Molecular docking of phenolic compounds and screening of antioxidant and antidiabetic potential of Olea europaea L. Ethanolic leaves extract

2.1 Reagents

Acarbose and alpha-amylase, and Folin–Ciocâlteu reagent were obtained from Sigma- Aldrich Corporations, USA. Ethanol, gallic acid, sodium carbonate and sodium phosphate buffer were purchased from Fouz Chemical Company, Saudi. DNS (3, 5-Dinitrosalicylic acid) and phosphate buffer were attained from Merck Millipore Corporation, USA. DPPH (2,2-diphenyl-1-picrylhydrazyl) was obtained from Cayman Chemical Company, USA. The remaining chemicals or reagents were of analytical grade.

2.2 Plant sample collection

Olea europaea L. matured leaves were gathered from Al-sqeer farms in the Qassim region, Saudi Arabia. The plant specimen was authenticated at the Department of Pharmacognosy, Qassim University, Kingdom of Saudi Arabia (Ref. No.: QA/FOP/03).

2.3 Preparation of plant extract

The plant leaves were cleaned thoroughly in lukewarm water and dried under shade. The leaves were ground and macerated accordingly. For maceration, the ratio of absolute ethanol and ground leaves, 3:1. Thus, 200 g ground leaves were added to 600 ml absolute ethanol in tightly sealed Erlenmeyer flasks (vessels). The mixture was macerated at 100 rpm on the shaker for approximate five days at room temperature. The supernatant liquid was filtered through a muslin cloth. Subsequently, the rotary evaporated at 70°c. The extract (OLE) was transferred to a china dish, kept on a water bath at 60°c, and freeze-dried (Murugan and Parimelazhagan, 2014).

2.4 Physicochemical and phytochemical studies

The physicochemical analysis was studied on OLE using the standard methods (Roy et al., 2013; Sharma et al., 2013).

In brief, for the total ash value method, the crucible weight was taken and recorded. The powdered sample material (2 g) was added to the pre-weighed crucible. Then, the sample was heated at 650°-700 °C until most carbons were burnt off, forming ash or white coloured powder. The sample was left to cool. Subsequently, the ash weight was taken. The total ash percentage was calculated as follows, Ash value %=[(Weight of crucible (g) + Dry ash(g))-Weight of empty crucible (g)]/Weight of sample taken (g) × 100

Loss on Drying (Gravimetric method), powdered sample material (1.5 g) was added into a flat thin porcelain dish and allowed to dry at 100°c in the oven. The sample weight was taken 5 min intervals after cooling until the consecutive weights did not vary by more than 0.5 mg. The loss identified the moisture content upon drying in terms of grams (g), calculated as follows, Moisture content (g) = Initial weight of the powdered sample(g)–Final sample weight after drying (g)

Foreign organic material method, powdered sample material (100 g) was spread on cleaned white cloth. The sample was inspected for any foreign organic material, including the remaining stem or other plant parts to be removed. Then, the sample powder was weighed after most of the foreign organic was removed, calculated as follows, Foreign organic matter(%) = [Sample weight difference(g)/Initial sample weight(g)]x100

Phytochemical analysis was carried out for the plant extracts as per the standard procedures. Qualitative tests on the sample were used to distinguish constituent presence, including carbohydrates, phenols, flavonoids, anthocyanins, proteins, saponins, glycosides, tannins and gums (Chigurupati et al., 2017; Majid et al., 2015; Roopashree et al., 2008).

• Test for Tannins:

The appearance of bluish-green coloration on the addition of 2 ml of OLE (1 mg/ml) to 3–4 drops of 5% iron (III) chloride solution indicated the tannin presence.

• Tests for Flavonoids:

The existence of the flavonoids was confirmed by the appearance of pink color after a few minutes when small pieces of magnesium ribbon and concentrated HCl were combined with OLE.

• Tests for Glycosides:

OLE was added with 2 ml of acetic acid and 2 ml of chloroform. The mixture was then cooled and Conc. H₂SO₄ was added. The green color appearance indicated the presence of aglycone, which is a steroidal part of glycosides.

• Test for Terpenoids:

The terpenoids were indicated via gray color formation when 2 ml of chloroform was added with the 5 ml OLE and then 3 ml of Conc. H₂SO₄ was added and boiled.

