In vitro and Molecular Docking Studies of DPPH with Phoenix dactylifera L. (Deglet-Nour) Crude Fruits extracts and Evaluation of their Antioxidant Activity
Samir Zeroual1*, Ismail Daoud 2,3, Randa Gaouaoui1, Said Ghalem3
2University Mohamed Khider, Department of Matter Sciences, BP 145 RP; (07000) Biskra, Algeria.
3Laboratory of Natural and Bio-actives Substances. Tlemcen University- Faculty of Science. P.O. Box 119-Tlemcen-Algeria.
*Corresponding Author E-mail: samir.zeroual@univ-biskra.dz
ABSTRACT:
Uses edible parts of date palm (Phoenix dactylifera L) fruits from Algeria were analyzed to determine their phenolic compound and antioxidant activities using DPPH, FRAP assay and molecular docking. In this paper, in-silico and in-vitro studies were used to study interaction Enzyme-Compounds and evaluate antioxidant activities of compounds. The results show that the date fruit rich in the total phenolic content was found Gallic acid, the flavonoid: quercetin and condensed tannins of catechin. The antioxidant activity ranged between 64, 46-91, 3 for DPPH (%) and 0,194-14,923µg AAE/ 0.5mg extract for FRAP. In-silico analysis was an agreement with in-vitro in which phenolic compounds showed more negative free energy against standard drug and bound with catalytic residues of Kinase. The findings provide evidence that date fruit has potential as a natural source of antioxidant with ethyl acetate showing to be the best extracting with corresponding higher antioxidant activities.
However, various studies have been conducted to determine the chemical composition of the date as: sugars, proteins, lipids, fibers and minerals.7-9 While studies on its content of secondary metabolites, especially polyphenols, remain few and do not concern most Algerian varieties, given their nutritional, economic and medicinal value and their implications in many biological activities (antimicrobial, anti-inflammatory, anticarcinogen and antiviral).10,11 Some studies have also shown the role of phenolic compounds in the antioxidant activity of the date.12,4,13 Our work is part of this framework, which we considered to conduct a study on a variety of Algerian dates (Deglet Nour) Commercial variety par excellence. It is a semi-soft date, considered to be the best date variety because of its appearance, its creaminess and its flavor. Therefore, in this review we attempt to combine two methods: in vitro and in silico for the purpose to realize different extractions with water and organic solvents with increasing polarity, to assay qualitatively and quantitatively the phenolic compounds in these extracts, to test in vitro the antioxidant activity of these latter.
In Vitro study:
1. Plant material:
2. Sample preparation:
Deglet Nour dates were pitted, cut into pieces and then reduced to paste using a blender.
3. Preparation of plant extract:
Organic extracts were prepared according to Diallo's method14 with small modifications. To 300 g of date paste for the variety. Deglet Nour, was added 1.8 l of a methanol-water mixture in proportions (80/20) (v/v). The preparation was stirred for 24 hours and filtered after this step was repeated three times. Methanol and water were recovered using a rotary evaporator. The hydro-methanolic extract obtained after evaporation was successively exhausted by four solvents with increasing polarity: hexane (Hex), dichloromethane (Dcm), ethyl acetate (ETAC) and n-butanol (n-BuOH). After evaporation of the solvents using a rotary vacuum evaporator maintained at 45°C, the various dry extracts were weighed and stored at 4°C.
The qualitative phytochemical screening of the fruits of Phoenix dactylifera L. (Deglet-Nour) was determined using the standard methods described by Sofowora and Trease and Evans.15,16 The filtrates were treated with Mayer's reagent (potassium mercury iodide). The formation of a yellow precipitate indicates the presence of alkaloids. The extracts were diluted with distilled water to 10ml and mixed well. The formation of foam indicates the presence of saponins. 0.25g of extract is stirred with 2ml of water. If the foam produced persists for 10 minutes, it indicates the presence of saponins. The extracts were treated with 3-4 drops of ferric chloride solution. The formation of a bluish black color indicates the presence of phenols or tannins. A 1% gelatin solution containing sodium chloride was added to the extract. The formation of a white precipitate showed the presence of tannins. The extracts were treated with a few drops of sodium hydroxide solution. The formation of an intense yellow color, which later became colorless upon the addition of dilute acid, indicated the presence of flavonoids. 5 ml (1 mg/ml) of the fraction with a few drops of chloroform was added and then 3 ml of concentrated H 2 SO 4 was added to the mixture. The reddish-brown color change revealed the presence of terpenoids. The extracts were treated with a few drops of conc. Nitric acid. The formation of the yellow color marked the presence of proteins. The test solution was mixed with a few drops of Benedict's reagent (alkaline solution containing a cupric citrate complex) and boiled in a water bath. The formation of reddish brown precipitate showed a positive result for the presence of carbohydrates.
