# Chemical Composition, Antibacterial, Antifungal and Antidiabetic Activities of Ethanolic Extracts ofOpuntia dillenii Fruits Collected from Morocco

Authors: EL Hassania Loukili; Btissam Bouchal; Mohamed Bouhrim; Farid Abrigach; Manon Genva; Kahina Zidi; Mohamed Bnouham; Mohammed Bellaoui; Belkheir Hammouti; Mohamed Addi; Mohammed Ramdani; Marie-Laure Fauconnier
Journal: Journal of Food Quality
 (2022)

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## Abstract

Opuntia dillenii (Ker Gawl.) Haw. belongs to the Cactaceae family and is native to the arid and semi-arid regions of Mexico and the southern United States. O. dillenii are now used as medicinal plants in various countries. In this study, we investigated the chemical composition of ethanolic extracts obtained from seeds, juice, and peel of O. dillenii fruits collected from Morocco, and we evaluated their antibacterial, antifungal, and antidiabetic activities. Phytochemical screening revealed high quantities of polyphenols (193.73 ± 81.44 to 341.12 ± 78.90 gallic acid eq [g/100 g dry weight]) in the extracts. The major phenolic compounds determined by HPLC were gallic acid, vanillic acid, and syringic acid. Regarding flavonoids, quercetin 3-O-β-D-glucoside and kaempferol were the predominant molecules. Juice extracts showed weak to moderate antibacterial activity against the bacteria species Listeria monocytogenes, Escherichia coli, and Salmonella braenderup. All tested extracts displayed a significant inhibitory effect on α-glucosidase and α-amylase activities in vitro, with the peel extracts showing the greatest inhibitory effects. Together, these findings suggest that O. dillenii fruits are a promising source for the isolation of novel compounds with antibacterial or antidiabetic activities. For the most abundant phytochemicals identified in O. dillenii peel ethanolic extract, molecular docking simulations against human pancreatic α-amylase enzyme were performed. These indicated the presence of bioactive compounds in the extract with a better potential to decrease the enzyme activity than the commercial drug acarbose.


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## Body

## 1. Introduction

Opuntia spp. are plants belonging to the Cactaceae family [1, 2] which are native to arid and semi-arid regions of Mexico and the southern United States [3, 4] and were introduced to Europe and North Africa in the 16th century [5, 6]. Among those, Opuntia dillenii (Ker Gawl.) Haw. is one of the most important cacti for agronomic and medicinal uses in many countries, as it represents a potential source of various chemical compounds of biological and pharmacological importance. Plant secondary metabolites display recognized therapeutic benefits. As plant materials have long been used as traditional medicines for healing without knowing the exact origin of their actions, the characterization of their secondary metabolites has been the subject of numerous research based on in vitro and in vivo studies. This is particularly the case for phenolic compounds, including polyphenols and flavonoids, which are the subject of the present study. These compounds are widely used in therapy due to their anti-inflammatory, enzyme inhibitory, antioxidant, and antiradical activities [7–9]. They are also used as additives in the food, pharmaceutical, and cosmetic industries [10]. Many researchers have focused their investigations on characterizing the properties of plants of the genus Opuntia to form a useful database for the prevention and treatment of chronic diseases [11, 12]. In particular, plants of this genus are known for their analgesic, antihyperglycemic [13], inflammatory [14], antifungal [15], anticancer [16, 17], hepatoprotective [18, 19], and antidiabetic activities [16, 20, 21]. Opuntia fruits are a rich source of dietary fiber, vitamins (B1, B2 and C) and natural colorings, i.e. betanin and indicaxanthin [22–27], which are responsible for the intense red coloring of the fruit [27], which are used to prevent certain diseases. Opuntia dillenii fruits are notably used for the treatment of gastritis, atherosclerosis and diabetes due to their high content of phenolic compounds, which provide antioxidant, anticancer and neuroprotective properties [9] and are of great interest for potential applications in human health and medicine. As efforts are underway in Morocco to develop cactus production and to increase its use in various common foods, the evaluation of the chemical composition of the pulp, peel, and seeds of edible Opuntia is of high interest.Previous studies have described the chemical composition ofO. dillenii fruits. However, no study has yet compared the chemical composition of different parts of the fruits as well as the effect of the content of these compounds on the associated biological activities. The present work aimed to compare the chemical compositions of different extracts of seeds, juice, and peel of O. illeniid fruits harvested from Morocco. Moreover, the biological activities of those extracts were evaluated. More specifically, the antimicrobial (antifungal and antibacterial) properties, as well as the pancreatic α-amylase and intestinal α-glucosidase inhibitory activities of the ethanolic extracts, were determined.


## 2. Experimental Section

### 2.1. Chemicals and Reagents

n-hexane, ethyl acetate, and ethanol were purchased from Merck (Darmstadt, Germany). All chemicals and reagents used for the determination of total phenolic compounds were of analytical grade and were purchased from Merck and Carl Roth GmbH (Karlsruhe, Germany). 3,5-Dinitrosalicylic acid (DNSA), acarbose,α-glucosidase, and α-amylase were purchased from Merck (Sigma-Aldrich, St. Louis, MA, USA). The kit necessary for the measurement of blood sugar was purchased from Biosystems (Barcelona, Spain). The bacterial strains were supplied by the Pasteur Institute of Casablanca, Morocco. The fungal strains were provided by Professor Adnane Sellam (Université Laval, Canada).Phenolic standards: gallic acid, vanillic acid, syringic acid, salicylic acid, 4-hydroxybenzoic acid, vanillin, ferulic acid, caffeic acid, p-coumaric acid, cinnamic acid, sinapic acid, quercetin, kaempferol, rutin, quercetin 3-O-β-D-glucoside, catechin, naringin, and chalcone were purchased from Merck and Carl Roth GmbH (Karlsruhe, Germany).


### 2.2. Sample Collection and Preparation

Mature purple fruits ofO. dillenii, the prickly pear plant, were collected in February 2017 in Morocco. The plant species was authenticated by Mohammed Fennan, a botanical expert from the Scientific Institute of Mohammed V University. The specimen was deposited at Mohammed First University, Oujda, Morocco, under the reference number HUMPOM 351 [2].The collected fruit specimens were washed with water to remove dust and spines, peeled and blended (Facilic glass LM310E; Groupe Seb, Mayerne, France), then the seeds were separated from the juice by passing the mixture through a sieve with a 2 mm mesh size (Figure1). The juice was dehydrated by heating at 40°C in an oven for 2 days. Seeds and peels were washed with distilled water, dried at 20°C for 1 week, weighed, and then reduced to a fine powder using a Moulinex coffee grinder (DPA241; Groupe Seb Lourdes, France). All components (seeds, juice, and peel) were stored at −20°C for a maximum of eight weeks.Figure 1
O. dillenii fruits (a), seeds (b), juice (c), and peel (d).
(a)(b)(c)(d)


### 2.3. Preparation ofOpuntia dillenii Fruit Extracts

One hundred grams of the ground seeds, juice, and peel ofO. dillenii fruits were macerated with 200 mL of n-hexane to remove fatty acids. The mixtures were stirred at 20°C for 24 h and filtered through a glass filter crucible (50 mL, Porosity 4; Isolab Wertheim, Germany). The extracts were then concentrated using a rotary evaporator (Laborota 4000; Heidolph Instruments, Schwabach, Germany) under reduced pressure at 40°C to obtain the corresponding powder. Then, ethyl acetate and ethanol were used successively to obtain the corresponding extracts by repeating the maceration process (Figure 2). The extracts were stored in dark bottles at 4°C until use.Figure 2
Maceration extraction process.


