Introduction

Citrus is an important fruit crop in the world (FAOSTAT, 2017). It includes citrons, lemons, limes, oranges, mandarins, grapefruits and pummelos etc. Many citrus species are valued for their medicinal and pharmacological activities such as antioxidant (He, Shao, Liu, & Ru, 2012), anticancer (Zhu et al., 2013), anti-inflammatory (Huang & Ho, 2010) and antimicrobial etc. The health benefits of citrus fruits have been attributed to the presence of bioactive compounds including antioxidants such as ascorbic acid, flavonoids,phenols, carotenoids etc. (Benavente-García & Castillo, 2008; Tripoli, Guardia, Giammanco, Majo, & Giammanco, 2007). The antioxidant capacities of fruits vary depending on contents of the above said compounds. Kinnow (Citrus reticulata Blanco), a hybrid of (Citrus nobalis x Citrus deliciosa), comes in mandarin group of citrus fruits and has a great demand in India especially in northern states.

Various studied have suggested that citrus phenols and flavonoids are protective against a variety of diseases such as coronary heart disease (Hertog et al., 1993; Majo et al., 2005) and some type of cancers. They also possess anti-inflammatory properties. These effects are probably due to their ability to inhibit lipid peroxidation and oxidation of LDL (Elangovan, Sekar, & Govindasamy, 1994; Martın et al., 2002; Stavric, 1994). These compounds have metal chelating properties, high redox potential and can act like hydrogen donors (Tsao & Yang, 2003).

Kinnow is an important natural source of antioxidants as it contains a number of significant biologically active compounds (Zhang, Yang, & Zhou, 2018). In order to effectively screen these compounds in Kinnow mandarin as a source of antioxidant, the present investigation focused on the evaluation of Total Phenol (TP), Total Flavonoid (TF) and antioxidant activity by DPPH free radical scavenging method in leaf extracts of Kinnow mandarin.

Materials and Methods

The plant parts under investigation were vegetative and fruit associated leaves of Kinnow. Leaves were collected from Dal farm house, Sriganganagar (Rajasthan) at three different times of the year: March, July and December, which corresponds to three different ripening stages of Kinnow fruit- early, mid and late. Leaves were dried for two weeks in free air and in dark and powdered. The methanolic extracts were prepared from dried and coarsely powdered plant materials.

Total phenols determination

Total phenolic content of the methanolic extracts was determined with FolinCiocalteu method (Mcdonald, Prenzler, Autolovich, & Robards, 2001). An aliquot of each plant extract (0.5ml 1:10 mg/l) or gallic acid (standard compound) was mixed with FolinCiocalteu reagent (5ml 1:10 diluted with distilled water) and 4ml of 1M Na₂CO₃ solution. The mixture was kept for 30 minutes at room temperature and absorbance was measured at 710nm. The values of Total phenolic content were reported as mg Gallic acid equivalent (GAE)/g fresh weight. All samples were analyzed in triplicates.

Total flavonoids determination

Aluminum Chloride method (Chang, Yang, Wen, & Chern, 2002) was used for Total Flavonoid Content determination. Methanolic solution of each leaf extract (0.5 ml of 1:10 g/lt) was mixed with 0.1 ml of 10% AlCl₃, 0.1 ml of 1M potassium acetate and 2.8 ml distilled water. The mixture was kept for 30 minutes at room temperature and absorbance was measured at 415nm. Total flavonoid content was expressed as mg quercitin equivalents (QE) per gram of fresh mass. Measurements were taken in triplicates for all samples.

Free radical scavenging assay by DPPH

DPPH free radical was used for determination of radical scavenging ability of the leaves extracts (Koleva, Beek, Linssen, Groot, & Evstatieva, 2002). DPPH prepared in methanol (0.004%) was mixed with different concentrations of each extract. The mixture was incubated for 15 minutes. The DPPH absorption at 517 nm was evaluated by spectrophotometer. The percentage discoloration was calculated by following formula.

Where AC₅₁₇ is absorbance of DPPH, AE₅₁₇ is the absorbance of DPPH and tested plant extract combination.

Results and Discussion

Total phenolic content

It is well known that methanol is an effective solvent for antioxidant extraction (Siddhuraju & Becker, 2003), therefore we used this solvent for extraction of bioactive compounds from Kinnow leaves extracts.

