Abstract

Our current lifestyle has encountered free radicals and toxic metabolites in our bodies. Free radicals have been reported to be responsible for several diseases. Citrus juices contain biologically active compounds which possess antioxidant activity. They are essential components of functional food. Darjeeling mandarin is an important crop grown in Darjeeling and Kalimpong District of West Bengal and needs compositional analyses The present investigation was undertaken to study the genetic variability in Darjeeling mandarin in terms of fruit biochemical compounds with respect to Total phenols and antioxidants. Significant differences were obtained in terms of Total Soluble Solids (TSS) which was found to be the highest in DS (12.00 ⁰B) and lowest in 8.5 Mile (8.51 ⁰B). The total acidity of the mandarin juice ranged from 0.46 % to 0.82 %. The total phenolic content was highest in SM 68.53 ± 6.66 µg GAE/ml to lowest in 8.5 Mile 38.29 ± 0.89 µg GAE/ml. The Antioxidant activities through DPPH, FRAP, and ABTS assay recorded highest 173.70 µg VCE/ml, 13.47 nmol/ml, 86.60 µM VCE/ml juice in Lower Bong Busty (SM) while Bong Busty (DK) recorded the lowest antioxidant through DPPH 0.43±0.05 µg VCE/ml, FRAP 9.86±0.65 nmol/ml and ABTS 85.62±0.89 µM VCE/ml juice.

1. Introduction

Free radicals have been claimed to play an important role in affecting human health by causing severe diseases including cancer, hypertension, heart attack, and diabetes. These free radicals are generated during metabolism. Citrus fruits are rich sources of natural antioxidants, which are now widely accepted as being beneficial to human health ¹. Some studies have suggested that in addition to Vitamin C (ascorbic acid) and carotenoids, phenolic compounds are found in abundance in citrus fruits, which play an important role primarily in citrus fruits ^{2, 3}. The major phenolic compounds detected in different citrus fruits are categorized as flavonoids and phenolic acids ^{4, 5}. Among various natural healthy fruits, citrus fruits prevail abundantly around the world, and mandarins are among the most popular citrus fruits. Mandarins are a good source of organic acid and phenolic compounds. Their nature and concentration largely affect taste characteristics and organoleptic quality ^{6, 7}. Another peculiar characteristic of citrus juice is the high concentration of flavanones. Flavanones in citrus fruits occur mainly under their glycoside form.

Citrus is the third most important fruit crop in India after banana and mango. The most important commercial citrus groups in India are the mandarin (Citrus reticulata Blanco), followed by sweet orange (Citrus sinensis Osbeck) and acid lime (Citrus aurantifolia Swingle). A few ecotypes of mandarin (Citrus reticulata Blanco) including Sikkim mandarin, Darjeeling mandarin, and Khasi mandarin are excellent in quality and have good export potential ⁸. The Total Phenolic, flavonoid content, and antioxidant capacity of Darjeeling Mandarin have not been investigated. Therefore, this study was conducted to determine the Total Phenolic, flavonoid content, and antioxidant capacity of Darjeeling Mandarin.

2. Materials and Methods

The fruits of Darjeeling mandarin were collected during the harvesting season November-December-January in mandarin growing areas. Ten trees were randomly selected and from those selected and tagged trees a minimum of five fruits were harvested for analysis. The details of fruit collection are given below in Table 1.

The fruits were washed in running tap water and dried at room temperature. Analysis was carried out at the Biochemistry Laboratory of IARI regional station, Kalimpong. The fruit was analyzed for Total Soluble Solids (TSS) ᵒBrix, Total Acidity (%), TSS/TA ratio, Total phenol content (µg GAE/ml Juice), Total Flavonoid content (µg Rutin/ ml Juice), DPPH scavenging activity (µgVitamin C Equivalent /ml of juice), FRAP assay(nmol/ml) and ABTS activity (µg Vitamin C Equivalent /ml of juice).