### 2.4. Total Polyphenol Content

The total phenol concentration in theO. dillenii ethanolic extracts was determined using the Folin-Ciocalteau method with some modifications. A standard curve was first plotted using gallic acid as a standard. To prepare the calibration curves and samples, 200 μL of ethanolic gallic acid solutions (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL) or 200 μL of O. dillenii ethanolic extracts were mixed up with 1000 μL of the Folin-Ciocalteu reagent and 800 μL (75 g/L) of sodium carbonate (Na2CO3). The mixture was incubated for 1 h, and the absorbance was measured at 765 nm against ethanol as a blank. All samples were analyzed in triplicate. Data obtained are the average of one analysis performed in triplicate. The results were expressed as mg gallic acid equivalent (GAE)/100 g of dry matter of the extract.


### 2.5. Total Flavonoid Content

Total flavonoids were measured according to the method described by Kim et al. with some modifications [28]. In brief, 200 μL of O. dillenii ethanolic extract, 1000 μL of distilled water and 50 μL of NaNO2 (5% W/V) were mixed up. After 6 min, 120 μL of AlCl3 (10% W/V) was added and incubated for 5 min, followed by the addition of 400 μL of NaOH (1 M) and 230 μL of distilled water. The calibration curve was constructed using quercetin standard solutions at different concentrations (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL). The absorbance was measured at 510 nm using ethanol as a blank. All measures were performed in triplicate. Data for the total phenol concentration were expressed as quercetin equivalents (QE) per milligram of dry matter of the extracted sample.


### 2.6. Quantification of Phenolic Compounds inO. dillenii Ethanolic Extracts by HPLC

The phenolic compounds in the ethanolic extracts were determined by HPLC using an Agilent 1200 (Agilent Technologies, Palo Alto, CA, USA) connected to a diode array UV detector (Bruker, Germany). Each extract (20μL) was injected into a Zorbax XDB-C18 (5 μm porosity, 250 × 4.6 mm; Agilent Technologies series 1100 system (Palo Alto, CA, USA)) column equipped with a 4 × 3 mm C18 cartridge precolumn (Agilent Technologies) with the following elution gradient: 0–25 min, 20% B; 25–30 min, 100% B and 30–35 min, 20% B. The mobile phases used for the elution of samples were A (water/0.5% phosphoric acid) and B (methanol), with a flow rate of 1 mL/min. Separation was performed at a constant temperature of 40°C. Spectrophotometric detection was performed at 254, 280, 320, 350, and 540 nm. Compounds were identified by comparison of their retention times and UV spectra with those of authentic standards. The quantification (Table 1) was performed using calibration curves with different concentrations of standards in ethanol (0.2; 0.4; 0.6; 0.8; 1 mg/mL). For each molecule, the limit of detection (LOD) and limit of quantification (LOQ) [29] were determined using the following formulas:(1)LOD=3.3∗standarddeviationofsignalintensityfromlow−concentrationsamplesslopeofthecalibrationcurve,(2)LOQ=10∗standarddeviationofsignalintensityfromlow−concentrationsamplesslopeofthecalibrationcurve.Table 1
Linearity of phenolic compounds standards and sensitivity of the HPLC-DAD method.
N°CompoundsConcentration range (mg/mL)Linear regressionraLODb (mg/mL)LOQc (mg/mL)1Gallic acid0.2–1y = 904.2x + 40.60.990.020.052Catechin0.2–1y = 2784.3x + 133.30.990.050.1634-Hydroxybenzoic acid0.2–1y = 3406.7x + 235.10.990.010.034Vanillic acid0.2–1y = 2040.9x + 124.80.990.030.095Caffeic acid0.2–1y = 24178x + 522.40.990.010.026Syringic acid0.2–1y = 14815x + 177.80.990.010.037Vanillin0.2–1y = 1622x + 91.90.990.020.078p-Coumaric acid0.2–1y = 4265.3x + 40.40.990.020.059Sinapic acid0.2–1y = 1052.4x + 34.20.990.030.0910Ferulic acid0.2–1y = 987.9x + 3.50.990.020.0611Naringin0.2–1y = 443.3x + 15.30.990.010.0312Rutin0.2–1y = 578.4x + 8.30.990.010.0413Salicylic acid0.2–1y = 815.1x + 12.20.990.030.0814Quercetin0.2–1y = 7989.6x + 116.20.990.020.0515Cinnamic acid0.2–1y = 828.1x − 18.10.990.050.1616Quercetin 3-O-β-D-glucoside0.2–1y = 1090.6x + 15.20.990.020.0717Kaempferol0.2–1y = 1000.9x + 12.50.990.030.0918Chalcone0.2–1y = 2666x + 5.10.990.010.04a, Correlation coefficients of the regression equation. b, LOD limit of detection. c, LOQ limit of quantification.Each calibration curve was repeated three times. Results were expressed as mg/100 g of dry matter. Analyses were performed in triplicate.


### 2.7. Antifungal Activity

The antifungal activity of the extracts was measured using liquid cell culture assays as described in the protocol by Bay et al. [30]. Four fungal species were used: Saccharomyces cerevisiae (BY4741), Candida albicans (SC5314), Candida krusei (ATCC6258), and Candida tropicalis (My070362). The growth rate of the yeast cells in the presence or absence of plant extracts was monitored by measuring the optical density at 600 nm (OD600) using a V-1200 spectrophotometer (Shanghai Mapada Instruments CO. LTD, Shanghai, China) as described by Bouchal et al. [31]. All experiments were repeated three times, with the residual extract of Fredolia aretioides used as a positive control (control +) according to Bouchal et al. [32].


### 2.8. Antibacterial Activity

Antibacterial activity was evaluated using a disc diffusion test as previously reported by Abrigach et al. [33]. Two Gram-positive bacteria species (Staphylococcus aureus and Listeria monocytogenes) and two Gram-negative bacteria species (Escherichia coli and Salmonella braenderup) were used in this study. Cells were first spread on Petri plates containing solid LB medium. Then, paper discs (6 mm in diameter) that had been impregnated with the tested compounds were placed on the inoculated agar plates and incubated at 37°C. All the extracts were used at a concentration of one milligram per disc. Twenty-four hours later, the antibacterial activity was assessed by measuring the diameter of the growth-inhibition zone in millimeters. The inhibition zones were measured three times for each extract, and the antibiotic streptomycin was used as a positive control.The minimum inhibitory concentration (MIC) was defined as the lowest concentration that inhibits bacterial growth following incubation for 24 h. The MIC was determined by visual observation of the red color indicator [34, 35]. All experiments were carried out in triplicate, and means were calculated.


