Investigation of biochemical components and antioxidant activity of karonda squash

Introduction

Karonda (Carissa carandas L.) a Apocynaceae member, is an indigenous, hardy plant, underutilized in mainstream horticulture. Thriving where other fruit crops struggle, this evergreen spiny shrub is resilient enough to yield satisfactory results even in marginal soil, unlike many other fruit varieties (Tripathi et al., 2014) [13] . Its dense, thorny growth habit has traditionally made it an effective bio-fence or hedge plant for protecting fields and gardens. While Karonda is gaining popularity due to its rich source of nutrients (notably Vitamin C and Iron) and antioxidants, its fresh consumption is limited. The fruit, particularly when unripe, is distinctly acidic and astringent, making it difficult to be relished in large quantities (Bajpai et al., 2015) [2] . This high acidity, however, makes the fruit an ideal candidate for value addition and processing. Converting the fruit into popular products like squash, jams, jellies, and pickles effectively mitigates its tartness and astringency. The processing helps in creating useful Products. For Socio-Economic Development in Rural India (Srivastava et al., 2017) [11] . Processing also helps in handling large scale production of Karonda fruits when used as a fence and also plays a role in income generation. The development and standardization of Karonda squash, therefore, represent a significant step in promoting the wider use of this resilient and nutritionally valuable underutilized fruit.

Material and Methods

The study investigating the potential of Karonda squash preparation was conducted in 2018. Research activities and analysis were carried out at College of Horticulture, Rajendranagar, Hyderabad, SKLTGHU and the Central Instrumentation Cell (PJTAU). Karonda fruits were sourced from the Fruit Research Station, Sangareddy, SKLTGHU, and all other necessary ingredients were purchased from the local market.

Total Carotenoids

Total carotenoids were determined following the method described by AOAC (2000) [1] . A one-gram (X) sample was first saponified with alcoholic KOH for 20 minutes at 37 °C. The carotenoids were then repeatedly extracted from the saponified mixture into petroleum ether using a separating funnel until the solvent layer became clear. The volume of the pooled petroleum ether was recorded (N ml), and its absorbance (M) was measured at 450 nm.

Total Phenols

The Folin-Ciocalteu Reagent (FCR) method, as described by Singleton et al., 1999 [10] , was used to quantify total phenols. The following procedures are involved in the colorimetric assay: 0.5 ml of diluted FCR (1:1), 10 ml of sodium carbonate, and 0.2 ml of the sample's methanol extract.

After making up to 12 ml with distilled water, the mixture is incubated for 60 minutes at 37 degrees Celsius. At 750 nm, the resultant solution was subsequently measured using spectrophotometry (Kamalaja and Prashanthi, PJTAU). A Gallic Acid standard was used to determine the results.

Sample OD x Standard concentration x Volume made up x 100 Total Phenols (GAE mg/100 g) = Sample weight (g) x Standard OD x Aliquot taken x 1000

Total Flavonoids

A colorimetric assay (Zhishen et al., 1999) [15] was used to measure the total flavonoids expressed as Rutin Equivalents per 100 g. After preparing a measured volume of the sample's methanol extract, it was diluted with distilled water to a final amount of 5 ml. 0.3 milliliters of sodium nitrite were added to this. 0.6 cc of 10% aluminum chloride was added and well mixed after a 5-minute wait. 2 milliliters of 1N sodium hydroxide and 2.1 milliliters of distilled water were added. After then, the mixture was well blended. Using a UV-visible spectrophotometer and a blank for references, the final pink solution's absorbance was measured at 510 nm (Kamalaja and Prashanthi, PJTAU).

Sample OD x Standard concentration Total Flavonoids (RE mg/100 g) = Sample weight (g) x Standard OD x Aliquot taken x 10

DPPH Radical Scavenging Activity

The 2, 2-diphenyl-1-picryl-hydrazyl radical scavenging assay (Brand-Williams et al. 1995) [3] , was used to measure antioxidant activity. This technique is predicated on the antioxidants in the sample reducing the stable, purplecolored DPPH radical, which results in a detectable color shift. A UV spectrophotometer is used to measure the solution of purple color intensity at 517 nm. By comparing the absorption of the resulting oxidized solution to a methanol blank, the reduction of the DPPH radical was ascertained. Trolox Equivalents mg/100 g sample was the measure of the sample's total antioxidant capacity as determined by the DPPH assay (Kamalaja and Prashanthi PJTAU).

