The potential use of citrus juice waste as sources of natural phenolic antioxidants

Article history: Received on: 29/08/2016 Revised on: 19/09/2016 Accepted on: 12/10/2016 Available online: 28/12/2016 The methanolic extract of citrus juice processing waste was evaluated for its potential applications in medicine or as nutraceutical materials. Determination of total phenolic and flavonoid contents was done by microplate reader, whereas UPLC-mass detection was used for the analysis of individual flavanone (naringin, hesperidin, hesperetin, neohesperedin and narirutin) and flavonol (rutin). In addition, free radicals (DPPH, O2 , H2O2 and NO) scavenging and ferrous ion chelating assays were used to determine the antioxidant capacity. The contents of total phenolic and flavonoid were more prevalent in citrus juice processing waste than the level found in the pulp of citrus fruit. The results of UPLC analyses indicate that citrus juice processing waste was rich in mainly hesperidin and neohesperedin, and it had different flavonoid composition in comparison with the pulp of citrus fruit. The citrus juice processing waste was also found to possess an evident antioxidant capacity. The results showed that citrus wastes could be economic and readily accessible source of natural antioxidants and as a possible food and pharmaceutical supplement.


INTRODUCTION
Citrus is the main fruit crop in the world, with a total production of 122 million tons in 2008 (Terol et al., 2010).In Korea, Jeju island is well known for its important production of citrus fruits (0.6 million tons in 2008/2009) (Department of Citrus Policy, Jeju Special Self-Governing Providence, Korea).Citrus species are rich sources of vitamin C, folate, dietary fibre, and minerals as well as many antioxidant phytophenolics, including flavonoids, amino acids, triterpenes, phenolic acids and carotenoids that can potentially protect health (Gypta et al., 2015).Recently, there is much biomedical interest in citrus fruits because their consumption appears to be associated with lower risk of colorectal, esophageal, gastric and stomach cancers and stroke (Roussos, 2011).Citrus fruits are utilized primary for juice recovery, where about half of the processed citrus including peels, segment membrane and seeds ends up as wastes.These solid residues are referred to as citrus wastes with estimated worldwide production of 15 million tons per year (Marı´n et al., 2007).Only a very small part of the wastes has been properly converted into useful or high-value products and most are disposed in landfills, constituting severe economic and environmental problems (Montgomery, 2004).In Jeju island, more than 50,000 tons of citrus wastes including citrus peels generated by juice processing facilities and canning industries, and premature fruit drops in orchards annually have been incinerated in disposal yards and dumped into the ocean (Yang et al., 2009).The by-products industry has a potential for growth since products have also been produced from citrus fruit residues.The peel of citrus fruits, the primary by-product, is a rich source of flavones as well as many polymethylated flavones, they could be which are very rare in other plants (Nogata et al., 2006).These beneficial actions suggest new value-added uses for these compounds as nutraceuticals and specialty ingredients, and studies of the occurrence of the polymethoxylated flavones in by-products generated from citrus juice processing or premature fruit drops caused by poor condition from climate change, some diseases and other stresses are currently of considerable interest to both pharmaceutical and food industries.Therefore, this study was performed to test the potentiality of citrus juice processing waste as a functional nutraceutical and pharmaceutical source on the basis of their biomolecules with antioxidant property.Here we report a comparative analysis of the polyphenolic compounds and in vitro antioxidant properties of the citrus pulp extract and citrus peel waste after juice extraction.

Plant material and extraction
The peel waste of citrus fruit (Citrus unshiu Marc.) after juice extraction was obtained from a local food processing company (Jeju Provincial Development Co., Jeju, Korea).Samples were extracted twice according to the method described by Im et al. (2014).

Analysis of total phenolic and flavonoid contents
Contents of total phenol and flavonoid were determined by the method described previously (Im et al., 2014).
The injected sample volume was 2 μL and the UV spectra by PDA were recorded between 210 and 410 nm.MS detection was performed directly after PDA measurements.The ESI source was optimized with positive ionization mode as follows: scan spectra from m/z 100 to 400, capillary voltage 3.30 kV, cone voltage 20 V, source temperature 120 °C and desolvation temperature 360 °C.Nitrogen was used as the desolvation and cone gas with a flow rate of 850 and 50 L/h, respectively.For quantitative determination of the five flavonoids, the MS detection was operated in negative ESI mode with multiple reaction monitoring (MRM).Argon was used as the collision gas at a pressure of 3.82 × 10 -3 mbar.Instrument control, data acquisition, and quantification were performed by Mass Lynx 4.1 software (Waters).

Statistical analysis
Comparisons of all results between citrus pulp and citrus waste samples were made by using a nonparametric test (Mann-Whitney U test) with p < 0.05 (SPSS, ver.12.0; SPSS Inc., Chicago, IL, US).For each measurement, three replicate samples were tested.A dose response curve was plotted to determine the IC 50 values which are defined as the concentration sufficient to obtain 50 % of a maximum scavenging capacity.