5. Evaluation of antioxidant activity:
a. DPPH radical scavenging assay:
Antioxidant activity was determined by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method.17 A 750μl portion of the DPPH solution (4mg/100ml MeOH) was incubated with 250μl of extract or standard, After 30 minutes in the dark at room temperature, he absorbance of the mixture was measured at 517 nm. The activity of scavenging (%) was calculated using the following formula:
OD control – OD sample
DPPH radical scavenging % =––––––––––––––––––––––––× 100.
OD control
The IC50 value, the inhibitory concentration as mg phenol of the test sample that decreases 50% of initial radical. The IC50 values were calculated from the dose response curves.
b. Ferric reducing antioxidant power assay (FRAP):
The ferric reducing power of the date extracts was determined according to the method of.18 0.5mL of extract or standard was mixed with 225µl phosphate buffer (0.2M pH 6.6) and 225µl of potassium ferricyanide , with agitation and the mixture was incubated in the dark for 20 minutes in a water bath whose temperature was 50°C, 225µl of TCA (trichloroacetic) was added to the mixture and then centrifuged for 10 min at 700rpm. 375μl of distilled H2O was mixed with 375μl of the supernatant and then75μl of FeCl3 was added. The absorbance was measured at 700nm against the blank, which the extract was replaced by methanol.
5. Statistical analysis:
All assays were conducted in triplicate. Statistical analysis of the results was performed with the ANOVA application was performed by the Origin Pro2016 64Bit software on the obtained values.
Molecular docking study:
1. Ligands Preparation
The ligands (Table 1) named L1-8 and LRef C15 H10 O7: (3,5,7,3',4' Pentahydroxyflavone) structures were optimized by using MM+19 molecular modeling and the semi-empirical AM1 method, both of which are implemented in Hyperchem 7.0 software (Version 7.0, Hypercube, USA, http://www.hyper.com). For these calculations, the Polak-Ribiere conjugate gradient algorithm was employed, with the RMS gradient set to 0.0001 kcal/(Å mol).
Table 1. Some chemical properties of ligands
|
Ligands |
Number of atoms |
Number of heavy atoms |
Molecular weight |
Flexible torsions |
|
L1(gallic acid) |
18 |
12 |
170.120 |
1 (out of 1) |
|
L2(Quercetin) |
32 |
22 |
302.236 |
1 (out of 1) |
|
L3(P.coumaric acid) |
20 |
12 |
164.158 |
2 (out of 2) |
|
L4(m annisic acid) |
19 |
11 |
152.147 |
2 (out of 2) |
|
L5(caffeic acid) |
21 |
13 |
180.157 |
2 (out of 2) |
|
L6(syringic acid) |
24 |
14 |
198.173 |
3 (out of 3) |
|
L7(trans-2.4dimetoxycinnamic acid) |
27 |
15 |
208.211 |
4 (out of 4) |
|
L8(Myriecetin) |
33 |
23 |
318.235 |
1 (out of 1) |
|
LRef (L2) |
32 |
22 |
302.236 |
1(out of 1) |
Table 2. Propriety of enzyme pdb: 2HCK
|
Chains |
Number of residues |
Number of atoms |
Number of heavy atoms |
Number of bonds |
Molecular weight |
|
A |
433 |
4192 |
3434 |
4275 |
46090.4 |
|
B |
433 |
4192 |
3434 |
4275 |
46090.4 |
From the results obtained it is found that the ligands L6 and L7 have an important number of torsion angles relative to other ligands, this shows that these ligands are more flexible. It is noted that the growth of the torsion angle depends on the binding number of the molecule (compound).
