Total Phenolic, Flavonoid Contents and Antioxidant Activity of Ethanolic Extracts (Leaves and Fruit) of Cucurbita maxima Duch. ex Lam

The genus Cucurbita is a member of the Cucurbitaceae family which includes five major species (Cucurbita moschata, Cucurbita pepo, Cucurbita argyrosperma, Cucurbita maxima and Cucurbita ficifolia). Hundreds of varieties and cultivars of Cucurbita  are grown around the world and are major agricultural commodities. The most important Cucurbit genus members include; gourd, melon, cucumber, squash and pumpkin, which are used as food and industrial crops (El-Adawy et al., 2001; Acquaah, 2004; Leffingwell et al., 2015). Pumpkin is cultivated worldwide for its nutritional and medicinal importance. Each pumpkin  part contains a significant amount of antioxidants,   tocopherols and carotenoids (Kim et al., 2012). Pumpkin pulp can be consumed both raw and after being processed, e.g., cooked, or as compotes, jams, purees and juices (Paris et al., 2006). Polyphenolic compounds are secondary metabolites and constitute the largest group of phytochemicals that promote health. It is known that these compounds have important antioxidants, exhibit anti-glycemic, anti-viral, anti-cancer and anti-inflammatory activities, anti-allergic and anti-microbial properties (Manach et al., 2004). The antioxidant activity of phenolic compounds depends on the number and position of their hydroxyl groups, which can easily donate their H+ to reactive oxygen species (ROS). ROS are generated due to the result of environmental effects, such as UV radiation, cigarette smoke and air pollutants (Okmen et al., 2009). Therefore, the main aim of study is to determine the antioxidant activity of ethanolic extracts of Cucurbita maxima.

Plant material
 
This study was carried out on both parts: leaves and fruits of a species of the family Cucurbitaceae called Cucurbita maxima. The plant was harvested in November 2018 in the region of N’gaous in Batna, The identification of the species was made by Dr Nouioua Wafa (Laboratory of Phytotherapy Applied to Chronic Diseases).
 
Preparation of ethanolic
 
The ethanolic extract of leaves and fruit was obtained by maceration in water/ ethanol mixture (20:80) for 5 days in the dark with agitation every 24 hours. The resultant extract was filtered through Wattman paper and the solvent was removed by rotary evaporator under reduced pressure at 45°C  (Lakic et al. 2010).

Determination of total phenolic content
 
Folin Ciocalteu method was used for determination of poylphenol content Li et al., (2007). The samples (0.2 mL) were mixed with 1 mL of the Folin-Ciocalteu reagent previously diluted with 10 mL of deionized water. The solutions were allowed to stand for 4 minutes at 25°C before 0.2 mL of a saturated sodium carbonate solution (75 mg/mL) was added. The mixed solutions were allowed to stand for another 120 minutes before the absorbance were measured at 765 nm. Gallic acid was used as a standard for the calibration curve. The total phenolic content was expressed as mg equivalent of Gallic acid per gram of extract (mg EAG/GE).
 
Determination of total flavonoids content
 
The flavonoids content in crude extract were estimated by the Aluminium chloride solution according to the method described by Bahorun et al., (1996). Briefly, 1 mL of the methanol solution of the extract was added to 1 mL of 2% AlCl3 in methanol. After 10 minutes, the absorbance was determined at 430 nm. Quercetin was used as a standard. Results were expressed as mg equivalent Quercetin per gram of extract (mg EQ/GE).
 
Determination of tannins content
 
The capacity to precipitate haemoglobin was determined by using bovine fresh blood according to the method described by Gharzouli et al., (1999). Briefly, a volume of leaves and fruits extracts were  mixed with an equal volume of hemolysed bovine blood (absorbance=1.6). After 20 min, the mixture was centrifuged at 4000 rpm for 10 min and the absorbance of the supernatant was measured at 756 nm. Results were expressed as mg equivalent tannic acid per gram dried weight (mg TAE/g DW).
 
Evaluation of antioxidant activity
 
DPPH assay
 
The donation capacity of extract was measured by bleaching of the purple-coloured solution of 1, 1-diphenyl-2-picrylhy drazyl radical (DPPH) according to the method of (Hanato et al., (1998). One mL of the extract at different concentrations was added to 0.5 mL of a DPPH-methanol solution. The mixtures were shaken vigorously and left standing at room temperature for 30 minutes in the dark. The absorbance of the resulting solutions were  measured at 517 nm. The antiradical activity was expressed as IC₅₀ (micrograms per millilitre). The ability to scavenge the DPPH radical was calculated using the following equation:  Where,
A₀:  Absorbance of the control at 30 minutes
A₁: Absorbance of the sample at 30 minutes. BHT was used  as standard Bettaieb Rebey et al. (2012).