### 2.9.In Vitro α-Amylase Inhibition Assay

The inhibition of pancreaticα-amylase activity by O. dillenii extracts was studied using the procedure described by Daoudi et al. with some modifications [36, 37]. The assay mixtures contained 200 μL of pancreatic α-amylase enzyme solution (13 IU), 200 μL of phosphate buffer (0.02 M; pH 6.9) and 200 μL of O. dillenii extracts (0.5 or 1 mg/mL) or acarbose (0.5 or 1 mg/mL). Acarbose is a specific inhibitor of intestinal alpha-glucosidase and pancreatic alpha-amylase enzymes [38]. The mixtures were pre-incubated at 37°C for 10 min. Then, 200 μL of starch (1% W/V) dissolved in phosphate buffer was added to each tube and incubated for 20 min at 37°C. To stop the enzymatic reaction, 600 μL of DNSA color reagent was added. Immediately afterwards, the tubes were incubated for 8 min at 100°C, then they were placed in an ice-cold water bath for a few minutes. The mixture was diluted by adding 1 mL of distilled water, and the absorbance was measured at 540 nm. The inhibition percentage was calculated using the following formula:(3)α−amylaseinhibitionpercentage=ODtest540nm−ODcontrol540nmODtest540nm×100.


### 2.10.In Vitro α-Glucosidase Inhibition Assay

The effects of theO. dillenii extracts on the intestinal α-glucosidase activity were quantified by monitoring the glucose released from sucrose degradation according to the protocol described by Ouassou et al. with some modifications [39, 40]. The assay mixtures contained 100 μL of sucrose (50 mM), 1000 μL of phosphate buffer (50 mM; pH 7.5) and 100 μL of intestinal α-glucosidase enzyme solution (10 IU). Then, 10 μL of distilled water (control), acarbose (positive control) or O. dillenii extract solutions at two different concentrations (166 or 328 μg/mL) were added to the mixture, which was incubated at 37°C in a water bath for 25 min. The mixture was then heated at 100C for 5 min to stop the enzymatic reaction, and the release of glucose was estimated by the glucose oxidase method using a commercial auto-kit (GLUCOSE, Biosystems). The absorbance was measured at 500 nm, and the inhibition percentage was calculated using the following formula:(4)α−glucosidaseinhibitionpercentage=ODcontrol500nm−ODtest500nmODcontrol500nm×100.


### 2.11. Molecular Docking Study

The structures of major compounds present inO. dillenii peel ethanolic extract, which were chosen as the representatives of different metabolites’ classes (phenolic acids, phenolic alcohols, flavonols, flavanols, flavanones, and chalcons), were downloaded from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The compounds were gallic acid (CID: 370), p-coumaric acid (CID: 637542), kaempferol (CID: 5280863), catechin (CID: 9064), naringin (CID: 442428), chalcone (CID: 637760). Acarbose (CID: 41774) was used as the standard drug. X-ray crystal structure of human pancreatic α-amylase (pdb code: 2qv4) [41] enzyme used in this study was retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/). The structure of the receptor was prepared before docking by eliminating unnecessary molecules (water molecules, inhibitors, cofactors). Missing polar hydrogen atoms were also added with the aid of BIOVIA Discovery Studio Visualizer 2020. PyRx 8.0 software [42] was utilized to perform the molecular simulations. The dimensions of the grid box were defined to enclose the active pocket of protein. The grid box was 30 × 30 × 30 grid points in size centered at 12.20 × 50.06 × 26.87. The binding affinity of compounds toward the receptor was evaluated based on Gibbs’s free energy values calculated by the docking process. Analysis and visualization of the results were carried out using the BIOVIA Discovery Studio Visualizer 2020.


## 2.1. Chemicals and Reagents

n-hexane, ethyl acetate, and ethanol were purchased from Merck (Darmstadt, Germany). All chemicals and reagents used for the determination of total phenolic compounds were of analytical grade and were purchased from Merck and Carl Roth GmbH (Karlsruhe, Germany). 3,5-Dinitrosalicylic acid (DNSA), acarbose,α-glucosidase, and α-amylase were purchased from Merck (Sigma-Aldrich, St. Louis, MA, USA). The kit necessary for the measurement of blood sugar was purchased from Biosystems (Barcelona, Spain). The bacterial strains were supplied by the Pasteur Institute of Casablanca, Morocco. The fungal strains were provided by Professor Adnane Sellam (Université Laval, Canada).Phenolic standards: gallic acid, vanillic acid, syringic acid, salicylic acid, 4-hydroxybenzoic acid, vanillin, ferulic acid, caffeic acid, p-coumaric acid, cinnamic acid, sinapic acid, quercetin, kaempferol, rutin, quercetin 3-O-β-D-glucoside, catechin, naringin, and chalcone were purchased from Merck and Carl Roth GmbH (Karlsruhe, Germany).


## 2.2. Sample Collection and Preparation

Mature purple fruits ofO. dillenii, the prickly pear plant, were collected in February 2017 in Morocco. The plant species was authenticated by Mohammed Fennan, a botanical expert from the Scientific Institute of Mohammed V University. The specimen was deposited at Mohammed First University, Oujda, Morocco, under the reference number HUMPOM 351 [2].The collected fruit specimens were washed with water to remove dust and spines, peeled and blended (Facilic glass LM310E; Groupe Seb, Mayerne, France), then the seeds were separated from the juice by passing the mixture through a sieve with a 2 mm mesh size (Figure1). The juice was dehydrated by heating at 40°C in an oven for 2 days. Seeds and peels were washed with distilled water, dried at 20°C for 1 week, weighed, and then reduced to a fine powder using a Moulinex coffee grinder (DPA241; Groupe Seb Lourdes, France). All components (seeds, juice, and peel) were stored at −20°C for a maximum of eight weeks.Figure 1
O. dillenii fruits (a), seeds (b), juice (c), and peel (d).
(a)(b)(c)(d)


## 2.3. Preparation ofOpuntia dillenii Fruit Extracts

One hundred grams of the ground seeds, juice, and peel ofO. dillenii fruits were macerated with 200 mL of n-hexane to remove fatty acids. The mixtures were stirred at 20°C for 24 h and filtered through a glass filter crucible (50 mL, Porosity 4; Isolab Wertheim, Germany). The extracts were then concentrated using a rotary evaporator (Laborota 4000; Heidolph Instruments, Schwabach, Germany) under reduced pressure at 40°C to obtain the corresponding powder. Then, ethyl acetate and ethanol were used successively to obtain the corresponding extracts by repeating the maceration process (Figure 2). The extracts were stored in dark bottles at 4°C until use.Figure 2
Maceration extraction process.


## 2.4. Total Polyphenol Content

The total phenol concentration in theO. dillenii ethanolic extracts was determined using the Folin-Ciocalteau method with some modifications. A standard curve was first plotted using gallic acid as a standard. To prepare the calibration curves and samples, 200 μL of ethanolic gallic acid solutions (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL) or 200 μL of O. dillenii ethanolic extracts were mixed up with 1000 μL of the Folin-Ciocalteu reagent and 800 μL (75 g/L) of sodium carbonate (Na2CO3). The mixture was incubated for 1 h, and the absorbance was measured at 765 nm against ethanol as a blank. All samples were analyzed in triplicate. Data obtained are the average of one analysis performed in triplicate. The results were expressed as mg gallic acid equivalent (GAE)/100 g of dry matter of the extract.


