2.5.11. Tests for Flavones and Flavonoids


#### *2.6. HPTLC Analysis*

HPTLC was used for the detection of the phenolic compounds. HPTLC is a highly efficient, reliable and cost-efficient separation technique that is ideally applicable for the analysis of herbal drugs as well as botanicals. A Camag HPTLC instrument with a Linomat V automatic spotter fitted with a 100 μL syringe connected to a nitrogen cylinder and a Scanner-III were used. The developing chamber used was a twin trough and the viewing cabinet was equipped with dual-wavelength UV lamps (Camag, Muttenz, Switzerland). The precoated HPTLC plates used were aluminium backed with silica gel 60 F254 and the thickness was 0.2 mm. The HPTLC plates were cleaned by predevelopment with methanol before the analysis and activated at 110 ◦C for 5 min for the removal of solvents. Specific mobile phases were used for the separation of a particular group of phytochemicals.

#### Sample Application

A total of 10 μL of the sample was spotted using Linomat V on a precoated TLC plate as a narrow bandwidth (8 mm) 10 mm from the bottom and 15 mm from left and right edges of the plates. The samples were applied under a continuous dry stream of nitrogen gas.

THF (toluene, formic acid and water with a ratio of 16:8:2:1) was used as mobile phase for the development of the plates spotted with the samples for the detection of the phenolic compounds. The method of development was linear ascending and it was carried out in a 10 × 10 cm twin trough glass chamber that was equilibrated with the mobile phase. It was saturated for 20 min at 25 ± 2 ◦C with a relative humidity of 60 ± 5%. The volume of the mobile phase used for the development was 10 mL (5 mL in a trough containing the plate and 5 mL in another trough). It was allowed to migrate up to a distance of 85 mm from the point of the sample application. The TLC plate was dried after the development and the chromatogram was viewed in a UV chamber at 254 nm and 366 nm to visualise the phenolic compounds. Fast blue salt B was used to detect the phenolic compounds.

#### *2.7. Total Antioxidant Activity Estimation*

Test solutions containing 50, 100 and 200 μg/mL of MECM in methanol were prepared and 0.3 mL of the solution was mixed with a reagent solution (3 mL) containing 0.6 mL H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate. The tubes containing the reaction solutions were incubated at 95 ◦C for 90 min and then cooled to room temperature. The absorbance of the resulting solution was measured at 695 nm against a blank. The solution of methanol (0.3 mL) was the control

The antioxidant activity was expressed as the number of grams equivalent to ascorbic acid. The antioxidant activity values were portrayed in standard graphs plotted with an OD of the standard against the various concentrations of ascorbic acid (10, 25, 50, 100, 250 and 500 μg/mL) treated similarly.

#### *2.8. DPPH Radical Scavenging Assay*

The free radical scavenging property of the extracts was estimated by a DPPH radical scavenging assay [16–19]. The hydrogen atom-donating capability of the plant extractives was determined by the decolourisation of the methanol solution of the DPPH (2,2-diphenyl-1-picrylhydrasyl) that produces a violet/purple colour in the methanol solution and fades to shades of yellow in the presence of antioxidants. A solution of 0.1 mM DPPH in methanol was prepared and 2.4 mL of this solution was mixed with 1.6 mL of the methanol extract at different concentrations (12.5–150 μg/mL). The reaction mixture was vortexed thoroughly and left in the dark at RT for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. The percentage DPPH radical scavenging activity was calculated by the following equation:

% DPPH radical scavenging activity = {(A0 − A1)/A0} × 100

where A0 was the absorbance of the control and A1 was the absorbance of the extractives/standard. The % of inhibition was then plotted against the concentration and, from the graph, the IC50 was calculated. The experiment was repeated three times at each concentration [20–24].

#### *2.9. Hydroxyl Radical Scavenging Activity*

The assay principle was based on the competition estimation between the MECM and 2-deoxyribose for the hydroxyl radical that was formed by the Fenton's reaction [24,25]. The underlying mechanism is the oxidation of ferrous ion to ferric ion by hydrogen peroxide and hydroxyl free radicals and hydroxide ions are also formed. The Fe3+ form is reduced back to the Fe2+ form by reacting with another molecule of H2O2, forming a superoxide radical and an H+. This reaction is essential in biological systems because transition metals such as iron and copper can donate or accept free electrons through various reactions taking place within the cells and can generate free radicals. The hydroxyl ion formed in the Fenton's reaction can react with barbituric acid and convert it into alloxan [26].

