ABSTRACT

Melon (Cucumis melo); an edible fruit all over the subcontinent, has very delicious flavor, high quantity of water and diverse range of bioactive components. The following work was performed to evaluate the total phenolic contents and antioxidant potential of melon peel and seeds. The extraction of phenolic antioxidant was carried out via enzyme-assisted solvent extraction. Total phenolic content were estimated using spectrophotometric method. The antioxidant potential of melon peel extracts was assessed by different in-vitro assays including 1,1-diphenyl-2-picrylhydrazy (DPPH) radical scavenging assay, Inhibition of peroxidation in linoleic acid system, Trolox equivalent antioxidant capacity assay (TEAC), Total antioxidant capacity (TAC) and measurement of Reducing power (RP). The obtained results were analysed by using Analysis of Variance technique (ANOVA). According to results percentage yield ranged from 20.94 to 39.09 for melon peel and from 4.35 to 19.30 for seeds. Total phenolic contents obtained ranged from 222.62 to 35.03 for peel and 19.75 to 86.42 for seeds. For DPPH free radical scavenging assay IC₅₀ values were calculated ranged from 2.89 to 10.59 for melon peel and 2.80 to 17.21 for seeds. In Linoleic acid peroxidation system results expressed in percentage inhibition ranged from 31.03 to 74.54 for melon peel and 3.25 to 87.32 for melon seeds. Trolox equivalent antioxidant capacity assay (TEAC) expressed results in mmol/g and range from 99.56 to 282.98 for melon peel and 57.30 to 282.06 for melon seeds. Similarly Total antioxidant capacity (TAC) was also measured in percentage ranged from 30.22 to 98.26 for melon peel and 18.43 to 97.94 for melon seeds. Reducing power of melon peel and seed sample was also measured, expressed in λmax. As the concentration increased absorption was also increased. Correlation among all the assays showed high correlation (P< 0.005) which indicates that as the enzyme concentration increases, the availability of antioxidants also increases.

KEYWORDS

Melon, Peel and Seed, Antioxidants, Phenolics

TO CITE THIS ARTICLE

Reena Islam and Bushra Sultana 2020. Investigation of the Antioxidant Activity in Melon Peel and Seeds (Cucumis melo). Journal of Applied Sciences and Research, 1: 1-6
URL: https://www.sciworldpub.com/article-abstract?doi=34-jasr-20

1. INTRODUCTION

Antioxidants slow down the oxidation process of oxidizing molecules. In oxidation process electrons or hydrogen is transferred from one species to an oxidizing agent. Free radical produced in the result of oxidation and chain reaction which cause damage or even death to the cell. Free radicals cause tissue damage and variety of pathogenic disorders including degenerative disorders of central nerves system suchas Alzheimer’s disease, diabetes mellitus, aging and hypertension [5]. Free radical also has the potential to oxidize the lipids present in membrane; destroy DNA and disrupt the cytoskeleton [6]. Antioxidants terminate these reactions and used to cure many diseases [4,16].

Plants in which fruits and vegetables are included considered as the rich source of antioxidants [7]. Phytochemicals specially, phenolics lessen the risk of several chronic diseases such as heart diseases and cancer [22,24]. Antioxidants considered as the compounds that delays the oxidation of biomolecules such as lipids, in this way they avoid thedamage of body cells. Phenolic compounds such as flavonoids known as dominant antioxidants while beta-carotenes, Vitamins C and E have less antioxidant potential [23].

Melon (C. melo) belongs to the Cucurbitaceae family that consists of edible and fleshy fruits. Melon pulp extract contain anti-inflammatory and high antioxidants potential [25] but the antioxidant estimation of different parts of C. melo is very limited [13]. Fruits skin is a potential source of antioxidants such as flavonoids, polyphenols and ascorbic acid. These have positive impact on human health [1]. There are many reports available on bioactivities of melon as it is anti-inflamatory, ureas inhibitory, antioxidative, pletelet inhibitory, diuretic and anthelmintic potential [14,28]. It was also reported that the extract of melon peel has great pharmacological importance as it is helpful in treatment of thyroid abnormalities, diabetes mellitus and cardiovascular diseases [17,18]. A previous study indicates that extract of melon peel possesses antioxidant and anti-inflammatory properties [25]. Thus, the main objectives of the study were to determine the phenolic content and antioxidant activities of melon peel and seeds using enzyme assisted solvent extraction technique.

