INTRODUCTION:

In recent years, the large agricultural area especially in the Middle East and North Africa countries has promote research efforts about the possibilities of utilization of natural source of bioactive compounds in vegetables, fruits, trees and herbs for the dietary management of certain chronic diseases such as diabetes, cardiovascular diseases, obesity, cancer etc ¹^,²^,³^,⁴.

The oxidative stress is one of the most common pathogens currently time, it produces of imbalance between the formation and neutralization of reactive free radicals, this last are continuously neutralized and produced in our body to maintain the constant internal environment redox state. Reactive free radicals include reactive oxygen species (ROS) like superoxide anion radical (O2.-), peroxyl radical (ROO.), hydroxyl free radical (.OH) or reactive nitrogen species (RNS)⁵^,⁶^,⁷.

Dates are produced in 35 countries worldwide and cultivates on about 2.9million acres of land. In Algeria is an important tree for people and plays principal roles in social, economic and environmental sectors. As production Algeria is one of the first producers of the fruits of the date in the world with 50000 t per year⁸^,⁹^,¹⁰.

The date palm (Phoenix dactylifera L.) is usually considered as the most important source of food that provides a wide range of essential nutrients with many potential health benefits for local population of Oued Souf. Several studies have indicated that dates contain a wide variety of carbohydrates, dietary fibers, certain essential vitamins and minerals and it acids, flavonoids and tannins¹¹^,¹²^,¹³^,¹⁴^,¹⁵.

Accordingly, the objectives of this study were to evaluate the phytochemical compositions (the total phenolic and flavonoids) of two fruits of date palm of Oued Souf region (South-East of Algeria) watered with different sources of water (Ground water and Tap water), as well as to evaluate their antioxidant activity.

MATERIAL AND METHODS:

Chemicals and reagents:

Chemicals utilized in this work were analytical grade. The solvents (methanol, chloroform, ethyl acetate, butanol and ethanol), Folin-Ciocalteu, sodium carbonate, gallic acid, aluminum trichloride, quercetin, sulfuric acid, sodium phosphate, ammonium molybdate, ascorbic acid, 2,2-diphenyl-2- picrylhydrazyl, Butylated hydroxytoluene, pyrogallol.

Collection of Plant Materials:

In this work, fruits from one date palm variety (Ali Ourached) grown in the Oued Soufregion (South-East of Algeria) were studied. These two date palm fruits with two different sources of water were collected at the final stage of fruit ripeness in the period from Mid-October 2018.

Preparation of the extract:

The 100g of the edible parts (Ali Ourached) of different samples were macerated with 300ml methanol 80% at room temperature. The methanolic extracts of fruit was additionally divided in separating funnel using chloroform, ethyl acetate and butanol. The extracts were concentrated under reduced pressure and then re-dissolved with minimum of methanol or water and kept at 4C°¹⁶.

Phytochemical Composition:

Measurement of Total Phenolic Content:

The amounts of phenolics in the study medicinal plant extracts were determined with Folin-Ciocalteu reagent using the method of Salvi with a little bit adjustment. Folin-Ciocalteu reagent 0.5ml (prediluted 10-fold with distilled water) was added to a solution containing 100µl of extract. The solution was mixed and after 5 min, 2ml of sodium carbonate solution (20g/L) was added. The blend was left for 30 min, and the absorbance was measured at 760nm¹⁷^,¹⁸^,¹⁹. The same procedure was repeated for all gallic acid concentrations solutions (0.03-0.3g/l). All tests were carried out in triplicate. The TPC was calculated from the standard curve and expressed as milligram gallic acid equivalent per 100g dry weight (mg GAE/100 g DW)²⁰.

Measurement of Total Flavonoid Content:

Total flavonoid contents (TFC) in the extracts were determined using the aluminum chloride colorimetric method described by Chang et al. 1.5ml of methanol or water extracts was mixed with 1.5 ml of 2% aluminum trichloride in ethanol. The absorption at 430 nm was read after 30 minutes²¹^,²²^,²³. Quercetin was used to calculate the standard curve (0.003 and 0.03g/L). All determinations were carried out in triplicates. The TF compound was calculated from the standard curve and expressed as milligram quercetin equivalent per 100g dry weight (mg CEQ/100 g DW)²⁴.