We note that compounds L2 and L8 have an important number of molecular weights and these L8 an important number of heavy atoms compared to other ligands and we note also that compounds L2 and L8 have a same number of 6-ring.
2. Target Preparation:
The Xray crystal structures of the kinase (HEMATOPOETIC CELL KINASE HCK) (PDB ID: 2HCK)20, were downloaded from RCSB Database.21 2HCK is a three dimensional structure with EC Number: 2.7.11.22, chains (A) and chains (B), resolution 3 Å and R-value 0.228. Computational analysis was carried out on chain A of 2HCK (Table 2). The eight 2HCK inhibitors (L1-8 and LRef) were selected to study the associated physical-chemical parameters and protein-ligands interactions.
3. Molecular Docking Simulations:
The initial kinase coordinates were obtained from the PDB (PDB ID: 2HCK). All solvent molecules and the co-crystallized inhibitor were removed from the structures to provide sterically unimpeded cavities for ligand docking. Docking was performed by using Molegro Virtual Docker (MVD)22 software package because this algorithm maintains a rigid macromolecule while allowing ligand flexibility.
The enzyme and compound structures were prepared using the same default parameter settings in the same software package (Score function: MolDock Score; Ligand evaluation: Internal ES, Internal HBond, Sp2–Sp2 Torsions, all checked; Number of runs: 10 runs; Algorithm: MolDock SE; Maximum Interactions: 1500; Max. population size: 50; Max. steps: 300; Neighbor distance factor: 1.00; Max. number of poses returned: 5). The docking procedure was performed using a GRID of 15 Å in radius and 0.30 in resolution to cover the ligand-biding site of the kinase structure.
The MolDock score [GRID] algorithm was used as the score function, and the MolDock search algorithm was used.23
RESULTS AND DISCUSSION:
In Vitro results:
a. Returns of extractions:
The determination of the yield rates of different extractions made it possible to calculate the contents of phenolic compounds, assayed in the extracts, with the fresh weight of the date. These yields are expressed as a percentage of the fresh material. The yields of hexane, dichloromethane and ethyl acetate extracts are very low, varying between 0.03 and 0.11%. While the alcoholic extracts (n-BuOH) is high (2.84%), and the crude extract (Water-MeOH) Degla-Nour which gave the highest value (64.28%).
b. Phytochemical screening:
The chemoprofiling of fruit extract in different solvents (hexane, dichlorométhane, ethyl acetate, 1-buthanol, methanol/ water, and water) indicated the presence of gold tannins Phenolic compounds, reducing sugar and Terenoids in the majority of extracts. While saponins and alkaloids were absent in all extracts. The presence of flavonoids in the dichloromethane and ethyl acetate extracts is relatively low and is absent in the n-butanol, hexane, hydroalcoholic and aqueous extracts. Our results of the phytochemical screening obtained are in agreement with those reported by24,25 analyzed the phenolic profile of seven Algerian date cultivars, including Deglet-Nour, reporting the presence of phenolic acids, such as cinnamic acid and its derivatives (p-coumaric acid, ferulic and sinapic acid and caffeoylshikimic acid). Daas Amiour confirms the absence of saponosides in extracts of the cultivar Deglet-Nour. Nour (Table 3).
Table 3. Chemoprofiling of Phoenix dactylifera L. fruits extracts in different solvents.
|
S. No |
Extracts |
||||||
|
|
Phytochemicals |
Hexane |
Dichloromethane |
Ethyl acetate |
n-butanol |
Methanol/Water |
Water |
|
1 |
Alkaloids |
- |
- |
- |
- |
- |
- |
|
2 |
Phenolics compounds or Tannins |
- |
++ |
++++ |
+++ |
+ |
- |
|
3 |
Saponins |
- |
- |
- |
- |
- |
- |
|
4 |
Flavonoids |
- |
± |
± |
- |
- |
- |
|
5 |
Terpenoids |
- |
++ |
++++ |
+++ |
+ |
+ |
|
6 |
Reducing sugar |
+ |
+++ |
++ |
+++ |
++++ |
+++ |
Depending on their intensity, the reactions that occur are ranked from negative (-) to positively positive (++++). ±: relatively low.