2. MATERIAL AND METHOD

2.1. Sample
Mature and healthy Melon fruits (C. melo) were collected from the local market of Faisalabad Pakistan. The fruit was identified and verified from Department of Botany, University of Agriculture; Faisalabad, Pakistan. Melons were washed with water to clean it.

2.2. Chemicals and reagents
All the chemicals, reagents and standards including 1,1-diphenyl-2-picrylhydrazyl radical (DPPH°), Butylatedhydroxytoluene (BHT), Gallic acid, Folin-Ciocalteu reagent and linoleic acid were purchased from Sigma-Aldrich (Chemie GmbH, Germany), whereas, potassium dihydrogen phosphate, ferrous chloride, ammonium thiocyanate, dipotassium hydrogen phosphate and sodium bicarbonate from Merck (Darmstadt, Germany).

2.3. Extraction
Air-dried samples of melon peel and seed were ground into a fine powder in a grinding mill (Tector–Cemotec 1090 sample mill, Hognas, Sweden). The collected powdered samples of melon peel and seeds were treated with Natuzyme enzymes and 80% methanol at temperature (45°C), pH (5.5) and incubation time (75 min). The extract was separated from the solids by filtration with Whatman No. 1 fillter paper. The remaining solids were extracted twice with the same solvent and extracts combined. The extracts were concentrated under reduced pressure at 60°C, in a rotary evaporator (EYELA, Tokyo, Japan). Concentrated extracts were stored in a refrigerator (-4°C) until analysed.

2.4. Determination of Total Phenolic Contents (TPC)
Amount of TPC was assessed using Folin-Ciocalteu reagent procedure as described by Chaovanalikit and Wrolstad [3]. Briefly, 50 mg of dry mass of melon peel extract was mixed with 0.5 mL of Folin-Ciocalteu reagent and 7.5 mL deionized water. The mixture was kept at room temperature for 10 min, and then 1.5 mL of 20% sodium carbonate (w/v) was added. The mixture was heated in a water bath at 40°C for 20 min and then cooled in an ice bath; finally absorbance at 755 nm was measured (HitachiU-2001 spectrophotometer, model 121-0032). Amounts of TPC were calculated using a calibration curve for gallic acid (10–100 ppm) (R²= 0.9986). The results were expressed as gallic acid equivalents (GAE) per dry matter. All samples were analyzed thrice and results averaged.

2.5. DPPH radical scavenging assay
Free radical scavenging activity of melon peel and seed extracts were assessed using procedure described by Iqbal et al., [9]. Briefly, to 110 μL of melon peel containing 1 mg/mL of dry matter in methanol, 100 μL of freshly prepared solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) at concentration 1 mg/mL, 0.1 mg/mL, 0.01mg/mL and 0.001mg/mL was added. Absorbance was measured at 517 nm using Hitachi U-2001 spectrophotometer, model 121-0032.

2.6. Antioxidant activity determination in Linoleic acid system
The antioxidant activity of melon peel and seed extracts were also determined by measuring percentage of oxidation of linoleic acid system [9]. Extracts, 0.5 mg of dry matter, were added to a solution of linoleic acid (0.13 mL), 99.8% ethanol (1 mL) and 0.5 mL of 0.2 M sodium phosphate buffer (pH 7). The mixture was made up to 2.5 mL with distilled water and absorbance was measured at 500 nm and incubated at 40°C for 360 hr. Extent of oxidation was measured by peroxide value applying thiocyanate method as described by Yen et al., [26]. Briefly, 1 mL of ethanol (75% v/v), 0.2 mL of aqueous solution of ammonium thiocyanate (30% w/v), 0.2 mL of sample solution and 0.2 mL of ferrous chloride (FeCl₂) solution (20 mM in 3.5% HCl; v/v) added sequentially. After shaking well, the absorption was measured at 500 nm using spectrophotometer. A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxyl toluene (BHT) was used as positive control. Percent of inhibition of linoleic acid oxidation were calculated with the Eq. 1:

(1)

2.7. Determination of Reducing Power
The reducing power of the extracts was determined according to the procedure described by Yen et al., [26], with modification. Equivalent volume of melon peel and seed containing 10.0–2.5 mg of dry matter was mixed with sodium phosphate buffer (0.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (0.5 mL, 1.0%); the mixture was incubated at 50°C for 20 min. Then 0.5 mL of 10% trichloroacetic acid added and then allowed to cool at low temperature for 10 min. The upper layer of the solution (0.5 mL) was diluted with 0.5 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm (Hitachi U-2001). The measurement was run in triplicate and results averaged.