## 2.5. Total Flavonoid Content

Total flavonoids were measured according to the method described by Kim et al. with some modifications [28]. In brief, 200 μL of O. dillenii ethanolic extract, 1000 μL of distilled water and 50 μL of NaNO2 (5% W/V) were mixed up. After 6 min, 120 μL of AlCl3 (10% W/V) was added and incubated for 5 min, followed by the addition of 400 μL of NaOH (1 M) and 230 μL of distilled water. The calibration curve was constructed using quercetin standard solutions at different concentrations (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL). The absorbance was measured at 510 nm using ethanol as a blank. All measures were performed in triplicate. Data for the total phenol concentration were expressed as quercetin equivalents (QE) per milligram of dry matter of the extracted sample.


## 2.6. Quantification of Phenolic Compounds inO. dillenii Ethanolic Extracts by HPLC

The phenolic compounds in the ethanolic extracts were determined by HPLC using an Agilent 1200 (Agilent Technologies, Palo Alto, CA, USA) connected to a diode array UV detector (Bruker, Germany). Each extract (20μL) was injected into a Zorbax XDB-C18 (5 μm porosity, 250 × 4.6 mm; Agilent Technologies series 1100 system (Palo Alto, CA, USA)) column equipped with a 4 × 3 mm C18 cartridge precolumn (Agilent Technologies) with the following elution gradient: 0–25 min, 20% B; 25–30 min, 100% B and 30–35 min, 20% B. The mobile phases used for the elution of samples were A (water/0.5% phosphoric acid) and B (methanol), with a flow rate of 1 mL/min. Separation was performed at a constant temperature of 40°C. Spectrophotometric detection was performed at 254, 280, 320, 350, and 540 nm. Compounds were identified by comparison of their retention times and UV spectra with those of authentic standards. The quantification (Table 1) was performed using calibration curves with different concentrations of standards in ethanol (0.2; 0.4; 0.6; 0.8; 1 mg/mL). For each molecule, the limit of detection (LOD) and limit of quantification (LOQ) [29] were determined using the following formulas:(1)LOD=3.3∗standarddeviationofsignalintensityfromlow−concentrationsamplesslopeofthecalibrationcurve,(2)LOQ=10∗standarddeviationofsignalintensityfromlow−concentrationsamplesslopeofthecalibrationcurve.Table 1
Linearity of phenolic compounds standards and sensitivity of the HPLC-DAD method.
N°CompoundsConcentration range (mg/mL)Linear regressionraLODb (mg/mL)LOQc (mg/mL)1Gallic acid0.2–1y = 904.2x + 40.60.990.020.052Catechin0.2–1y = 2784.3x + 133.30.990.050.1634-Hydroxybenzoic acid0.2–1y = 3406.7x + 235.10.990.010.034Vanillic acid0.2–1y = 2040.9x + 124.80.990.030.095Caffeic acid0.2–1y = 24178x + 522.40.990.010.026Syringic acid0.2–1y = 14815x + 177.80.990.010.037Vanillin0.2–1y = 1622x + 91.90.990.020.078p-Coumaric acid0.2–1y = 4265.3x + 40.40.990.020.059Sinapic acid0.2–1y = 1052.4x + 34.20.990.030.0910Ferulic acid0.2–1y = 987.9x + 3.50.990.020.0611Naringin0.2–1y = 443.3x + 15.30.990.010.0312Rutin0.2–1y = 578.4x + 8.30.990.010.0413Salicylic acid0.2–1y = 815.1x + 12.20.990.030.0814Quercetin0.2–1y = 7989.6x + 116.20.990.020.0515Cinnamic acid0.2–1y = 828.1x − 18.10.990.050.1616Quercetin 3-O-β-D-glucoside0.2–1y = 1090.6x + 15.20.990.020.0717Kaempferol0.2–1y = 1000.9x + 12.50.990.030.0918Chalcone0.2–1y = 2666x + 5.10.990.010.04a, Correlation coefficients of the regression equation. b, LOD limit of detection. c, LOQ limit of quantification.Each calibration curve was repeated three times. Results were expressed as mg/100 g of dry matter. Analyses were performed in triplicate.


## 2.7. Antifungal Activity

The antifungal activity of the extracts was measured using liquid cell culture assays as described in the protocol by Bay et al. [30]. Four fungal species were used: Saccharomyces cerevisiae (BY4741), Candida albicans (SC5314), Candida krusei (ATCC6258), and Candida tropicalis (My070362). The growth rate of the yeast cells in the presence or absence of plant extracts was monitored by measuring the optical density at 600 nm (OD600) using a V-1200 spectrophotometer (Shanghai Mapada Instruments CO. LTD, Shanghai, China) as described by Bouchal et al. [31]. All experiments were repeated three times, with the residual extract of Fredolia aretioides used as a positive control (control +) according to Bouchal et al. [32].


## 2.8. Antibacterial Activity

Antibacterial activity was evaluated using a disc diffusion test as previously reported by Abrigach et al. [33]. Two Gram-positive bacteria species (Staphylococcus aureus and Listeria monocytogenes) and two Gram-negative bacteria species (Escherichia coli and Salmonella braenderup) were used in this study. Cells were first spread on Petri plates containing solid LB medium. Then, paper discs (6 mm in diameter) that had been impregnated with the tested compounds were placed on the inoculated agar plates and incubated at 37°C. All the extracts were used at a concentration of one milligram per disc. Twenty-four hours later, the antibacterial activity was assessed by measuring the diameter of the growth-inhibition zone in millimeters. The inhibition zones were measured three times for each extract, and the antibiotic streptomycin was used as a positive control.The minimum inhibitory concentration (MIC) was defined as the lowest concentration that inhibits bacterial growth following incubation for 24 h. The MIC was determined by visual observation of the red color indicator [34, 35]. All experiments were carried out in triplicate, and means were calculated.


## 2.9.In Vitro α-Amylase Inhibition Assay

The inhibition of pancreaticα-amylase activity by O. dillenii extracts was studied using the procedure described by Daoudi et al. with some modifications [36, 37]. The assay mixtures contained 200 μL of pancreatic α-amylase enzyme solution (13 IU), 200 μL of phosphate buffer (0.02 M; pH 6.9) and 200 μL of O. dillenii extracts (0.5 or 1 mg/mL) or acarbose (0.5 or 1 mg/mL). Acarbose is a specific inhibitor of intestinal alpha-glucosidase and pancreatic alpha-amylase enzymes [38]. The mixtures were pre-incubated at 37°C for 10 min. Then, 200 μL of starch (1% W/V) dissolved in phosphate buffer was added to each tube and incubated for 20 min at 37°C. To stop the enzymatic reaction, 600 μL of DNSA color reagent was added. Immediately afterwards, the tubes were incubated for 8 min at 100°C, then they were placed in an ice-cold water bath for a few minutes. The mixture was diluted by adding 1 mL of distilled water, and the absorbance was measured at 540 nm. The inhibition percentage was calculated using the following formula:(3)α−amylaseinhibitionpercentage=ODtest540nm−ODcontrol540nmODtest540nm×100.