In Vitro Antioxidant Studies:

Total Antioxidant Using Phosphomolybdenum Assay:

The total antioxidant capacity of the methanolic or water extracts was assessed by the phosphomolybdenum method according to the procedure depicted by Prieto et al. 300µl from each sample extract were combined with 3mL of reagent solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min. The absorbance was measured at 695nm after cooling at room temperature²⁵^,²⁶^,²⁷. The experiment was performed in triplicate. Methanol (0.3mL) in the place of extract was used as the blank and the same with water. The standard curve was constructed using different concentrations of ascorbic acid. The AA was expressed as the number of equivalents of ascorbic acid (mmol of ascorbic acid/g of extract)²⁸.

DPPH Radical Scavenging Activity:

The free radical scavenging activity of the extracts, based on the scavenging activity of the stable 2,2-diphenyl-2- picrylhydrazyl (DPPH) free radical. Different dilutions of the methanolic and water extracts were prepared for each fruits. An aliquot of 1.5mL of a diluted sample was added to 1.5mL of a (6×10⁵ M) DPPH methanol solution and immediately vortexed. The mixture was then incubated at room temperature in the dark for 30 min, the absorbance of the samples was read at 517nm²⁹^,³⁰^,³¹. Methanol and were used to zero to spectrophotometer and DPPH• solution was used as blank sample. The percentage inhibition (I %) of the DPPH radical was evaluated according to the formula:

I % = (A blank – A sample/A blank) × 100

Where A blank is the absorbance of the control reaction (containing methanol or water except the test extract), and A sample is the absorbance of the test extract. The same procedure was iterated for BHT concentrations solutions (0.0075-0.06g/l) as positive control. All tests were realized in triplicate. The AA of the extracts was expressed as IC50, this value denotes the concentrations (in g/l) of extracts required to scavenge 50% of DPPH radicals and was obtained by interpolation from linear regression analysis.

Statistical Analysis:

All analyses were expressed in triplicate and data were detailed as means ± SD.

RESULTS AND DISCUSSION:

Phytochemical Composition:

Total Phenolic Content:

Results of the total phenolic content showed that maximum phenolic content was found in crude extract of fruits (AG) (124.2469±0.0000 mg GAE/100g of DW) which was followed by crude extract of fruits (AT) (93.4840±0.0264mg GAE/100g of DW). Whereas, in case of fractions, the higher phenolics was found in butanol fraction (AT) (20.3728±0.0000mg GAE/100gm of DW) as compared to butanol fraction (AG) (16.9774±0.0012mg GAE/100gm of DW). While the smaller value was chloroform fraction (AG) (1.9513±0.0002 mg GAE/100gm of DW) (Table 1). From the results it is evident that Ali Ourached with watering by a ground water are rich in phenolics more than the other The total phenolics content reported by Biglari. Al Karkhi et al¹⁸. ranges between (2.89 –141.35mg GAE/100gm of DW) which looks like our observation. Moreover, in our study we have demonstrated that Watering has an important role in the amount of compounds found in date fruits.

Total flavonoid Content:

Flavonoids content in crude extract of fruits and organic fractions of tested date palm cultivars are shown in
Table 1. Small variation in flavonoids concentration existed within the same cultivar with different watering water as well as between our fractions.

The chloroform fraction (AG) showed the lowest flavonoid content (0.0631±0.0001 mg CEQ /100 g DW) less than chloroform fraction (AG) (0.1177±0.0001mg CEQ/100g DW). The crude extract of fruits (AT) that exhibited the highest flavonoids with (4.3717±0.0016 mg CEQ/100g DW) as compared to crude extract of fruits (AG) (3.4781±0.0027 mg CEQ/100g DW). These results were in correspond with the results reported by Biglari et al¹⁸. who announced that the TFC of Iranian date cultivars are extended among 1.62 and 4.71 mg CEQ/100g DW. Flavonoids are generally found in food products derived from plant sources and they have different biological activities³²^,³³^,³⁴.

Antioxidant Activity:

Phosphomolybdenum Method:

The total AA of the methanolic extracts was estimated using phosphomolybdenum method that was based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and the formation of green Mo (V) complexes with a maximal absorption at 695 nm.

As can be shown from Table 2, ethyl acetate fraction (AT) showed the lowest level (40.4555±1.9596 mmol of ascorbic acid/g of extract) compared to ethyl acetate fraction (AG) (41.8416±2.2754 mmol of ascorbic acid/g of extract), while crude extract of fruits (AG) showed the highest value (303.1836±2.3229 mmol of ascorbic acid/g of extract) compared to crude extract of fruits (AT) (239.5746±5.0161 mmol of ascorbic acid/g of extract). Higher activity in methanol extract and our fractions may be due to the interferences of other compounds present in this extract or fraction, and it has been accounted that solvents used for extraction have a dramatic effect on the chemical species.