$$\mathrm{Fe}^{2+} + \mathrm{H}\_{2}\mathrm{O}\_{2} \rightarrow \mathrm{Fe}^{3+} + \mathrm{OH}\mathrm{.} + \mathrm{OH}^{-}.$$

$$\mathrm{Fe}^{3+} + \mathrm{H}\_{2}\mathrm{O}\_{2} \rightarrow \mathrm{Fe}^{2+} + \mathrm{HOO}\mathrm{.} + \mathrm{H}^{+}.$$

#### *2.10. Nitric Oxide Generation and the Assay of Nitric Oxide Scavenging*

Nitric oxide (NO) was generated from sodium nitroprusside and measured by the Greiss reaction as described previously. Sodium nitroprusside in an aqueous solution at a physiological pH spontaneously generates NO, which interacts with oxygen to produce nitrite ions that can be estimated by the Greiss reagent [27,28]. Scavengers of NO compete with oxygen, leading to a reduced production of NO. Sodium nitroprusside (5 mM) in phosphate-buffered saline was mixed with different concentrations of various plant extracts dissolved in suitable solvent systems and incubated at 258 ◦C for 150 min and reacted with the Greiss reagent (1% sulphanilamide, 2% H3PO4 and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotisation of the nitrite with sulphanilamide and the subsequent coupling was noted.

#### *2.11. Evaluation of the Reducing Power Activity*

Concentrations of 50, 100 and 200 μg/mL of MECM and standard ascorbic acid of 50, 100 and 200 μg/mL each were mixed with 2.5 mL of phosphate buffer (pH 6.6). To the resultant solution, 1% potassium ferricyanide (2.5 mL) was added and boiled for 20 min at 50 ◦C. To that mixture, TCA (2.5 mL) was added and centrifuged for 10 min at 2000 rpm. The supernatant was collected and distilled water (1 mL) and 0.1% ferric chloride (250 μL) were added. The absorbance of the solution was then measured at 700 nm. The reducing power activity was indicated by the increase in the optical density [29,30].

#### **3. Results**

#### *3.1. Qualitative Screening Tests of the Methanolic Extract of Muskmelon*

The qualitative analysis of the methanolic extract of *C. melo* fruit showed the presence of various phytochemical constituents such as carbohydrates, alkaloids, sterols, phenolic compounds, terpenes and flavonoids (Table 1). The Rf value obtained from HPTLC analysis of the MECM for phenolic compounds were 0.80, 0.83 and 0.86 (Figure 2).


**Table 1.** Qualitative chemical tests of the methanolic extract of muskmelons.

+ Present, − Absent.

**Figure 2.** HPTLC of the phenolic compounds: UV 366 nm, UV 254 nm and derivatised under visible light using a fast blue salt B reagent.

#### *3.2. Estimation of the Total Antioxidant Activity*

The antioxidant activity of MECM in concentrations of 50, 100 and 200 μg were equated to the antioxidant activity of 3.3 ± 0.1732, 6.867 ± 0.5457 and 13.63 ± 0.8295 μg of ascorbic acid, respectively (Figure 3).

**Figure 3.** Evaluation of the total antioxidant activity: a standard curve of ascorbic acid.

#### *3.3. DPPH Scavenging Activity*

DPPH, 2,2-Diphenyl-1-picrylhydrasyl, is a dark-coloured compound composed of stable free radical molecules. The purple colour of DPPH decays in the presence of antioxidants. The change in absorbance at 517 nm in the presence of antioxidants can be equated with the antioxidant potential of the compound.

The % scavenging activity = 100 × [(OD control − OD test)/OD control].

The standard employed was ascorbic acid. Both the extract and the ascorbic acid were tested in concentrations of 100, 250 and 500 μg. It was observed that the calculated percentage scavenging activity of ascorbic acid at these concentrations was 29.24 ± 0.8712, 36.76 ± 1.3 and 52.06 ± 0.7963, respectively (mean ± SEM). The percentage inhibition for MECM calculated at similar concentrations was 11.79 ± 0.5469, 16.50 ± 1.065 and 22.45 ± 1.131; when compared with the control, these values were significant (*p* < 0.001). The results are summarised in Table 2 and Figure 4.



\*\*\* Significant, *p* < 0.001, ANOVA, Dunnett's multiple comparison with control, F = 356.3.

**Figure 4.** The DPPH scavenging activity of MECM using ascorbic acid as a standard. Compared with ascorbic acid in its pure form, MECM showed a significant DPPH scavenging activity (\*\*\* Significant, *p* < 0.001).