## 2.10.In Vitro α-Glucosidase Inhibition Assay

The effects of theO. dillenii extracts on the intestinal α-glucosidase activity were quantified by monitoring the glucose released from sucrose degradation according to the protocol described by Ouassou et al. with some modifications [39, 40]. The assay mixtures contained 100 μL of sucrose (50 mM), 1000 μL of phosphate buffer (50 mM; pH 7.5) and 100 μL of intestinal α-glucosidase enzyme solution (10 IU). Then, 10 μL of distilled water (control), acarbose (positive control) or O. dillenii extract solutions at two different concentrations (166 or 328 μg/mL) were added to the mixture, which was incubated at 37°C in a water bath for 25 min. The mixture was then heated at 100C for 5 min to stop the enzymatic reaction, and the release of glucose was estimated by the glucose oxidase method using a commercial auto-kit (GLUCOSE, Biosystems). The absorbance was measured at 500 nm, and the inhibition percentage was calculated using the following formula:(4)α−glucosidaseinhibitionpercentage=ODcontrol500nm−ODtest500nmODcontrol500nm×100.


## 2.11. Molecular Docking Study

The structures of major compounds present inO. dillenii peel ethanolic extract, which were chosen as the representatives of different metabolites’ classes (phenolic acids, phenolic alcohols, flavonols, flavanols, flavanones, and chalcons), were downloaded from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The compounds were gallic acid (CID: 370), p-coumaric acid (CID: 637542), kaempferol (CID: 5280863), catechin (CID: 9064), naringin (CID: 442428), chalcone (CID: 637760). Acarbose (CID: 41774) was used as the standard drug. X-ray crystal structure of human pancreatic α-amylase (pdb code: 2qv4) [41] enzyme used in this study was retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/). The structure of the receptor was prepared before docking by eliminating unnecessary molecules (water molecules, inhibitors, cofactors). Missing polar hydrogen atoms were also added with the aid of BIOVIA Discovery Studio Visualizer 2020. PyRx 8.0 software [42] was utilized to perform the molecular simulations. The dimensions of the grid box were defined to enclose the active pocket of protein. The grid box was 30 × 30 × 30 grid points in size centered at 12.20 × 50.06 × 26.87. The binding affinity of compounds toward the receptor was evaluated based on Gibbs’s free energy values calculated by the docking process. Analysis and visualization of the results were carried out using the BIOVIA Discovery Studio Visualizer 2020.


## 3. Statistical Analysis

All data are presented as the mean ± SD. Multiple comparisons of mean values were set up using one‐way parametric analysis of variance (ANOVA). The degree of statistical significance of the data was considered with a probability of (p<0.05) using STATISTICA software (version 7.1).


## 4. Results

### 4.1. Yields of the Ethanolic Extracts

The yields of the n-hexane, ethyl acetate and ethanolic extracts ofO. dillenii seeds, juice and peel obtained by maceration in the present study are presented in Table 2. The highest yields were obtained with ethanol extracts (8.41 ± 5.19%). The n-hexane extract of seeds was more abundant in fatty acids than those that were from the juice and peel (8.41 ± 5.19%; 0.52 ± 0.14% and 1.33 ± 0.38), respectively. In addition, the lowest yields were obtained with ethyl acetate extracts (between 0.53 ± 0.30 and 1.07 ± 0.23%). Regarding the ethanolic extracts, the highest yields were obtained with the juice (20.30 ± 0.58%) in comparison with the other parts of the fruits. Therefore, the extraction yield depended on the polarity of the solvent.Table 2
Yields of the n-hexane, ethyl acetate and ethanolic extracts ofO. dillenii.
Yield (%)SolventsSeedsJuicePeelN-hexane8.41 ± 5.190.52 ± 0.141.33 ± 0.38Ethyl acetate1.07 ± 0.230.53 ± 0.301.00 ± 0.10Ethanol0.66 ± 0.1720.30 ± 0.5810.11 ± 0.29


### 4.2. Phytochemical Analysis of the Ethanolic Extract

#### 4.2.1. Total Polyphenol and Flavonoid Contents

According to the results obtained using theFolin-Ciocalteu method (Table 3), the total phenolic content of the ethanolic extracts obtained from O. dillenii harvested in Morocco ranged between 193.73 ± 81.44 and 341.12 ± 78.90 mg GAE/100 g DW. The seeds extract had the highest total phenolic content (341.12 ± 78.90 mg GAE/100 g DW) while the juice extract and the peel extract had the lowest phenolic contents (193.73 ± 81.44 and 240.91 ± 99.70 mg GAE/100 g DW, respectively). Concerning the flavonoid analysis, results showed low flavonoid content in the extracts, as the highest flavonoid concentration measured in the juice extract of O. dillenii fruits was 11.93 ± 11.72 mg QE/100 g DW. The lowest flavonoid concentration was measured in the peel and seed extracts (6.57 ± 1.32 and 6.63 ± 2.50 mg QE/100 g).Table 3
Phytochemical analysis of ethanolic extracts of seeds, juice and peel ofO. dillenii fruits.
SamplesTotal polyphenol content (mg GAE/100 g DW)Total flavonoid content (mg QE/100 g DW)Seeds341.12 ± 78.906.63 ± 2.50Juice193.73 ± 81.4411.93 ± 11.72Peel240.91 ± 99.706.57 ± 1.32