AC-AE Percent inhibition =

x 100 AE Where, AC-Absorption of control AE-Absorption of extract or standard Volume made up x std. conc. x sample % inhibition TAC by DPPH assay TE mg/100 g = Aliquot taken x sample weight (g) x sample % inhibition x 10

Ferric Reducing Antioxidant Power Assay

The Ferric Reducing Antioxidant Power assay, following the methodology outlined by Benzie and Strain (1996) [4] and Tadhani et al. (2007) [12] , was employed to assess the Ferric Reducing Antioxidant Power of the Karonda squash.The reduction of ferric-2,4,6-tris (2-pyridyl-striazine) complex (Fe3+-TPTZ) to its intensely blue ferrous form (Fe2+-TPTZ) at an acidic pH by antioxidants of the sample is the principle of assay. The procedure entailed diluting a specified volume of the methanol extract to 0.3 ml using distilled water, subsequently incorporating 1.8 ml of FRAP reagent. The resulting solution after 10-minute incubation at 37 °C, is measured for absorbance at 593 nm. Ferric Reducing Antioxidant Power of Karonda squash is expressed as Trolox Equivalents mg/100 g (Kamalaja and Prashanthi).

Statistical Analysis

Completely Randomized Design is used to analyze data for interpretation.

Results

The changes observed in carotenoid levels throughout the six-month storage duration of karonda squash are shown in Table 1. The peak content was identified in karonda squash immediately after preparation quantified at 0.312 mg/100 g. The overall carotenoid content observed substantially decreased from the second month of storage to the sixth month. The minimum concentration was documented at the end of the six-month duration, at 0.254 mg/100 g. Carotenoids are more prone to auto-oxidation during storage period might be a reason for a fall in carotenoid content. The unsaturated chemical structure makes it more susceptible to thermal breakdown and oxidation.

Similarly Harshita et al. (2016) [5] conducted an analysis of mango ready-to-serve drinks and squash, examining alterations in their chemical composition over a three-month storage period at monthly intervals. They reported a decline in total carotenoids in both beverages as the storage duration increased.

The alterations observed in the total phenolic content during the six-month storage of karonda squash are detailed in Table 1. The highest level was noted in freshly processed karonda squash, measuring 34.96 GAE mg/100 g. A significant decline in total phenol content was recorded starting from the third month of storage, continuing until the sixth month, where it reached 31.54 GAE mg/100 g. The reduction in phenolic content may be attributed to oxidation and polymerization reactions. Comparable findings were documented by Sharma et al. (2012) in their study of guavajamun blended squash. Karpagavalli and Amutha (2015) [8] indicated a gradual decline in total polyphenols in pomegranate squash throughout the storage period. Additionally, Harshita et al. (2016) [5] noted a decrease in total phenols in storage of ready-to-serve mango drink and squash.

The total content of flavonoids of karonda squash throughout the six-month storage is documented in Table 1.

The maximum concentration of total flavonoids (128.68 RE mg/100 g) was recorded in karonda squash immediately after preparation, with no significant alterations detected over a two-month storage period. However, a notable decline was recorded in the latter part of the storage period, reaching a minimum of 113.96 RE mg/100 g. Karpagavalli and Amutha (2015) [8] similarly noted a gradual reduction in total flavonoids in pomegranate squash over a storage period of 180 days.

Ferric Reducing Antioxidant Power Assay (FRAP Assay) (TE mg/100 g) Ferric Reducing Antioxidant Power capacity was assessed throughout the six-month storage with findings displayed in Table 1. The peak FRAP value (13.56 TE mg/100 g) was observed at the 0 month and exhibited a notable decrease from the second month of storage to the sixth month (6.64 TE mg/100 g). The decline in ascorbic acid, carotenoids, phenols, and flavonoids during the storage might be responsible for decrease in Ferric Reducing Antioxidant Power capacity of karonda squash. Karpagavalli and Amutha (2015) [8] showed a reduction in antioxidant activity during storage.

The DPPH radical scavenging activity expressed as Trolox equivalents, of karonda squash was quantified over a sixmonth storage period, with findings presented in Table 1.

The peak DPPH radical scavenging activity was seen at 0 months (67.85 TE mg/100 g), with a notable decline from the third month to the sixth month of storage (54.20 TE mg/100 g). Diminished levels of ascorbic acid, carotenoids, phenols, and flavonoids might be the reason for decrease in DPPH radical scavenging activity. Karpagavalli and Amutha (2015) [8] showed a reduction in free radical scavenging capacity ranging from 36 to 83 percent during storage. Moreover, Yang et al. (2007) [14] reported a decline in the antioxidant activity of noni juice.

Conclusion

The squash kept a lot of its biochemical composition and antioxidant capacity as shown by DPPH radical scavenging activity and FRAP though there is decline with increase in storage duration. The results revealed that it is possible to process Karonda fruit into squash.

Acknowledgement

AcknowledgmentsThe authors gratefully acknowledge College of Horticulture, Rajendranagar, SKLTGHU, the Central Instrumentation Cell and the Quality Control Laboratory, PJTAU for providing the necessary facilities and technical support required for this research.

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