The juice was extracted by cutting the fruit into two halves and careful hand squeezing to obtain the juice. The juice was passed through a strainer to remove pulp and seeds. The freshly squeezed juice was filtered through Whatman no 1 and kept overnight for filtration. The clear juice was stored at -20°C until further analysis.

Total soluble solids (TSS) were measured as °Brix using a refractometer (HI96801 Hanna Instrument).

The total titrable acidity (TA) was assessed by titration with sodium hydroxide (0.1 N) using 2-3 drops of phenolphthalein indicator and expressed as %.

Where:

m = molarity of NaOH

t = titre of NaOH required (mL)

v = volume of sample used (mL).

Sodium hydroxide (NaOH) pellets, phenolphthalein indicator .

Folin-ciocalteau (FC reagent) obtained from Merck (Darmstadt, Germany), Sodium carbonate, monohydrate from Himedia, Gallic acid monohydrate from Himedia.

Aluminium chloride, Sodium nitrate, methanol, sodium hydroxide

2,2-diphenyl-1-picryl hydrazyl (DPPH) was obtained from Sigma-Aldrich (St. Louis, MO, Ascorbic acid, methanol.

Sodium acetate, acetic acid, 2,4,6-Tripyridyl-s-triazine (TPTZ), Hydrochloride acid (HCL), ferric chloride FeCl₃

2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), was obtained from Himedia

Spectrophotometric measurements were performed on an ultraviolet UV/visible (UV-Vis) spectrophotometer (Eppendorf BioSpectrometer basic) equipped with 10mm quartz cells.

Total phenolic content was determined using the method of Thimmaiah ⁹. In one hundred µl of sample, 150µl of FCR (diluted 10 times solution was added and the solution was given a through spin. The solution was then kept for 5 to 10 min at room temperature. Then, 150 µl of 7.5 % of Sodium carbonate (Na₂CO₃) was added and incubated for 20 min at Room temperature. Absorbance was taken at 765 nm.

Calculations: Total Phenolic content was expressed as gallic acid equivalent (µg GAE/ml) through the calibration curve of gallic acid. The linearity range of the calibration curve was 10 to 100 µg/ml. The results were expressed as µg of gallic acid equivalents (GAE) per millimetre of extract (µg GAE/ml). All tests were performed in triplicate.

The aluminium chloride colorimetric method was used to measure the flavonoid content ¹⁰ with minor modifications. To 1ml of the juice, 1.25ml of distilled water was added. One ml of 5% Sodium nitrate (NaNO₂) was then added to the mixture followed by incubation for 5 minutes after which 0.5 ml of 10 % Aluminium chloride was added. The mixture was allowed to stand for 6 min at room temperature. Then 1ml of 1M sodium hydroxide (NaOH) was added to the solution diluted with 0.5ml distilled water. The mixture was incubated for 30 min at room temperature and absorbance was measured at 510 nm with a UV/VIS spectrophotometer immediately.

Calculations: Total flavonoid content was expressed as rutin equivalents (mg rutin/ml juice). The linearity range of the calibration curve was 10 to 100 µg/ml.

Many different methods have been reported for the antioxidant capacity evaluation of plant samples ¹¹. In this study, antioxidant activity was measured by DPPH, ABTS, and FRAP methods.

The 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) assay was performed according to the method developed by Brand-Williams et al. ¹² as slightly modified by Kim et al. ¹³. A solution of 1 mM DPPH in 80% (v/v) methanol was stirred for 40 min. The absorbance of the solution was adjusted to 0.650 at 517 nm using 80% (v/v) methanol. Then, 50 ml of the standard or test were mixed with 2.95 ml of DPPH solution and incubated for 30 min in the dark by covering the samples with aluminium foil. The decrease in absorbance was monitored at 517 nm at 30 min by a UV-Visible spectrophotometer. A control consisted of either 50 ml of distilled deionized water in 2.95 mL of DPPH solution for vitamin C standard or 50 mL of 50% (v/v) methanol in 2.95 ml of DPPH solution for samples. The DPPH scavenging activities of samples were expressed as mg vitamin C equivalent (VCE)/100 ml.