#### 4.2.2. HPLC Analysis ofO. dillenii Ethanolic Extracts

The chemical composition ofO. dillenii fruit ethanolic extracts (Table 4 and Figure 3) was determined by HPLC-DAD. Four families of phenolic compounds were detected in the extracts (Table 5). Phenolic acid derivatives were the most abundant molecules (38.50 ± 14.17–60.07 ± 8.74%), followed by flavonoids (28.03 ± 4.96–44.33 ± 10.51%), phenolic alcohols (5.77 ± 0.65–24.55 ± 7.70%) and chalcones (1.18 ± 0.63–3.25 ± 1.01%). Gallic acid, vanillic acid, and syringic acid were the major constituents detected in the extracts. Results also showed that the concentrations of different phenolic compounds depended on the extracted part of the O. dillenii fruit. In all extracts, the juice extract presented the highest gallic acid concentration (447.63 ± 292.59 mg/100 g) followed by the peel extract (183.04 ± 215.78 mg/100 g), while the gallic acid concentration found in seeds extract was the poorest (4.95 ± 1.87 mg/100 g). The seeds presented the highest amount of syringic acid (252.74 ± 139.85 mg/100 g) followed by the peel (101.88 ± 73.78 mg/100 g). Salicylic acid was more abundant in the peel, with a level of 117.53 ± 73.88 mg/100 g. Finally, the flavonoid profile, quercetin 3-O-β-D-glucoside was highly dominant in seeds extract (199.66 ± 212.15 mg/100 g), followed by peel extract (153.15 ± 8.09 mg/100 g). Kaempferol was also abundant in almost all the extracts, but the peel presented the highest amount (166.68 ± 54.85 mg/100 g). On the other hand, chalcone was present at low concentrations.Table 4
HPLC-DAD analysis of the four families of phenolic compounds; phenolic acids, phenolic alcohols, flavonoids and chalcons; which were detected in seeds, juice and peel of ethanolic extracts ofO. dillenii fruits harvested in Morocco.
Sample (mg/100 g)Retention time (Tr) (min)SeedsJuicePeelPhenolic acidsGallic acid2.404.95 ± 1.87447.63 ± 292.59183.04 ± 215.78Vanillic acid6.91139.87 ± 80.1093.80 ± 35.4368.94 ± 15.24Syringic acid6.72252.74 ± 139.8563.07 ± 44.13101.88 ± 73.78Salicylic acid11.3029.79 ± 23.658.88 ± 2.26117.53 ± 73.884-Hydroxybenzoic acid5.8940.54 ± 20.7459.43 ± 42.2426.46 ± 29.13Vanillin7.1125.99 ± 17.788.54 ± 2.3111.52 ± 6.13Phenolic alcoholsFerulic acid8.462.76 ± 2.6013.40 ± 6.3118.55 ± 3.45Caffeic acid6.4015.14 ± 9.4134.63 ± 50.4971.13 ± 8.44p-Coumaric acid8.157.70 ± 11.9813.10 ± 10.42109.22 ± 78.73Cinnamic acid12.4026.03 ± 12.5910.64 ± 1.2026.05 ± 14.88Sinapic acid8.4111.96 ± 7.0950.60 ± 23.6462.59 ± 4.61FlavonolsQuercetin12.2138.95 ± 20.6316.27 ± 9.5720.78 ± 4.85Kaempferol13.72104.78 ± 94.6494.90 ± 21.33166.68 ± 54.85Rutin9.64100.96 ± 54.9218.10 ± 9.2631.28 ± 3.39Quercetin 3-O-β-D-glucoside9.60199.66 ± 212.1557.80 ± 21.55153.15 ± 8.09FlavanolsCatechin4.0715.18 ± 14.42113.67 ± 103.4232.27 ± 18.07FlavanonesNaringin9.3928.42 ± 16.1920.69 ± 8.2613.28 ± 7.49ChalconesChalcone17.3935.30 ± 16.7010.70 ± 1.1018.73 ± 6.95Figure 3
Example of a HPLC-DAD chromatogram obtained following analysis ofO. dillenii ethanolic extract at 280 nm and of standards (Std; gallic acid 1, catechin 2, 4-hydroxybenzoic acid 3, vanilic acid 4, caffeic acid 5, syringic acid 6, vanillin 7, p-coumaric acid 8, sinapic acid 9, ferulic acid 10, naringinin 11, rutin 12, salicylic acid 13, quercetin 14, cinnamic acid 15, quercetin 3-O-β-D-glucoside 16, kaempferol 17, trans-chalcon 18).Table 5
Families of phenolic compounds in ethanolic extracts ofO. dillenii seeds, juice and peel. Values are means of three determinations ± standard errors of the mean (SEM).
Families of phenolic compoundsSeedsJuicePeelPhenolic acids (%)46.64 ± 11.2660.07 ± 8.7438.50 ± 14.17Phenolic alcohols (%)5.77 ± 0.6510.74 ± 3.6824.55 ± 7.70Flavonoids (%)44.33 ± 10.5128.03 ± 4.9635.39 ± 6.44Chalcones (%)3.25 ± 1.011.18 ± 0.631.54 ± 0.36


## 4.1. Yields of the Ethanolic Extracts

The yields of the n-hexane, ethyl acetate and ethanolic extracts ofO. dillenii seeds, juice and peel obtained by maceration in the present study are presented in Table 2. The highest yields were obtained with ethanol extracts (8.41 ± 5.19%). The n-hexane extract of seeds was more abundant in fatty acids than those that were from the juice and peel (8.41 ± 5.19%; 0.52 ± 0.14% and 1.33 ± 0.38), respectively. In addition, the lowest yields were obtained with ethyl acetate extracts (between 0.53 ± 0.30 and 1.07 ± 0.23%). Regarding the ethanolic extracts, the highest yields were obtained with the juice (20.30 ± 0.58%) in comparison with the other parts of the fruits. Therefore, the extraction yield depended on the polarity of the solvent.Table 2
Yields of the n-hexane, ethyl acetate and ethanolic extracts ofO. dillenii.
Yield (%)SolventsSeedsJuicePeelN-hexane8.41 ± 5.190.52 ± 0.141.33 ± 0.38Ethyl acetate1.07 ± 0.230.53 ± 0.301.00 ± 0.10Ethanol0.66 ± 0.1720.30 ± 0.5810.11 ± 0.29


## 4.2. Phytochemical Analysis of the Ethanolic Extract

### 4.2.1. Total Polyphenol and Flavonoid Contents

According to the results obtained using theFolin-Ciocalteu method (Table 3), the total phenolic content of the ethanolic extracts obtained from O. dillenii harvested in Morocco ranged between 193.73 ± 81.44 and 341.12 ± 78.90 mg GAE/100 g DW. The seeds extract had the highest total phenolic content (341.12 ± 78.90 mg GAE/100 g DW) while the juice extract and the peel extract had the lowest phenolic contents (193.73 ± 81.44 and 240.91 ± 99.70 mg GAE/100 g DW, respectively). Concerning the flavonoid analysis, results showed low flavonoid content in the extracts, as the highest flavonoid concentration measured in the juice extract of O. dillenii fruits was 11.93 ± 11.72 mg QE/100 g DW. The lowest flavonoid concentration was measured in the peel and seed extracts (6.57 ± 1.32 and 6.63 ± 2.50 mg QE/100 g).Table 3
Phytochemical analysis of ethanolic extracts of seeds, juice and peel ofO. dillenii fruits.
SamplesTotal polyphenol content (mg GAE/100 g DW)Total flavonoid content (mg QE/100 g DW)Seeds341.12 ± 78.906.63 ± 2.50Juice193.73 ± 81.4411.93 ± 11.72Peel240.91 ± 99.706.57 ± 1.32