• Test for Steroids:

Added 5 ml of OLE with 2 ml of H2SO4 concentrated and chloroform. The presence of steroids was shown as red color in the chloroform layer.

• Test for saponins:

1 ml OLE and 5.0 ml of distilled water were mixed in a test tube and shaken. Then the frothing was mixed with olive oil drops and vigorously shaken. The appearance of foam indicated the presence of saponins.

• Test for gum:

10 ml OLE (1 mg/ml) was added portion by portion in a slow manner into 25 ml of absolute alcohol and moved lightly. If precipitation occurs, it indicates the point to the gum presence.

• Test for protein:

2 ml of OLE (1 mg/ml) was added to 2 ml of 4% sodium hydroxide solution. The combination was mixed lightly, then added 1% copper sulfate solution. The protein presence was confirmed by the form of violet color.

• Test for carbohydrates:

2 ml of OLE (1 mg/ml) was added with about 3–4 drops of Molisch’s reagents and mixed appropriately. Then, added to the solution 2 ml of HCl. The presence of carbohydrates is verified by the appearance of a purple reddish violet ring on the interface zone.

• Tests for phenol:

2 ml (1 mg/ml) of OLE was prepared then added 5% of ferric chloride solution. The formation of a dark green color indicated phenol presence.

• Test for Alkaloid:

OLE dissolved in dilute hydrochloric acid and filtered. The presence of alkaloids was confirmed by a yellow-colored precipitate when filtrates of OLE were treated with potassium mercuric iodide (Mayer’s reagent).

2.5 Total phenolic content estimation

The phenolic content was estimated by the Folin–Ciocalteu technique. The samples and Gallic acid (GA) as standard drug (Lins et al., 2018) were prepared at various concentrations (10–1,000 µg/ml). Sample (1 ml), Folin-Ciocalteau reagent (1 ml) and 2.5% sodium carbonate (2 ml) solution were added to the test tube. The mixture was incubated for 30 min at room temperature in dark conditions. The absorbance was taken at 760 nm. The absorbance reading of the samples was calculated and analysed accordingly. The sample's quantifications' phenolic content was determined by a standard curve of GA and was expressed as mg GAE/g (mg of GA equivalent per gram) (Baba and Malik, 2015).

2.6 Total flavonoid content estimation

The flavonoid content was quantified by the aluminium chloride (AlCl₃) colourimetric method. The sample and Rutin (RE) as standard drugs were prepared at various concentrations ranging from 10 − 1,000 mg/ml, and 2% aluminium chloride (3 ml) was added. The mixtures were kept for 60 min at room temperature, followed by absorbance taken at 415 nm. The RE calibration curve was plotted accordingly to the absorbance value. The extracts' flavonoid concentration is mg RUE/g (mg of RUE equivalent per gram dry weight) (Baba and Malik, 2015).

2.7 DPPH radical scavenging activity

DPPH solution was formulated using Molyeux and Blois technique with slight modifications for antioxidant assay. The extract and standard ascorbic acid were prepared in different concentrations using absolute ethanol (10–1000 µg/ml). 0.1 mM DPPH solution (0.5 ml) was added to the sample (0.5 ml) and incubated at room temperature in dark conditions for approximate 20 min. Accordingly, the absorbance was measured at 517 nm (Chigurupati et al., 2016). 0.5 ml Absolute ethanol and 0.5 ml DPPH solution are used as control. The percentage (%) of the free radical scavenging inhibitory assay was computed as follows:

% Inhibition = (absorbance _control – absorbance _sample)/ absorbance _control × 100

Absorbance _control is the absorbance of the control reaction, and the absorbance _sample is the absorbance of the test sample. The sample concentration providing 50% inhibition (IC50) was computed from the plotted graph percentage of radical scavenging versus sample concentration.