Table 4. Total phenolics, total flavonoids and total condensed tannins contents of date varietie Degla-Nour
|
Extracts |
Total flavonoids (mg QE/100 g DW) |
Total condensed tannins (mg CE/100 g DW) |
Total phenolics (mg GAE/100 g DW) |
|
Hexane |
98,35±2,35 |
416,25±19,45 |
1389,5±207 |
|
Dichloromethane |
175,6±3,15 |
58,75±1,75 |
1668±136,5 |
|
Ethyl acetate |
208,35±3,9 |
377,5±99 |
5971,5±434 |
|
n-butanol |
116,65±6,3 |
88,75±1,75 |
946,5±75,5 |
These tests were carried out to highlight the presence or absence of certain chemical groups that may be responsible for the biological activities studied.
Total bioactive compound:
A comparison of the bioactive compounds of the Degla-Nour date extracts is shown in Table 4. Significant differences in the total content of phenolic compounds were observed between the dates extracts studied. The contents of the different extracts in flavonoids are shown in the same table. Deglet Nour contains a low total flavonoid content (98.35 ± 2.35-208.35 ± 3.9mg EQ/ 100%). g DW). Our results are spiked to the results of Benmeddour et al26 and Bouhlali et al27 who found that total flavonoids ranged from 15.22 to 299.74 mg EQ/ 100g of date and (68.88 208.53 mg of rutin equivalent (RE)/100 of DW).
The four extracts of Degla-Nour contained a high content of condensed tannins ranged between (58.75 ± 1.75 to 416.25 ± 19.45mg CE / 100 DW g), the results found in this study are considerably more higher than values of 100 g of date and (68.88 to 208.53 mg of rutin equivalent (RE) /100 of DW) reported by the results of published papers.27,28 They showed that the tannin content varied from (57,564 to 92,141 mg EC / 100g DW) and from (5.29 to 152.15 EC / 100 g DW) in the Moroccan cultivar.
The total polyphenol content of the date fruit varieties examined showed significant differences, as shown in Table 4. Deglet Nour had (946.5 ± 75, 5-5971.5 ± 434mg GAE / 100g DW). Benmeddour et al26. Found that the total phenol content ranged from (199.43 to 576.48mg GAE / 100g) of ten cultivars dated from the Tolga station (Biskra, Algeria) to approximately our results. However, the current results are much higher than those reported by Mansouri et al.25 who found that the TP content of methanolic extracts from seven different date palms in Ghardaia, Algeria, ranged from (2.49 to 8.36 mg GAE / 100 g body weight). Different factors may be responsible for the observed differences such as variety, growing conditions, maturity, geographical origin, soil type, storage conditions, climatic conditions and the use of different phenolic acid standards.29,30
c. Antioxidant activity:
The antioxidant activity of the date variety was investigated by FRAP and DPPH radical scavenging method. The fruits contain different polyphenols, compounds, which have different antioxidant activities. In the present study, two methods: ferric reducing power (FRAP) and free radical scavenging activity assay (DPPH) were used to evaluate the antioxidant activity of the date fruit extracts.
Figure 1. Antioxidant activity expressed as percentage inhibition in the presence of the various extracts, BHA and BHT.
The DPPH radical is one of the most used substrates for a direct, fast and reliable evaluation of antioxidant activity because of its stability and the simplicity of analysis. The antioxidant activity is determined by spectrophotometric analysis at 517 nm, following the reduction of DPPH. which results in the change from purple to yellow. The DPPH is characterized by its adaptation to several samples in a short time, so it is sensitive enough to detect active ingredients at low concentrations, For this purpose, it has been used for screening anti - radical activities of plant extracts.31
The antioxidant capacity of the different extracts was determined from the IC50, this is the concentration needed to reduce 50% of the DPPH radical. The smaller the IC50 value, the greater the antioxidant activity of the test extract.25 The antioxidant activity of the various extracts of this date variety was evaluated using the DPPH method and examined by comparing it to the controls (BHA and BHT). The highest oxidation inhibitor was achieved by dichloromethanolic extract (91.3%) followed by ethyl acetate (77.69%), n-butanol (76.62%) and hexane antioxidant activity (64.46%) (Fig 1).