The ability of MECM to scavenge hydroxyl radicals generated by Fenton's reaction was tested in this study. The test depends upon the formation of the coloured product by the reaction of hydroxyl radicals with thiobarbituric acid and the measurement of its optical density by a colorimetric assay. A reduction in the OD correlated with the ability of MECM to scavenge the hydroxyl radicals from the reaction mixture.

The standard employed was ascorbic acid. Both the extracts and the ascorbic acid were tested in concentrations of 50, 100 and 200 μg. It was observed that the calculated percentage inhibition by ascorbic acid at these concentrations was 36.09 ± 0.296, 52.4 ± 0.387 and 65.98 ± 0.589, respectively (mean ± SEM). The percentage inhibition for MECM calculated at similar concentrations was 19.56 ± 0.194, 24.92 ± 0.194 and 33.3 ± 0.194; when compared with the control, these values were significant (*p* < 0.001). The results are summarised in Table 3 and Figure 5.

The IC50 calculated for the ascorbic acid was 108.95 μg whereas that of MECM was 385 μg. A comparison of the inhibitory activities of ascorbic acid and MECM is shown in Figure 4.


**Table 3.** Evaluation of the hydroxyl radical scavenging activity of MECM.

\*\*\* Significant, 0.001, ANOVA, post-hoc test by Dunnett's multiple comparisons, all groups with control, F = 470.

**Figure 5.** Comparison of the hydroxyl radical scavenging activity of MECM and ascorbic acid. Compared with ascorbic acid in its pure form, MECM showed a significant hydroxyl radical scavenging activity (\*\*\* Significant, *p* < 0.001).

#### *3.4. Nitric Oxide Scavenging Activity*

We evaluated the nitric oxide scavenging activity of MECM. The standard used for the comparison was gallic acid. The increased concentration of nitric oxide generated was indicated by the increase in the optical density. Thus, the decrease in the optical density evaluated the nitric oxide scavenging activity (Table 4).


**Table 4.** Nitric oxide scavenging activity of MECM.

\*\*\* Significant, ANOVA and Dunnett's multiple comparison against control, F = 1026, N = 3.

The observed mean OD of the control group was 0.201 ± 0.007, which corresponded with a zero % inhibition. As the exposed concentrations of gallic acid were increased from 50, 100 and 250 μg/mL, there was a significant reduction in the OD (*p* < 0.05). Hence, the inhibitory effect on the nitric oxide generation had to be considered inhibited in a corresponding gradation. The percentage inhibition calculated (with reference to the control OD) for the optical density values of 0.099, 0.064 and 0.029 was 51.8 ± 0.744, 68.83 ± 0.562 and 85.7 ± 0.342% for the gallic acid standard. These values were statistically significant at 0.05 levels.

The OD values of MECM for concentrations of 50, 100 and 200 μg/mL were 0.184 ± 0.004, 0.163 ± 0.003 and 0.130 ± 0.002, respectively. These values corresponded with the percentage inhibition of 10.1 ± 1.39, 20.13 ± 0.281 and 36.5 ± 1.55. When compared with the inhibition produced by gallic acid at the same concentrations, these values were significantly low (*p* < 0.05). Moreover, although the decrease in the OD was significant at 100 and 200 μg/mL concentrations, the effect of 50 μg was found to be insignificant at *p* < 0.05.

The IC50 for gallic acid was calculated to be 30.74 μg and that for MECM was 275.29 μg (Figure 6).

**Figure 6.** Evaluation of the nitric oxide scavenging activity of MECM: a comparison with gallic acid (\*\*\* Significant, *p* < 0.001).

#### *3.5. Evaluation of the Reducing Power Activity*

The evaluation of the reducing power activity was conducted by a simple colorimetric method. The change in the absorbance of a reaction system containing trichloroacetic acid, ferric chloride and potassium ferricyanide was extrapolated to calculate the reducing power activity of a compound.

The percentage increase in the absorbance with respect to the control was taken as the percentage reducing power. The result of the study is tabulated in Table 5.

The reducing power of ascorbic acid at concentrations of 50, 100 and 200 μg was found to be (mean ± SEM) 40.42 ± 1.35, 53.98 ± 0.2405 and 69.14 ± 0.2309 whereas that of MECM in similar concentrations was 11.8 ± 0.6132, 19.37 ± 0.8192 and 24.78 ± 0.8110 (Figure 7).


**Table 5.** Evaluation of the reducing power of MECM.

\*\*\* Significant, *p* < 0.001, ANOVA and Tukey's multiple comparison (comparison of all pairs of columns) *N* = 7, F: 1108.