2.8 Alpha-Amylase inhibitory activity

Extract and standard Acarbose were prepared in various concentrations using absolute ethanol (10–1,000 µg/ml). Accordingly, the samples (0.5 ml) were added to 0.5 mg/ml alpha-amylase solution (0.5 ml) prepared in 0.2 mM phosphate buffer (pH 6.9). Then, the mixtures were incubated at 25 °C for 10 min. 1% Starch solution (0.5 ml) prepared in 0.02 M sodium phosphate buffer is added then incubated for another 10 min at 25 °C. DNS (1 ml) is added and bring to boil for approximate 5 min. The tubes were cooled at room temperature. Distilled water (10 ml) was added accordingly. Control contains all the same solution mixture except the sample. Subsequently, absorbance was measured at 540 nm (Noreen et al., 2017). The sample's enzymatic inhibitory activity for the antidiabetic assay was computed as follows: %Inhibition=(absorbance _control – absorbance _sample)/absorbance _controlx100

Absorbance _control is the absorbance of the control reaction, and the absorbance _sample is the absorbance of the test sample. The sample concentration providing 50% inhibition (IC₅₀) was computed from the plotted graph percentage of enzyme inhibition versus sample concentration.

2.9 Kinetic analysis

Kinetic analysis of free radical scavenging activity was carried out by the modified Levenberg–Marquardt method (Benmehdi et al., 2017). In brief, the DPPH Stock solution was prepared by dissolving 1 mg of DPPH in 25 ml of methanol and stored in the dark at room temperature. Simultaneously, 5 mg of extract was dissolved in 4 ml methanol and from this, different concentrations were prepared (0.6250; 0.3125; 0.1562; 0.0780 mg/ml) to perform kinetic studies. A mixed equal volume of stock solution with extract solution (1:1 ratio) and absorbance was noted every 0.5 min until it became constant. The spectrophotometric data were taken until the disappearance of DPPH in the presence of the crude extracts occurred. The remaining (DPPH^.) present was calculated using the following equation: %(DPPH^.)₌[(DPPH)_]t=T/[(DPPH^.]_t=0)X100

[(DPPH^.]_t=0 represents the initial concentration of DPPH; [(DPPH^.]_t=T represents the concentration of DPPH at a steady-state

2.10 Statistical analyses

The samples were evaluated individually in triplicate, and data were shown as mean ± SEM (Standard error mean). The IC₅₀ value for the antidiabetic assay was computed by Graph Pad Prism Software (Version 5.03) by a non-linear regression graph calculated between the percentages of enzyme inhibition versus sample concentration. Similarly, the IC₅₀ values for the antioxidant assay were computed by Graph Pad Prism Software (Version 5.03) by non-linear regression graph calculated between percentages of radical scavenging versus sample concentration. With p < 0.001, the difference in the IC₅₀ between Ascorbic acid and OLE is significant for both antioxidant and antidiabetic studies. The kinetic analysis is implemented in Origin v.6.0 Programme for Windows.

2.11 Molecular docking

The X-ray crystal structure of the human pancreatic α-amylase in complex with mini-montbretin A (PDBID: 5E0F) was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank (available online: https://www.rcsb.org/structure/) (Alam et al., 2019). The structures of Phenolic compounds of Olea europaea L. were retrieved from the PubChem database (Kim et al., 2019). The mini-montbretin A, and the water atoms were deleted, while the nonpolar hydrogens were merged. The protein and ligands were prepared and optimised by using AutoDock Tool (ADT), bundled with the MGLTools package (version 1.5.6) (Morris et al., 2009) to add charges, polar hydrogen atoms, and set up rotatable bonds. At that moment, ADT was operated to make PDBQT files for the protein and ligands. The molecular docking was performed using AutoDock Vinav1.1.2 (Kim et al., 2019) in PyRx 30.8. The active binding site of the α-amylase was chosen as the grid centre and obtained by removing the ligand. The centre of grid box dimensions was selected to include all atoms of the ligand set. (Kalinowsky et al., 2018). The site of the grid box in α-amylase was set at −7.946, 10.438, and −21.863 Å (for ×, y and z) by means of a grid of 40, 40, and 40 points (for ×, y and z). Then, we created a configuration text file (config.txt) to run AutoDock Vina 1.1.2. This configuration file involves the receptor and ligand PDBQT file and the centre grid box coordinates. The docking scores were resulted in the generated .log files. The output docking scores were defined as affinity binding (Kcal/mol). The various protein–ligand interactions, such as hydrogen bonds, cation − π bonds, π − π bonds, and alkyl-π interactions between amino acids of the active sites and compounds were studied. The ligands protein interactions were created by using the Discovery Studio 2020 client. (Dassault Systèmes BIOVIA, 2020).