Reductive power method
Table 5. The reducing power of the varietie Degla-Nour in μg in ascorbic acid equivalent/0.5mg of extract
|
Extract |
Deglet-Nour |
|
Hexane |
14,923 |
|
Dichloromethane |
0,194 |
|
Ethyl acetate |
59,385 |
|
N-butanol |
8,769 |
The FRAP is extensively used in the assessment of the antioxidant activity. It measures the conversion of a Fe3+ ferricyanide complex to the ferrous form. The reduced capacity of a compound is considered by many authors to be an indicator of its antioxidant potential n.32 All date extracts exhibited a good reducing power which varied significantly (Table 5). The reduction power results obtained from a calibration of different concentrations of ascorbic acid expressed as μg EAA/0.5mg of extract. The best reducer was obtained by ethyl acetate extract (59.39μg EAA/0.5mg extract), while hexane, n-butanol and dichloromethane showed the lowest potency with values is 14.92 ,8.77 and 0.19μg EAA/0.5mg extract respectively.
Individual phenolic compounds:
The contents of phenolic compounds that were identified by HPLC analysis are presented in Table 4. Eight phenolic acids and flavonoids were determined and quantified in the studied date fruits by comparison with retention times. Six phenolic acids: Gallic acid, P.coumaric acid, m annisic acid, caffeic acid, syringic acid, trans-2.4dimetoxycinnamic acid and two flavonoids: Quercitine, and Myricetin were detected. It was observed that caffeic acid was present in the 2 extracts dichlorométhane and éthyle acétate.
In Silico results (Molecular docking):
a. Active site residues:
To obtain better potential binding sites in the 2HCK, a maximum of two cavities was detected using default parameters. The volume and surface of cavities are shown in Table 6.
Table 6. Volume and Surface of five cavities detected by MolDock Score
|
Cavities |
Volumes (Å3) |
|
Surfaces (Å2) |
|
1 |
860.16 |
|
2383.36 |
|
2 |
478.72 |
|
1251.84 |
|
3 |
183.808 |
|
586.24 |
|
4 |
52.736 |
|
172.8 |
|
5 |
33.792 |
|
140.8 |
It found that the ligand co-crystallize selective inhibitor (C15 H10 O7) of 2HCK is fixed in cavity 2 (V=478.72Å3, S=1251.84Å2). Out of the detected cavities, cavity 2 was selected for further studies (see Figure 2).
b. EnzymeLigand Interaction:
The scoring functions of the compounds were calculated from minimized E-L complexes. In order to compare the binding affinity of the newly ligands, we docked eight compounds into the empty binding active site of kinase. Based on the determination of two parameters: Energy score and the distances of the existing interactions between the studied ligands and the active site residues, five top poses for each ligand were returned in the simulation, out of which one best pose for each ligand was selected on the basis of their MolDock score.
Figure 2. Cavities detected by MolDock Score (green color)
Figure 3. The top binding pose obtained by molecular docking simulations for all complexes formed and generated by Ligplot+ program.34
Table 7. Docking results of ligands L1 to 8 and LRef with kinase in the active site.