(%) Scavenging activity = Absorbance (control)-Absorbance (sample)/Absorbance (sample) X 100

Where control refers to the DPPH solution in methanol and sample is the absorbance of the sample.

All tests were performed in triplicate.

Antioxidant activity was also measured using an improved ABTS method as described by Re et al. ¹⁴ and Pellegrini et al. ¹⁵. The ABTS radical cation solution was prepared through the reaction of 7mm ABTS and 2.45 mm potassium persulfate, after incubation at 23°C in the dark for 12-16h. The ABTS solution was then diluted with 80% ethanol to obtain an absorbance of 0.700±0.005 at 734 nm. Added 10µl of the sample to 1ml of diluted ABTS Solution. The reaction mixture was allowed to stand at 23°C for 10 min and the absorbance at 734nm was immediately recorded. A standard curve was obtained by using Vitamin C standard solution at various concentrations in 80% ethanol. The absorbance of the reaction samples was compared to that of the vitamin C standard and the results were expressed in terms of vitamin C equivalents (VCE).

The working FRAP reagent was prepared by mixing 10 volumes of 300 mM acetate buffer, pH 3.6, with 1 volume of 10mM TPTZ (2,4,6-Tripyridyl-s-triazine) in 40mM HCl and with 1 volume of 20mM FeCl₃ ×6H₂O (ferric chloride hexahydrate). The prepared working FRAP reagent was warmed to 37°C. In a 6 ml FRAP reagent, 0.2 ml of sample and 0.6 ml of deionized water were added and the absorbance was taken at 593 nm against reagent blank after 4min ¹⁶. FRAP value was expressed as nmol/ml juice.

All analyses were run in triplicate and the results were expressed as mean ± standard error mean SEM. Statistical significance between groups was analyzed by applying a student's t-test. Values of P less than 0.001 were considered statistically significant.

3. Result and Discussion

The highest Total Soluble Solids (TSS) ᵒBrix of the fruit juices were reported from 7^th Mile Kalimpong-DS (12.00 ᵒBrix) followed by Bong Busty-DK (11.76ᵒBrix), whereas the lowest was recorded in 8.5 Mile of 8.51ᵒBrix (Table 2). Kundan et al., ¹⁷ reported a maximum TSS of 12.00ᵒBrix in Sikkim mandarin from the eastern district and the lowest of 10.7ᵒBrix from the northern district of Sikkim. Dorji and Yapwattanaphun ¹⁸ also recorded the highest TSS (greater than 12ᵒBrix) from the accession of Zhemgang and Trongsa district of Bhutan to TSS of an intermediate-range (10-12ᵒBrix) from other locations of Bhutan. The variation in TSS might be due to genetic and environmental variation.

The total acidity (%) of the Darjeeling mandarin is given in Table 2. It was found to be minimum in Barbot-YK of 0.46 % followed by 7^th-mile Kalimpong-DS (0.48 %) whereas maximum acidity was recorded in 8.5 Mile of (0.82 %). The total acidity was 1.04, 0.89, and 1.02 % in Satsuma, Robinson, and Fremont, respectively ¹⁹. The total acidity in juices of different mandarin varieties ranged from 0.94 to 1.87 % ²⁰. Nagy and Smoot ²¹ reported concentrations of total acidity (%) from 0.77 to 1.11. The higher acidity in the fruit may be due to less synthesis of polyphenol oxidase and peroxidase activities which increased the synthesis of organic acids like citric acid ^{17, 22}.

The TSS/TA ratio is an important parameter, associated with the quality characteristics of citrus fruits. 7^th Mile Kalimpong (DS) had the highest TSS/TA of 25 while 8.5 Mile had the lowest TSS/TA of 10.38 (Table 2). The ratios obtained for the studied samples were in good agreement with data previously reported by Yu et al. ²³ and Xu et al. ²⁰.