RESULTS AND DISCUSSION
Phenolic compounds act as free radical terminator, and mostly include flavonoids, phenolic acids, stilbenes, coumarins and tannins (Winkel-Shirley, 2002).In many cases, flavonoids may increase antioxidant activity as part of a general stress response (Winkel-Shirley, 2002).The mechanisms of action of flavonoids are through scavenging or chelating process (Kessler et al., 2003).In the present study, citrus juice processing waste extract (41.6 and 13.2 g/100 g) had significantly higher total phenolic and flavonoid contents than the pulp extract of citrus fruit (8.3 and 5.7 g/100 g), respectively (p < 0.05) (Figure 1).Flavonoids are a widely distributed group of phenolic compounds, which have a wide range of biological effects, such as inhibition of key enzymes in mitochondrial respiration, protection against coronary heart disease and anti-inflammatory, antitumor, and antimicrobial activities (Wang et al., 2008).Four types of flavonoids occur in citrus: flavanones, flavones, flavonols and anthocyanidins (Benavente-García et al., 1997).Citrus fruit contains high levels of the flavanones, as well as flavonol, which are very rare in other plants (Gattuso et al., 2007).Some of these flavanones and flavonol are of commercial interest because they are used in the pharmaceutical and food industries.In this study, obtained results showed that naringin, hesperidin, hersperetin, neohesperedin, neohesperedine and narirutin were the main flavanones in citrus juice processing waste extract (Table 1).
In comparison with the pulp of citrus fruit, citrus waste contained significantly lower concentration of hesperidin, narirutin and rutin (p < 0.05), but naringin and neohesperedin were present only; the flavanone glycoside pattern consisted predominantly of hesperidin (0.104 g/100 g) and neohesperedin (0.105 g/100 g) (Table 1).
The interest in natural antioxidants has grown in the recent years with the awareness of several fatal diseases.Antioxidants act in the various ways, binding metal ions, scavenging radicals and decomposing peroxides.Often more than one mechanism is involved.In the present study, citrus juice processing waste extract and the pulp extract of citrus fruit were evaluated for their antioxidant capacity by free radical scavenging (DPPH, superoxide, hydrogen peroxide and nitric oxide) and ferrous ion chelating assays, as shown in Table 2.The citrus juice processing waste extract showed slightly higher or similar hydrogen peroxide and superoxide scavenging activities compared with those of the pulp extract of citrus fruit (Table 2).
Other antioxidant assays need to be undertaken to verify this point and to understand the full potential of citrus juice processing waste extract as an antioxidant agent.Therefore, we examined the DPPH and ferrous ion chelating activities of citrus juice processing waste extract (Table 2).In the DPPH radical scavenging assay, IC 50 value of the citrus juice processing waste extract (1.1 mg/mL) was significantly lower than that of the pulp extract of citrus fruit (2.2 mg/mL) (p < 0.05) (Table 2).The DPPH assay is often used to evaluate the ability of antioxidants to scavenge free radicals which are known to be a major factor in biological damages caused by oxidative stress.DPPH radical involves a hydrogen atom transfer process (Kaviarasan et al., 2007).In this assay, the antioxidant activity on DPPH radicals of the citrus juice processing waste extract may be attributed to a direct role in trapping free radicals by donating hydrogen atom.
The methanolic extract of the citrus juice processing waste was also tested for ferrous ion chelating activity (Table 2).It exhibited 1.0 mg/mL IC 50 value, they were similar to that of the pulp extract of citrus fruit, indicating that the reductive capacity of antioxidants may affect ion, especially Fe 2+ and Cu 2+ (Senevirathne  et al., 2010).Ferrous is a vital substance for normal physiology, but an excess of it may result in cellular damage.If iron undergoes the Fenton reaction, these reduced metals may form highly reactive hydroxyl radicals and thereby contributing to oxidative stress (Senevirathne et al., 2010).
The results from the present work, citrus juice processing waste possessed the higher total phenolic content and exhibited strong antioxidant activity and it, therefore, may be developed as natural antioxidant for food industry and other fields.The bioactive components and antioxidative actions of citrus juice processing waste extract warrant further studies in both in vitro and in vivo models.1.1 ± 0.08 * 1.6 ± 0.03 2.0 ± 0.18 * 2.8 ± 0.10 1.0 ± 0.02 IC50 was obtained by interpolation from linear regression analysis.Each values is expressed as mean ± standard deviation (n = 3).

Fig. 1 :
Fig. 1: Total phenolic and flavonoid compositions of the methanolic extracts obtained from citrus pulp and citrus juice processing waste.Each value is expressed as mean ± standard deviation (n = 3).* p < 0.05, compared to pulp.

Table 1 :
Flavonoid content (g/100 g dry weight) of citrus pulp and citrus juice processing waste.Values are significantly different from corresponding pulp (p < 0.05). *

Table 2 :
IC50 value in free radical scavenging and ferrous ion chelating properties of citrus pulp and citrus juice processing waste.