### 4.2.2. HPLC Analysis ofO. dillenii Ethanolic Extracts

The chemical composition ofO. dillenii fruit ethanolic extracts (Table 4 and Figure 3) was determined by HPLC-DAD. Four families of phenolic compounds were detected in the extracts (Table 5). Phenolic acid derivatives were the most abundant molecules (38.50 ± 14.17–60.07 ± 8.74%), followed by flavonoids (28.03 ± 4.96–44.33 ± 10.51%), phenolic alcohols (5.77 ± 0.65–24.55 ± 7.70%) and chalcones (1.18 ± 0.63–3.25 ± 1.01%). Gallic acid, vanillic acid, and syringic acid were the major constituents detected in the extracts. Results also showed that the concentrations of different phenolic compounds depended on the extracted part of the O. dillenii fruit. In all extracts, the juice extract presented the highest gallic acid concentration (447.63 ± 292.59 mg/100 g) followed by the peel extract (183.04 ± 215.78 mg/100 g), while the gallic acid concentration found in seeds extract was the poorest (4.95 ± 1.87 mg/100 g). The seeds presented the highest amount of syringic acid (252.74 ± 139.85 mg/100 g) followed by the peel (101.88 ± 73.78 mg/100 g). Salicylic acid was more abundant in the peel, with a level of 117.53 ± 73.88 mg/100 g. Finally, the flavonoid profile, quercetin 3-O-β-D-glucoside was highly dominant in seeds extract (199.66 ± 212.15 mg/100 g), followed by peel extract (153.15 ± 8.09 mg/100 g). Kaempferol was also abundant in almost all the extracts, but the peel presented the highest amount (166.68 ± 54.85 mg/100 g). On the other hand, chalcone was present at low concentrations.Table 4
HPLC-DAD analysis of the four families of phenolic compounds; phenolic acids, phenolic alcohols, flavonoids and chalcons; which were detected in seeds, juice and peel of ethanolic extracts ofO. dillenii fruits harvested in Morocco.
Sample (mg/100 g)Retention time (Tr) (min)SeedsJuicePeelPhenolic acidsGallic acid2.404.95 ± 1.87447.63 ± 292.59183.04 ± 215.78Vanillic acid6.91139.87 ± 80.1093.80 ± 35.4368.94 ± 15.24Syringic acid6.72252.74 ± 139.8563.07 ± 44.13101.88 ± 73.78Salicylic acid11.3029.79 ± 23.658.88 ± 2.26117.53 ± 73.884-Hydroxybenzoic acid5.8940.54 ± 20.7459.43 ± 42.2426.46 ± 29.13Vanillin7.1125.99 ± 17.788.54 ± 2.3111.52 ± 6.13Phenolic alcoholsFerulic acid8.462.76 ± 2.6013.40 ± 6.3118.55 ± 3.45Caffeic acid6.4015.14 ± 9.4134.63 ± 50.4971.13 ± 8.44p-Coumaric acid8.157.70 ± 11.9813.10 ± 10.42109.22 ± 78.73Cinnamic acid12.4026.03 ± 12.5910.64 ± 1.2026.05 ± 14.88Sinapic acid8.4111.96 ± 7.0950.60 ± 23.6462.59 ± 4.61FlavonolsQuercetin12.2138.95 ± 20.6316.27 ± 9.5720.78 ± 4.85Kaempferol13.72104.78 ± 94.6494.90 ± 21.33166.68 ± 54.85Rutin9.64100.96 ± 54.9218.10 ± 9.2631.28 ± 3.39Quercetin 3-O-β-D-glucoside9.60199.66 ± 212.1557.80 ± 21.55153.15 ± 8.09FlavanolsCatechin4.0715.18 ± 14.42113.67 ± 103.4232.27 ± 18.07FlavanonesNaringin9.3928.42 ± 16.1920.69 ± 8.2613.28 ± 7.49ChalconesChalcone17.3935.30 ± 16.7010.70 ± 1.1018.73 ± 6.95Figure 3
Example of a HPLC-DAD chromatogram obtained following analysis ofO. dillenii ethanolic extract at 280 nm and of standards (Std; gallic acid 1, catechin 2, 4-hydroxybenzoic acid 3, vanilic acid 4, caffeic acid 5, syringic acid 6, vanillin 7, p-coumaric acid 8, sinapic acid 9, ferulic acid 10, naringinin 11, rutin 12, salicylic acid 13, quercetin 14, cinnamic acid 15, quercetin 3-O-β-D-glucoside 16, kaempferol 17, trans-chalcon 18).Table 5
Families of phenolic compounds in ethanolic extracts ofO. dillenii seeds, juice and peel. Values are means of three determinations ± standard errors of the mean (SEM).
Families of phenolic compoundsSeedsJuicePeelPhenolic acids (%)46.64 ± 11.2660.07 ± 8.7438.50 ± 14.17Phenolic alcohols (%)5.77 ± 0.6510.74 ± 3.6824.55 ± 7.70Flavonoids (%)44.33 ± 10.5128.03 ± 4.9635.39 ± 6.44Chalcones (%)3.25 ± 1.011.18 ± 0.631.54 ± 0.36


## 4.2.1. Total Polyphenol and Flavonoid Contents

According to the results obtained using theFolin-Ciocalteu method (Table 3), the total phenolic content of the ethanolic extracts obtained from O. dillenii harvested in Morocco ranged between 193.73 ± 81.44 and 341.12 ± 78.90 mg GAE/100 g DW. The seeds extract had the highest total phenolic content (341.12 ± 78.90 mg GAE/100 g DW) while the juice extract and the peel extract had the lowest phenolic contents (193.73 ± 81.44 and 240.91 ± 99.70 mg GAE/100 g DW, respectively). Concerning the flavonoid analysis, results showed low flavonoid content in the extracts, as the highest flavonoid concentration measured in the juice extract of O. dillenii fruits was 11.93 ± 11.72 mg QE/100 g DW. The lowest flavonoid concentration was measured in the peel and seed extracts (6.57 ± 1.32 and 6.63 ± 2.50 mg QE/100 g).Table 3
Phytochemical analysis of ethanolic extracts of seeds, juice and peel ofO. dillenii fruits.
SamplesTotal polyphenol content (mg GAE/100 g DW)Total flavonoid content (mg QE/100 g DW)Seeds341.12 ± 78.906.63 ± 2.50Juice193.73 ± 81.4411.93 ± 11.72Peel240.91 ± 99.706.57 ± 1.32


## 4.2.2. HPLC Analysis ofO. dillenii Ethanolic Extracts

The chemical composition ofO. dillenii fruit ethanolic extracts (Table 4 and Figure 3) was determined by HPLC-DAD. Four families of phenolic compounds were detected in the extracts (Table 5). Phenolic acid derivatives were the most abundant molecules (38.50 ± 14.17–60.07 ± 8.74%), followed by flavonoids (28.03 ± 4.96–44.33 ± 10.51%), phenolic alcohols (5.77 ± 0.65–24.55 ± 7.70%) and chalcones (1.18 ± 0.63–3.25 ± 1.01%). Gallic acid, vanillic acid, and syringic acid were the major constituents detected in the extracts. Results also showed that the concentrations of different phenolic compounds depended on the extracted part of the O. dillenii fruit. In all extracts, the juice extract presented the highest gallic acid concentration (447.63 ± 292.59 mg/100 g) followed by the peel extract (183.04 ± 215.78 mg/100 g), while the gallic acid concentration found in seeds extract was the poorest (4.95 ± 1.87 mg/100 g). The seeds presented the highest amount of syringic acid (252.74 ± 139.85 mg/100 g) followed by the peel (101.88 ± 73.78 mg/100 g). Salicylic acid was more abundant in the peel, with a level of 117.53 ± 73.88 mg/100 g. Finally, the flavonoid profile, quercetin 3-O-β-D-glucoside was highly dominant in seeds extract (199.66 ± 212.15 mg/100 g), followed by peel extract (153.15 ± 8.09 mg/100 g). Kaempferol was also abundant in almost all the extracts, but the peel presented the highest amount (166.68 ± 54.85 mg/100 g). On the other hand, chalcone was present at low concentrations.Table 4
HPLC-DAD analysis of the four families of phenolic compounds; phenolic acids, phenolic alcohols, flavonoids and chalcons; which were detected in seeds, juice and peel of ethanolic extracts ofO. dillenii fruits harvested in Morocco.
Sample (mg/100 g)Retention time (Tr) (min)SeedsJuicePeelPhenolic acidsGallic acid2.404.95 ± 1.87447.63 ± 292.59183.04 ± 215.78Vanillic acid6.91139.87 ± 80.1093.80 ± 35.4368.94 ± 15.24Syringic acid6.72252.74 ± 139.8563.07 ± 44.13101.88 ± 73.78Salicylic acid11.3029.79 ± 23.658.88 ± 2.26117.53 ± 73.884-Hydroxybenzoic acid5.8940.54 ± 20.7459.43 ± 42.2426.46 ± 29.13Vanillin7.1125.99 ± 17.788.54 ± 2.3111.52 ± 6.13Phenolic alcoholsFerulic acid8.462.76 ± 2.6013.40 ± 6.3118.55 ± 3.45Caffeic acid6.4015.14 ± 9.4134.63 ± 50.4971.13 ± 8.44p-Coumaric acid8.157.70 ± 11.9813.10 ± 10.42109.22 ± 78.73Cinnamic acid12.4026.03 ± 12.5910.64 ± 1.2026.05 ± 14.88Sinapic acid8.4111.96 ± 7.0950.60 ± 23.6462.59 ± 4.61FlavonolsQuercetin12.2138.95 ± 20.6316.27 ± 9.5720.78 ± 4.85Kaempferol13.72104.78 ± 94.6494.90 ± 21.33166.68 ± 54.85Rutin9.64100.96 ± 54.9218.10 ± 9.2631.28 ± 3.39Quercetin 3-O-β-D-glucoside9.60199.66 ± 212.1557.80 ± 21.55153.15 ± 8.09FlavanolsCatechin4.0715.18 ± 14.42113.67 ± 103.4232.27 ± 18.07FlavanonesNaringin9.3928.42 ± 16.1920.69 ± 8.2613.28 ± 7.49ChalconesChalcone17.3935.30 ± 16.7010.70 ± 1.1018.73 ± 6.95Figure 3
Example of a HPLC-DAD chromatogram obtained following analysis ofO. dillenii ethanolic extract at 280 nm and of standards (Std; gallic acid 1, catechin 2, 4-hydroxybenzoic acid 3, vanilic acid 4, caffeic acid 5, syringic acid 6, vanillin 7, p-coumaric acid 8, sinapic acid 9, ferulic acid 10, naringinin 11, rutin 12, salicylic acid 13, quercetin 14, cinnamic acid 15, quercetin 3-O-β-D-glucoside 16, kaempferol 17, trans-chalcon 18).Table 5
Families of phenolic compounds in ethanolic extracts ofO. dillenii seeds, juice and peel. Values are means of three determinations ± standard errors of the mean (SEM).
Families of phenolic compoundsSeedsJuicePeelPhenolic acids (%)46.64 ± 11.2660.07 ± 8.7438.50 ± 14.17Phenolic alcohols (%)5.77 ± 0.6510.74 ± 3.6824.55 ± 7.70Flavonoids (%)44.33 ± 10.5128.03 ± 4.9635.39 ± 6.44Chalcones (%)3.25 ± 1.011.18 ± 0.631.54 ± 0.36


