolic acids in the peel. Food Chem. 2012;134(2):686-695.
    CrossRef
34. Mullen, W., Stewart, A. J., Lean, M. E., Gardner, P., Duthie, G. G., Crozier, A. Effect of freezing and storage on the phenolics, ellagitannins, flavonoids, and antioxidant capacity of red raspberries.  Agric. Food Chem. 2002;50(18):5197-5201.
    CrossRef
35. Foti, M. C., Daquino, C., & Geraci, C. Electron-transfer reaction of cinnamic acids and their methyl esters with the DPPH• radical in alcoholic solutions.  Org. Chem. 2004;69(7):2309-2314.
    CrossRef
36. Polinati, R., Oliveira, L., Fialho, E. Bioactive compounds, vitamin C and antioxidant capacities of fresh and industrialized frozen pulps of guava (Psidium guajava). Nat. Prod. J. 2012;2(3):196-204.
    CrossRef
37. Oh, C. W., Li, M., Kim, E. H., Park, J. S., Lee, J. C., Ham, S. W. Antioxidant and radical scavenging activities of ascorbic acid derivatives conjugated with organogermanium.  Korean Chem. Soc. 2010;31(12):3513-3514.
    CrossRef
38. Sir Elkhatim, K. A., Elagib, R. A., Hassan, A. B. Content of phenolic compounds and vitamin C and antioxidant activity in wasted parts of Sudanese citrus fruits. Food Sci. Nutr. 2018;6(5):1214-1219.
    CrossRef
39. Ghasemi, K., Ghasemi, Y., Ebrahimzadeh, M. A. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. J. Pharm. Sci. 2009;22(3):277-281.
40. Lagha-Benamrouche, S., Madani, K. Phenolic contents and antioxidant activity of orange varieties (Citrus sinensis and Citrus aurantium L.) cultivated in Algeria: peels and leaves. Ind. Crops Prod. 2013;50:723-730.
    CrossRef