|
Compounds |
S-score (kcal/mol) |
Bonds between atoms of compounds and residues of active site |
||||
|
Atom of compound |
Involved receptor atoms |
Involved receptor residues |
Type of interactions bond |
Distances (Å) |
||
|
L1 |
-73.595 |
O2 |
OG1 |
THR429 |
H-BOND |
3.10 |
|
|
|
O5 |
N |
LEU387 |
H-BOND |
2.63 |
|
L2 (Lref) |
-112.051 |
O3 |
N |
MET341 |
H-BOND |
2.67 |
|
L3 |
-78.377 |
O1 |
OD1 |
ASP404 |
H-BOND |
3.10 |
|
|
|
O2 |
N |
ALA408 |
H-BOND |
3.02 |
|
L4 |
-68.009 |
/ |
/ |
/ |
/ |
/ |
|
L5 |
-84.006 |
O2 |
OG |
SER447 |
H-BOND |
2.43 |
|
|
|
O1 |
OG |
SER447 |
H-BOND |
2.87 |
|
|
|
O2 |
O |
TRP428 |
H-BOND |
2.89 |
|
|
|
O1 |
O |
TRP428 |
H-BOND |
2.97 |
|
|
|
O1 |
N |
LEU387 |
H-BOND |
3.12 |
|
L6 |
-78.549 |
O3 |
OD1 |
ASP386 |
H-BOND |
3.04 |
|
|
|
O2 |
N |
LEU387 |
H-BOND |
2.88 |
|
|
|
O4 |
N |
ALA430 |
H-BOND |
2.73 |
|
L7 |
-85.996 |
O1 |
N |
ALA408 |
H-BOND |
3.09 |
|
|
|
O2 |
O |
ALA408 |
H-BOND |
3.25 |
|
L8 |
-112.329 |
O7 |
N |
LYS295 |
H-BOND |
2.71 |
|
|
|
O6 |
N |
ASP404 |
H-BOND |
2.85 |
|
|
|
O2 |
OG1 |
THR338 |
H-BOND |
2.69 |
|
|
|
O4 |
N |
MET341 |
H-BOND |
2.74 |
Table 7 presents the different Interaction between the ligand and active site residues of the enzyme and Score Energy for the selected complexes formed by these ligands and kinase.
The table above shows that the energy MolDock Score values are ranked in the following order:
L8 ˂ L2(Lref) ˂ L7 ˂ L5 ˂ L6 ˂ L3 ˂ L1 ˂ L4
The complex formed by L8 (2HCK-L8) has the lowest energy score compared to the other complexes. This complex gave a very low energy value score compared to the value of the complex formed by Lref (native ligand) (table 7 and Figure 3)
Essentially based on hydrogen bonds, In general we have:
2.5 Å <d <3.1 Å => Strong interaction
3.1 Å <d <3.55 Å => Average interaction
d > 3.55 Å => weak interaction.33
We note that this compound establish four hydrogen bonds with active site residues of enzyme kinase, the first one H-bonds (O7-LYS295=2.71 Å), second H-bonds (O6- ASP404= 2.85 Å), third H-bonds (O2- THR338= 2.69 Å) and last H-bonds (O4- MET341= 2.74 Å), all these interactions bonds belong to the range of strong interactions.
In other hand, Lref (L2) forms one hydrogen bond with active site residues of the enzyme kinase (O3- MET341=2.67) belong to the range of strong interactions (see Table 7).
Analysis of the L8 structure shows that this compound contains three rings (6-ring) and eight atoms of oxygen , but the Lref contains three rings (6-ring) and seven atoms of oxygen (see Figure 3), thus justifying the presence of an important number of hydrogen bond interactions in the compound L8 relative to the Lref compound.
CONCLUSION:
In vitro antioxidant activity and molecular docking study of Deglet Nour date were determined and presented in this paper. This variety of dates can be a good source of natural antioxidant. Knowing that these antioxidant activities were evaluated using different methods, such as DPPH radical scavenging activity and FRAP assay. These methods were undertaken to evaluate the effect of the substituent on the antioxidant activities. In addition, Dichlorométhane and éthyle acétate extract of Deglet-Nour fruits displayed most effective inhibition against the Kinase (2HCK) enzyme. This enzyme activity showed that fruit extract suggested inhibition with Kinase. The further molecular docking study confirmed that flavonoid compound (L8: Myricetin) binds with the catalytic residues of Kinase by four hydrogen bonding interaction. This inhibition of the enzyme might be either due to the synergistic effect of the phytochemical constituents of flavonoids present in it or acting separately.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
ACKNOWLEDGMENT:
We are grateful to our colleagues for their comments and suggestion about the manuscript.
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Received on 29.11.2019 Modified on 31.12.2019
Accepted on 25.01.2020 ©AJRC All right reserved
Asian J. Research Chem. 2020; 13(1): 52-59.
DOI: 10.5958/0974-4150.2020.00012.7