3.1 Phytochemical screening

In the current study (Table 2), the OLE phytochemical screening showed the presence of saponin, flavonoid, glycoside, tannin, phenol, and carbohydrate are inferring that blood glucose lessening the plant extract activity because of these phytoconstituents.

The total ash value commonly used characterise the purity or quality of crude extract, mostly in powder form. It excludes all traces of organic substances in vegetable drugs. After burning, the ash content of crude extracts the resulting residue due to naturally inorganic salt presences. The total ash value and foreign organic matter of OLE was 16.5 % and 0.2 %, respectively. As for loss on drying (Gravimetric method), the moisture content was 2 g.

3.2 Total phenol and flavonoid contents

The flavonoid content was denoted in Rutin equivalent; the macerated ethanolic extract of O. europaea exhibited the flavonoid content of 855 mg RUE/g. As for phenolic content, it was estimated using Folin-Ciocalteu reagent in terms of Gallic acid equivalent. The macerated ethanolic extract of O. europaea has total phenolic content of 490 mg GAE/g.

3.3 Antioxidant assay

The DPPH assay is based on the measurement of the scavenging capacity of antioxidants towards it. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine (Sahoo et al., 2013). As depicted in Fig. 1, OLE (IC₅₀ ± SEM: 38.37 ± 0.26 µg/ml) portrayed antioxidant activity comparable to the ascorbic acid (IC₅₀ ± SEM: 30.37 ± 0.17 µg/ml).

Close

Fig. 1

DPPH radical scavenging effects of OLE and standard, Ascorbic acid (n = 3).

Export to PPT

3.4 Kinetic analysis

Kinetic analysis of OLE was carried out to calculate the scavenging activity of DPPH· as a function of time. In this study, the effect of the concentration of ethanolic extracts (0.625; 0.312; 0.156 and 0.078 mg/ml) on scavenging free radicals was studied. Free DPPH· radical is relatively stable for more than 110 min at 25 °C in the reaction medium. So, we allowed the evaluation of the free radical scavenging activity of the extracts within that time. The free radical trapping was measured by calculating the remaining DPPH· concentration at the end of the reaction.

With the help of kinetic study, the steady-state time, half-life (t^1/2) and the remaining percentage DPPH were calculated for different concentrations of extract (Table 3). The results showed clearly that the ethanolic extract exhibited a significant antioxidant effect the steady-state time were 5.5; 16.1; 117.1 and 20.6 min at extract concentration of 0.625; 0.312; 0.156 and 0.078 mg/ml respectively. Further, we calculated the remaining DPPH· concentration (9.7; 20.0; 28.4 and 33.4 %). The effectiveness of extract was expressed by half-life t^1/2, which was shorter when concentration increases.

3.5 In vitro antidiabetic assay

As depicted in Table 4, the standard Acarbose and OLE were examined at various concentrations ranging from 10 − 1,000 µg/ml. In the alpha-amylase inhibitory activity, Acarbose and OLE showed enzymatic inhibition with an IC₅₀ value of 20.06 ± 0.19 µg/ml and IC₅₀ value of 37.99 ± 0.15 µg/ml, respectively.

3.6 Molecular docking studies

Upon conducting molecular docking studies, the most of the reported phenolic compounds of OLE showed good binding affinity against the human pancreatic α-amylase (HPA) (Table 5) (Sabbah et al., 2010).The docking scores for cynaroside and apigenin 7-O-glucoside are − 9.80 kcal/mol and − 9.70 kcal/mol, respectively while the docking scores for oleuropein and verbacoside are – 8.20 kcal/mol and − 8.10 kcal/mol, respectively. The binding interactions of the phenolic compounds of OLE versus Acarbose share hydrogen bonds with E233, D300, and H305 and π - π stacking with W56. The hydroxyl groups of flavone glycosides form H-bond with Q63, D197, and E233, and the aromatic ring of cynaroside induces π – anion with D300. The ligand–protein interactions of oleuropein and acteoside are similar to flavone glycosides (Fig. 3). The oleuropein and acteoside have extra H-bond with H305.