2.8.Trolox Equivalent Antioxidant Capacity (TEAC) assay and Total antioxidant capacity
ABTS radical scavenging activity of the test compounds was determined using an ABTS decolorisation assay [12]. To the ABTS liquid substrate system 2.45 mM potassium per sulphate was added in a stoichiometric ratio of 1:0.5 (v/v). The mixture was allowed to stand in the dark at room temperature for 8 hr. In the ABTS⁺ solution (100 μL) different concentrations of test compounds/extracts (10 μL) and 90 μL methanol were added. The reaction mixture was incubated for 6 min at room temperature and the absorbance was measured at 734 nm. The change in absorbance with respect to the control (containing ABTS solution only without sample, expressed as 100% free radicals) was calculated as percentage scavenging and the IC₅₀ value was calculated. The known antioxidants Trolox and ascorbic acid were used as positive controls.

2.9. Statistical analysis
Three samples of each part (peel and seed) were assayed. Each sample was analyzed individually in triplicate and data is reported as mean ± SD. Data was analyzed using one-way analysis of variance ANOVA using Minitab 2000 Version 13 statistical software (Minitab Inc. Pennysalvania, USA) at 5% significance level.

3. RESULTS AND DISCUSSION

3.1. Total Phenolic Contents (TPC)
Recently, much attention has been paid to understand the role of fruits in the promotion of human health and prevention of chronic diseases. Epidemiological studies have repeatedly shown the association between high fruit intake and the lower incidence of chronic diseases [15]. The chemoprotective properties of fruits have been partly attributed to phenolics such as gallic acid and chlorogenic acid, vitamin C and vitamin E. The phenolic content (PC) generally correlates with antioxidant capacity (AC) for various types of fruits. Phenolic antioxidants can scavenge free radical species, inhibit free radical formation, and prevent damage to cellular components and subsequent damage or death. Phenolics and polyphenolic compounds contribute directly to antioxidative action and they constitute the main class of natural antioxidants present in plants [2] therefore it is valuable to revalorize the fruit residues into medicinally important phenolic compounds.

TPC were calculated using a calibration curve. Total phenolic contents in C. melo as determined by enzymetic assisted solvent extraction were calculated using gallic acid calibration curve ranged (10-100ppm) (R²= 0.9986).

TPC in melon peel were ranged 35.03±0.41 to 222.62±7.15 mgGAE/g. Similarly TPC in melon seed ranges from19.75±0.13 to 86.42±0.66 mg GAE/g. The results of present study was higher than those (4.70 ± 0.23 mgGAE/g 2.85 ± 0.21 mg GAE/g, respectively) observed by Ismail et al. [11] who extracted phenolic compounds from melon fruit residues using aqueous methanol. This ultimately showed that incorporation of enzymes during extraction processes enhanced the availability of phenolic antioxidant. The increase in yield of TPC might be attributed to the composition of natuzyme cocktail, especially the presence of cellulase, pectinase and α-amylase initiated the release of glycosidically linked phenolic metabolites.

3.2. DPPH radical scavenging activity
The anti-radical activities of extracts were assessed in terms of their DPPH free radical scavenging ability. DPPH radical is commonly used for the assessment of antioxidant activity in-vitro and is foreign to biological systems [27]. DPPH is a very stable organic free radical with deep violet color which gives absorption maxima within 515–528 nm range. Upon receiving proton from any hydrogen donor, mainly from phenolics, it loses its chromophore and become yellow. As the concentration of phenolic compounds or degree of hydroxylation of the phenolic compounds increases their DPPH radical scavenging activity also increases, and can be defined as antioxidant activity [20]. Because these radicals are very sensitive to the presence of hydrogen donors, the whole system operates at very low concentration with it. It can allow a large number of samples to be tested in a short time [10,27]. Absorbance in this assay was recorded after incubation of 15 min time interval.

DPPH radical scavenging capacity (IC₅₀ mg/mL) of C. melo peel and seed extracts in range of 2.89±0.10 to 10.48±0.19 mg/mL and 2.80±0.10 to17.21±0.01 mg/mL, respectively.