Reducing power
 
The reducing power was determined according to the method of (Oyaizu ,1986). The extract (2.5 mL) was mixed with 2.5 mL of 200 mmol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 10 mg/mL potassium ferricyanide. The mixtures were incubated at 50°C for 20 minutes; after cooling, 2.5 mL of 100 mg/mL trichloroacetic acid were added and the mixtures were centrifuged for 10 minutes. The upper layer (5 mL) was mixed with 5 mL of deionized water and 1 mL of 1 mg/mL ferric chloride and the absorbance was measured at 700 nm against a blank. A higher absorbance indicates a higher reducing power. EC50 value (mg extract/mL) is the effective concentration at which the absorbance was 0.5 for reducing power and was obtained by interpolation from linear regression analysis. BHA (Butylated hydroxytoluene) was used as standard (Huang and Mau, 2006).
 
β-carotene bleaching assay
 
In this test, the antioxidant capacity of the The ethanolic extract of leaves and fruit  extract is determined according to the method of Dapkevicius et al., (1989). Briefly, A stock solution of β-carotene-linoleic acid mixture was prepared as follows: 0.5 mg β-carotene was dissolved in 1 ml of chloroform and 25 μl linoleic acid and 200 mg Tween 40. Chloroform was completely evaporated and then, 100 ml distilled water saturated with oxygen (30 min, 100 ml/min) were added with vigorous shaking. 2500 μl of this reaction mixture were dispensed into test tube and 350 μl of seeds extract, prepared at 2 mg /ml concentrations, were added and the emulsion system was incubated for 48 h at room temperature. The same procedure was repeated with synthetic antioxidant BHT as positive control and blanks (MeOH and H₂O). The absorbance of the mixture was measured at 490 nm after; 0, 1, 2, 4, 6 and 24 hour. The antioxidant activity was measured in terms of successful bleaching of β-carotene by using the following equation:  A sample: Absorbance in the presence of the extract.
A BHT: Absorbance in the presence of positive control BHT.

The total flavonoids content of Cucurbita maxima extracts was determined using the aluminium chloride method and the total amount of flavonoid compounds was determined in mg of quercetin equivalents/gin (Table 1). The extract of the fruit has given the highest rate in phenolic compounds which is of the order (50.13±4,49 EAG/g of dry extract), relative to the leaves (27.37±0, 95 mg EAG/g of dry extract). A similar study conducted by Hashash et al., (2017) showed that the total Polyphenols of the ethanolic extract of the fruit of the same family was (49.80 0.44 mg EAG/g of extract), as higher than that of our study. Studies carried out by Dissanayake et al., (2018) the total polyphenol content of the ethanolic extract of the leaves was  (7.0±0.2 mg EAG/g of extract) is less than that found in this study  and the flavonoid content was (4.5±0.2 mg Eq/g of extract), is small to that found in this study. The fruits and leaves are very rich in tannins and the results are very close and are of the order of (477.05±6.67mg EAT/g dry extract) and (481.69±5.77 mg EAT/g dry extract). Studies by Ebana et al., (2019) have shown that flavonoids in the fruit and leaves of the same family are (1.58±0.02 mg EAT/g dry extract) and (0.22±0.01 mg EAT/g dry extract, respectively), which are very close to that obtained in this study.


 
Antioxidant activity evaluation
 
DPPH radical scavenging activity
 
The scavengingeffect of the DPPH radical was evaluated spectrophotometrically according to the reduction of this radical which is accompanied by its passage of purple color to the measurable yellow color at 517 nm and the results are present in (Table 2). The antioxidant activity of ethanolic fruit extract (0, 18±0,06 μg/ml) is higher than that found by Hashash et al. (2017) from the same family (643.09±6.31 μg/ml). And leaves (0.41±4.20. 10 -3 μg/ml) are superior to that found by Sayed et al., (2017) of the same family (625.02±8.80 μg/ml). Phenolic compounds used as antioxidants reduce the odd electron of the nitrogen atom in the DPPH radical by giving it a hydrogen atom (Contreras-guzman and Strong,1982). Flavonoids are one of the phenolic compounds found in plants, they are associated with various pharmacological activities including anti-inflammatory and antitumor properties and they are capable of acting as antioxidants that shield the cells from destructive effects of free radicals (Moriasi et al ., 2020; Kibiti and  Afolayan, 2015).


 
β-carotene/linoleic acid bleaching assay
 
In the present study, the potential of the plant to inhibit linoleic acid oxidation was evaluated using the β-carotene/linoleic acid test system the resultofa show in (Table 3). The ethanolic extracts of leaf and fruit of Cucurbita maxima demonstrated lower inhibition percentages compared to the positive control BHT (99.51±1.15). However The oxidation of β-carotene from the leaves (70.13±3.84%) is higher than that obtained from the fruit (67.69±3.82%). The inhibitory power of β-carotene oxidation in leaves is higher than that obtained by Marzouk et al., (2016) be the same family, which is equal to (31.92 2.30%). These results are related to the presence of phenolic compounds, the effect of either inhibiting the peroxidation of linoleic acid or trapping hydroperoxide radicals formed during peroxidation of linoleic acid (Tepe et al., 2005).

The ethanol extracts (leaves and fruit) of Cucurbita maxima found with the highest frequency in antioxidant activity. It is clear that they have a good potential to be used in therapies as well as leaf vegetables for daily consumption. The total phenolic content of this study supports the hypothesis that phenolic compounds are a significant contributor to total antioxidant capacity.