DPPH Free Radical-Scavenging Activity:

The DPPH radical is a free extreme intensify that has been generally used to test the free radical rummaging capacity of different samples.

DPPH is a stable free radical and accepts an electron or hydrogen radical to turn into a stable diamagnetic molecule.

The normal purple color of DPPH will transform into yellow when its singlet electron will be matched up, thus decreasing the intensity of the absorption at 517nm. A linear curve was plotted for each extract to determine the IC50. IC50 values of the all extracts are listed in Table 2.

The study revealed that Ethyl acetate fraction (AT) exhibited the highest antioxidant activity with an average of 0.1121±0.0043g/l, whereas the lowest DPPH radical quenching was observed in Crude extract of fruits (AT) (3.1784 ± 0.3637g/l).

The AA in the extracts decreases in the order: Ethyl acetate fraction (AT) > Ethyl acetate fraction (AG)> Chloroform fraction (AT) > Chloroform fraction (AG) >Butanol fraction (AT) > Butanol fraction (AG) > Crude extract of fruits (AG) >Crude extract of fruits (AT).

However, the IC50 value for extracts were much lower

than positive control, BHA (0.0449± 0.0011mg/ml) used in the present study. Similar IC50 values (0.1121±0.0043–3.1784 ± 0.3637 mg/mL).

Polyphenol and flavonoids possess ideal structural chemistry for radical scavening, this property emerge from its high reactivity as hydrogen or electron donator, and the stability and delocalization of the hindered electron of polyphenol-derived or flavonoids radical.

It is accounted that synthetic antioxidants have several side effects, for example, risk of liver damage and carcinogenesis in laboratory animals Ishurda and Kennedy³⁵. Recent epidemiological evidence suggests that natural antioxidants have health-promoting properties that may reduce the risk related with chronic diseases such as cardiovascular disease³⁶.

Table 2: Phosphomolybdenum assay and DPPH scavenging and Superoxide anion radical scavenging activity.

Extract	Phosphomolybdenum assay (mmol of ascorbic acid/g)	DPPH (IC50 in mg/mL)
Crude extract of fruits	303.1836 ± 2.3229	1.5658 ± 0.0142	AG
Crude extract of fruits	239.5746 ± 5.0161	3.1784 ± 0.3637	AT
Chloroform fraction	138.3018 ± 5.1623	0.2161± 0.0089	AG
Chloroform fraction	99.9835 ± 4.5447	0.1351 ± 0.001	AT
Ethyl acetate fraction	41.8416 ± 2.2754	0.114 ± 0.0039	AG
Ethyl acetate fraction	40.4555 ± 1.9596	0.1121±0.0043	AT
Butanol fraction	213.5793 ± 8.0422	0.5862 ± 0.1313	AG
Butanol fraction	176.3021 ± 5.4179	0.273 ± 0.0446	AT
ascorbic acid	0.702 ± 0.0045	-
BHT	-	0.0449± 0.0011

CONCLUSION:

This study represents an overview of the Antioxidant profile of Ali Ourached date fruit with different irrigation water. Results showed dissimilarity of content of polyphenols and flavonoid in Fruit date palm and our fractions.

According to the evaluation of percentage of free radical-scavenging activity of fruit and our fractions, the ethyl acetate and chloroform were found to have a higher antioxidant activity than other extracts. Phosphomolybdenum assay value, the Crude extract of fruits showed the best activity.

The discernible variability of bioactive phytochemical antioxidant activity denotes the distinction between genotypes of the same fruits date palm with different watering water.

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Received on 18.09.2019 Modified on 30.11.2019

Asian J. Research Chem. 2020; 13(1): 28-32.

DOI: 10.5958/0974-4150.2020.00007.3

particulars	crude extract of fruits	chloroform fraction	ethyl acetate fraction	butanol fraction
TPC (mg of GAE/100 g of DW)	124.2469±0.0000	1.9513±0.0002	1.9910±0.0001	16.9774±0.0012	AG
TPC (mg of GAE/100 g of DW)	93.4840±0.0264	3.5324±0.0000	2.2358±0.0010	20.3728±0.0000	AT
TFC (mg of CEQ/100 gm of DW)	3.4781±0.0027	0.0631±0.0001	0.1934±0.00006	2.1372±0.0005	AG
TFC (mg of CEQ/100 gm of DW)	4.3717±0.0016	0.1177±0.0001	0.1853±0.00002	2.6725±0.0003	AT