**Figure 7.** A comparison of the reducing power of MECM with that of ascorbic acid at concentrations of 50,100 and 200 micrograms. The x-axis shows the concentrations exposed and the y-axis shows the percentage reducing activity. Compared with ascorbic acid in its pure form, MECM showed a significant reducing power.

#### **4. Discussion**

Oxidative stress plays an important role in the development and pathophysiology of many diseases [1]. The *C. melo* extract showed good activities in hydroxyl, nitric oxide radical and DPPH radical scavenging activity [11]. Moreover, the antioxidant potency of MECM was evaluated by estimating the total antioxidant activity and reducing power activity [12]. Our finding indicates that MECM is a good source of antioxidant constituents, which may be due to the presence of a series of components acting synergistically by different mechanisms. Natural antioxidants are considered better than synthetic compounds due to minimum adverse effects and *C. melo* fruit could be a dietary supplement that could be recommended to prevent oxidative stress.

The phytochemical analysis revealed the presence of a series of phytochemicals in MECM and the HPTLC analysis revealed the presence of polyphenolic compounds. The study mainly aimed to evaluate the antioxidant activity of MECM. Hence, by comparing the antioxidant activity with the standard ascorbic acid in its pure form, we demonstrated that MECM had an antioxidant activity. Ascorbic acid was used in its pure form and is a standard and potent antioxidant. Specifically, the study proved that MECM had an activity compared with the control, with a significance level of a *p*-value less than 0.05.

### *4.1. DPPH Radical Scavenging Activity of MECM*

This method is one of the most popular procedures to test the antioxidant potential of a plant extract. DPPH is a relatively stable radical that acts as an antioxidant or free radical scavenger by donating hydrogen ions to the compounds in the oxidised state [31]. The estimation of the free radical scavenging activity in food and plant-based drugs is extensively performed utilising this assay and the fruit extract had an excellent activity [32,33].

#### *4.2. Hydroxyl Radical Scavenging Potential of MECM*

The neutral form of a hydroxide ion (OH−) is a hydroxyl radical and is highly reactive. Therefore, it exists only for a short period. Several biological membranes including DNA can be damaged by hydroxyl radicals. To neutralise this radical, no endogenous enzymatic scavenging pathways are present inside the body and most often it is neutralised by several endogenous molecules such as glutathione, melatonin and antioxidants supplemented through diet [34]. MECM showed a statistically significant hydroxyl radical scavenging potential and the fruit could act as an excellent dietary source to produce this activity.

#### *4.3. Nitric Oxide Scavenging Potential of MECM*

Nitric oxide (NO), which is an important bioactive molecule, possesses several physiological functions [35]. It assists in maintaining vascular homeostasis, fights against pathological microorganisms and it is known to have an anti-cancer activity. NO can also act on the blood vessels, producing vasodilatation, altering the vascular endothelial permeability and acting as a neurotransmitter. When combined with a superoxide radical, it generates a peroxynitrite anion and becomes highly reactive and causes severe damage to intracellular components. The substances that scavenge nitric oxide could have a cytoprotecting property [35].

The methanolic extract of the F1 Hybrid variety of *C. melo* was tested for its ability to scavenge nitric oxide using gallic acid as a standard. In the in vitro study, the significant capacity of MECM to reduce the formation of NO in the reaction system was established. It can act as a potent scavenger of free radicals, thus inferring its cytoprotective effect [36–38].

#### **5. Conclusions**

*Cucumis melo Linn.* (*C. melo*) is an important horticultural crop cultivated worldwide. In the present study, we evaluated the antioxidant activity of a methanolic extract of *Cucumis melo Linn* (MECM) quantitatively and qualitatively using standard protocols. The qualitative analysis showed that MECM contained carbohydrates, alkaloids, sterols, phenolic compounds, terpenes and flavonoids. The total antioxidant activity of the concentrations of 50, 100 and 200 μg was 3.3 ± 0.1732, 6.867 ± 0.5457 and 13.63 ± 0.8295 μg of ascorbic acid, respectively. Significant nitric oxide and DPPH scavenging activities as well as a reducing power activity were also observed.

**Author Contributions:** Conceptualization, S.P.I., R.S.R.; methodology, R.S.R., S.P.I.; investigation, R.S.R.; data curation, P.P.N., M.E.-M.; writing—original draft preparation, R.S.R.; writing—review and editing, R.S.R., P.P.N., M.E.-M., M.S.K., G.S.A.; visualization, R.S.R.; supervision, S.P.I.; funding acquisition, R.S.R., P.P.N., M.E.-M., M.S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia for funding this work through the Research Group Program under Grant No: RGP 2/191/42.

**Conflicts of Interest:** There is no conflict of interest.