The total phenolic content is given in Table 3, the data on total phenolic content ranged from 68.53 to 38.28 µg GAE/ml. The highest total phenolic content was recorded in Lower Bong Busty-SM followed by Bong Busty (DK), Icchey Busty (AK), and Thapa Goan (DS) while the lowest phenolics content was recorded in 8.5 Mile. Similar results were also obtained by Prakash et al, ²⁴ and Srivastava et al, ²⁵. Ghasemi et al. ²⁶ reported the total phenolic contents in the fruit tissue was 66.5 mg GAE/g of the extract powder in Citrus unshiu var. Sugiyama to as high as 396.8 mg GAE/g of the extract powder in Citrus reticulata var. Clementine. The total phenolic contents in the juice of different citrus genotypes ranged from 7.28 to 34.03mg (gallic acid equivalent (GAE)g^-1 DW. These differences could be caused by the difference in citrus species or different growing conditions ²⁷. Also, Ghafar et al, ²⁸ reported differences in the results might be due to different extraction methods adapted and also due to different environmental conditions.

Flavonoids as one of the most diverse and widespread groups of natural compounds are probably the most important natural phenolics. These compounds possess a broad spectrum of chemical and biological activities including radical scavenging properties. The flavonoids in the juice combine with aluminum to form a complex flavonoid-aluminum that could be measured at 430 nm ²⁹. The total flavonoid content of Darjeeling mandarin juices is given in Table 3. Flavonoids ranged from 173.70 to 96.13 µg Rutin/ml juice.

DPPH scavenging methods have been used to evaluate the antioxidant activity of the compounds due to simple, rapid, sensitive, and reproducible procedures. Table 4 presents the results of the antioxidant capacity. Antioxidant activity was recorded highest in Lower Bong Busty (SM) at 0.76µg VCE/ml and lowest in Bong Busty (DK) at 0.43µg VCE/ml. The high antioxidant activity could be related to the high phenolic activity. Similar findings were reported by Elkhatim ³⁰ and Quijada ³¹.

ABTS is frequently used by the food industry and agricultural researchers to measure the antioxidant capacities of food. Antioxidant activity of Darjeeling mandarin through ABTS assay ranged from 87.92 to 82.34 µg VCE/ml represented in Table 4. The highest value was measured in Thapa Goan (DS) and Lowest was measured in 8.5 Mile (IARI). Similar findings were reported by Kelebek and Selli ¹⁹ who reported antioxidant activity of mandarin juice from 44.6 to 59.3 mg VCE/100 ml and 21.3 to 35.6 mg VCE/100ml for wines.

Frap assay was expressed in nmol/ml and represented in Table 4. The highest FRAP content was recorded in the DK genotype (13.74) followed by the SM genotype (13.30). DK genotype of Darjeeling mandarin recorded lowest of 9.86 nmol/ml.

4. Conclusion

In the present study ascorbic acid, total acidity, total phenolic, flavonoid content, and antioxidant capacity of Darjeeling Mandarin (Citrus reticulata Blanco) grown in Darjeeling and Kalimpong district of West Bengal, India was evaluated. Antioxidant activity by the DPPH method ranged from 0.76 µg VCE/ml to 0.43 µg VCE/ml. Antioxidant activity through ABTS assay ranged from 87.92 to 82.34 µg VCE/ml. The result shows that Darjeeling mandarin has an ample amount of antioxidants. The area of Darjeeling mandarin can be increased owing to its health benefits. The study was the first report about the antioxidant capacity of Darjeeling mandarin, study highlights its importance as a therapeutic food ingredient in health promotion and disease prevention.

Acknowledgements

I would like to Acknowledge Director, Indian Agricultural Research Institute (IARI) New Delhi, Principal Scientist and Incharge Indian Agricultural Research Institute (IARI) Regional Station, Head, Division of Fruits and Horticultural Technology, Indian Agricultural Research Institute (IARI), New Delhi for their valuable suggestion and contribution towards the manuscript.

References