The results of our study exhibited a considerable diversity in TPC among Kinnow leaves (Table 1 ). The TPC from leaves extracts screened in present investigation ranged from 5.86 mg GAE/g dw (phase I fruit associated leaf) to 9.49 mg GAE/g dw (phase II fruit associated leaf). For the two tested tissues, the average of the phenolic content when ordered from high to low was as follows: Fruit associated Leaves phase II 9.49 ± 0.075> Veg leaf phase I 8.98 ± 0.028>Fruit associated Leaves phase III 8.25 ± 0.05> Veg leaf phase III 7.74 ± 0.025> Veg leaf phase II 6.75 ± 0.05>Fruit associated leaves phase I 5.86 ± 0.09. The trend of TPC results in leaf is much lower than those (21.45 mg GAE/g dw) of reported by (Nasri, Bedjou, Porras, & Martínez-Flórez, 2017) and those reported by (Lagha-Benamrouche & Madani, 2013), but was higher than those (2.48±0.07mg GAE/g dw: C. grandis, 3.83±0.78:C. limon) published by (Khettal et al., 2017). These differences in TPC could be due to the different ecological conditions. In addition, the variation in TPC trend could also be due to the geographical origin, species, cultivar and age of the plant as well as drying and extraction methods of the individual laboratory.

Total flavonoid content

The results show the presence of flavonoid compound in vegetative and fruit leaves with varying proportions (Table 1). The TFC from leaves evaluated ranged from1.12 mg QE/g dw (phase I vegetative leaf) to 7.74 mg QE/g dw (phase II fruit associated leaf). For the two tested tissues, the average of the flavonoid content when ordered from high to low was as follows: Fruit associated leaves phase II 7.74 ± 0.125>Fruit associated leaves phase III 6.25 ± 0.011 >Fruit associated leaves phase I 6.24 ± 0.075> Veg leaf phase III 5.01 ± 0.028> Veg leaf phase II 2.5 ± 0.05>Veg leaf phase I 1.12 ± 0.025. The trend of TFC results in leaf is in accordance with results obtained for various Citrus species by (Khettal et al., 2017) , but was higher than those (1.38) published by (Nasri et al., 2017). Different ecological conditions, genetic variability of plants and experimental setup are some of potential factors responsible for the variation in TPC.

Antioxidant capacity analysis

Results obtained for antioxidant capacity by DPPH free radical scavenging method in Kinnow leaves are presented in (Table 1). It was found that antioxidant activity ranged from 28.49 to 31.49 mg AA/g dw. Phase I fruit associated leaves had highest (31.49 mg AA/g dw) whereas phase I veg leaf (28.49 mg AA/g dw) had lowest values of it. The average values of antioxidant capacity from higher to lower order is as follows: fruit associated leaves phase I (31.49 mg AA/g dw) >fruit associated leaves phase II (31.25 mg AA/g dw) >fruit associated leaves phase III (30.5 mg AA/g dw) >veg leaf phase I (30 mg AA/g dw) >veg leaf phase II stage (29.5 mg AA/g dw) >veg leaf phase I (28.49 mg AA/g dw)

Figure 1 summarizes the results of TP, TF and antioxidant capacity while Figure 2 and Figure 3 represents DPPH Discoloration % and IC 50 Values respectively of the experiment conducted.

Conclusions

The findings from this study indicated that there was an increasing trend in antioxidant activity for vegetative leaves but the fruit leaves showed the reverse trend. There was an increase in concentration of flavonoids in vegetative leaves while fruit leaves have an irregular pattern for both Phenols and flavonoids. Our Results suggested that Kinnow mandarin is a potential source of antioxidant in methanol extract and could be used as a natural anti-oxidant. Present study also provided novel information regarding variation of TPC, TFC and its antioxidant potential and future utilization of Kinnow mandarin germplasm in India. Further phytochemical screening is required to isolate phytoconstituents that can show broad spectrum of natural compounds. It can be concluded that this plant is able to produce and accumulate many medicinally valuable secondary metabolites.

Abbreviations

AA- Ascorbic Acid, DPPH-1, 1 diphenyl-2-picryl hydrazyl, dw -dry weight, GAE– Gallic acid equivalents, QE -Quercitin equivalents

Conflict of Interest

None.

Funding support

None.