According to previous studies DPPH free radical scavenging activity of melon peel methanolic extracts showed IC₅₀ value for peel and seed sample 9.58 ± 0.37 mg/mL and 25.44 ± 2.83 mg/mL, respectively. In this study, DPPH radical scavenging activity of melon peel and seed extracts is highly related to the amount of phenolic compounds present in the extracts [11]. The present work showed more refined results because DPPH (IC₅₀) directly depends upon total phenolic contents. Higher the amount of total phenolic contents lower the IC₅₀ value which shows higher %age inhibition.

3.3. Antioxidant activity of melon peel extracts in the linoleic acid peroxidation system
Plant antioxidants have been repeatedly and successfully used as food preservatives in addition to their extensive biological applications. The phenolic compounds present in plant material scavenge oxidants and break the chain of oxidation reactions. Therefore, inhibition of peroxidation linoleic acid system was used to evaluate the antioxidant activity of the melon peel and seed extract using thiocyanate method [26]. Linoleic acid is a polyunsaturated fatty acid, upon oxidation peroxides are formed which oxidize Fe²⁺ to Fe³⁺, the later forms complex with SCN, concentration of which is determined spectrophotometrically by measuring absorbance at 500 nm. Higher the absorbance higher will be the concentration of peroxides formed during reaction, consequently lower will the antioxidant activity.

A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxytoluene (BHT) was used as positive control.

Comparing percent antioxidant activity values before incubation and after incubation it was noted that the percentage activity of antioxidants decreasesed with increase in incubation time. Higher theabsorbance, higher the concentration of peroxides formed during reaction due to lower antioxidant activity. Linoleic acid peroxidase showed maximum percentageinhibition 74.54±0.78 for melon peel and 87.32±4.25 for melon seed.

3.4. Reducing Power (RP) of the melon peel and seed extracts
Measurement of reducing potential (RP) can reflect some aspects of antioxidant activity of extracts or compounds. In this method ferric ions are reduced to ferrous ions and with change in color from yellow to bluish green. The intensity of color depends on the reducing potential of the compounds present in the medium. Greater the intensity of the color, greater will be the absorption, consequently, greater will be the antioxidant activity [29]. Data was measured for extract concentrations 2.5, 5, 7.5 and 10.0 mg/g, respectively, and a general increase in RP was observed with increase extract concentration. Melon peel extract showed maximum absorption (λ max) 1.28±0.02, while melon seed showed maximum absorption (λ max)1.94±0.03.

3.5. Trolox Equivatent Antioxidant Capacity (TEAC) assay and total antioxidant capacity assay:
The method described gives a measure of the antioxidant activity of the range of carotenoids, phenolics and some plasma antioxidants, determined by the decolorization of the ABTS•⁺, through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm. The standard reference compound, compared with glutathione, uric acid, ascorbic acid, α-tocopherol and the flavonoid aglycone antioxidants, kaempferol, and cyanidin. The results demonstrate that the reaction with ABTS•⁺ is complete by 1 min, except for cyanidin and glutathione that show a further small inhibitory effect up to 4 min reaction.

The antioxidant capacities of test compounds are determined by measuring decreases in the intensity of the blue colour as a result of between the ABTS⁺ radical and the antioxidant compounds in the sample [19].

Table 1.	Comparison among different antioxidant assays

3.6. Comparison among different antioxidant assays
Linear correlation coefficients between the results from the various antioxidant tests are presented in Table 1. Significant correlations (P < 0.05) were detected in all cases for the total phenolics assay, the DPPH assay, the reducing power assay, the linoleic acid system assay and the TEAC assay and similar correlations were reported by Shahidi et al. [21]. All antioxidant assays used in this study measured the radical scavenging capacities of primary antioxidants [8]. This accounts for the significant correlations TPC between antioxidant tests.

4. CONCLUSION

The findings of this study indicates that enzyme assisted extraction was found more efficient than conventional solvent extraction or any other extraction in extracting natural antioxidants from melon peel and seed. Any of the antioxidant capacity assays employed in this study may be employed in assessing the antioxidant capacity of melon peel and seed extracts. This work suggests that both melon peel and seed showed highest antioxidant potential. It can be used as safe and effective source of natural antioxidants.

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