## 5. Antifungal and Antibacterial Activities ofO. dillenii Ethanolic Extracts

A previous study already showed thatO. dillenii fruit extracts have no antifungal activity against Candida albicans, which is the most common human fungal pathogen [17]. In the present work, we evaluated the antifungal effects of O. dillenii ethanolic fruit extracts on other Candida species involved in invasive fungal infections. These extracts were also evaluated for their antifungal activity against the budding yeast Saccharomyces cerevisiae, which is an excellent model system for studying antifungal compounds. As shown in Figure 4, all ethanolic extracts (peel, seeds, and juice) of O. dillenii harvested from Morocco showed no antifungal activity against any of the fungi species when used at 1 mg/mL.Figure 4
Antifungal activity ofO. dillenii fruit ethanolic extracts (peel, seeds and juice). Cells were cultured in the presence or absence of 1 mg/mL of each extract and the growth rate of cells was monitored every 2 h by measuring the OD600, as described in the materials and methods section. The residual extract of Fredolia aretioides was used as a positive control due to its recognized antifungal activity [32] and DMSO was used as a negative control. All experiments were carried out in triplicate. Error bars are shown; when they are not visible, they are shown smaller than the data symbols.The antibacterial activity ofO. dillenii ethanolic fruit extracts was evaluated against four different bacterial strains, as described in the Materials and Methods section. As shown in Table 6, the ethanolic extracts from O. dillenii peel or seeds showed no activity against any of the tested strains. Similarly, the ethanolic juice extracts of O. dillenii showed no activity against Staphylococcus aureus. Interestingly, ethanolic extracts from the juice of O. dillenii showed weak to moderate antibacterial activities against the Gram-positive bacteria Listeria monocytogenes and the two Gram-negative bacteria species Escherichia coli and Salmonella braenderup. The MIC value of this extract against these bacterial strains was higher than 2.5 mg/mL.Table 6
Antibacterial activity of ethanolic extracts ofO. dillenii on Listeria monocytogenes (a), Staphylococcus aureus (b), Salmonella braenderup, (c) and Escherichia coli (d).
Extracts/AntibioticInhibition area (mm)aEscherichia coliSalmonella braenderupStaphylococcus aureusListeria monocytogenesSeedsndndndndPeelndndndndJuice7.33 mm7.33 mmnd8.66 mmStreptomycin20 mm18 mm14 mm11 mma, Mean of three repetitions; nd, not detected.


## 6. Pancreaticα-Amylase and Intestinal α-Glucosidase Inhibitory Activities

The effects of theO. dillenii peel, seeds, and juice ethanolic extracts on the in vitro inhibition of pancreatic α-amylase and intestinal α-glucosidase activities are presented in Figure 5. Acarbose was used as a positive control. All the extracts had a significant inhibitory effect on the pancreatic α-amylase activity and this effect was dose-dependent. In general, the peel extracts of O. dillenii showed the greatest effect, followed by the juice and seed extracts. Concerning the in vitro intestinal α-glucosidase inhibition assay, results showed that the peel of O. dillenii also had the greatest inhibitory effect, followed by the juice and seeds.Figure 5
Inhibitory activity ofO. dillenii peel, seed and juice ethanolic extracts against pancreatic α-amylase (a) at two different concentrations (0.5 and 1 mg/mL) and intestinal α-glucosidase (b) at two different concentrations (166 and 332 μg/mL). ∗∗∗p value <0.001 as compared to the control. Values are means of three determinations.
(a)(b)


## 7. Molecular Modeling Studies

To rationalize the antidiabetic activity results obtained and to investigate the putative mechanisms of action of the most abundant bioactive compounds present inO. dillenii peel ethanolic extracts, molecular docking studies against human pancreatic α-amylase enzyme were carried out. This method is considered as one of the most popular and powerful theoretical tools utilized to provide an atomistic insight of the binding mode of a molecule into the active site of a given target protein/receptor [43, 44]. This knowledge plays a crucial role in the determination and understanding of the bioactivity efficiency of the molecule for use as a therapeutic agent [45]. The method is based on the calculation of the free-binding energy of the molecule-receptor complex. The complex with the lowest binding energy indicates the most putative active inhibitor.


### 7.1. Validation of Docking Protocol

The reliability of the docking protocol was checked by docking validation experiments. The native ligand (cocrystallized ligand) was redocked to the catalytic active site of the crystal structure ofα-amylase. The validation results showed that cocrystallized and redocked ligands were almost superimposed and had similar conformational poses in the binding pocket of α-amylase protein with a RMSD value of 1.172 Å, which is lower than 2.0 Å (the maximum allowable RMSD value). These findings clearly indicate the validity of the docking method used in this work. The validation results are displayed in Figure 6.Figure 6
Validation docking test results. Cocrystalized ligand (native ligand) is in green color, redocked ligand is in red color.


### 7.2. Docking Analysis for the Inhibitory Mechanism againstα-Amylase

The docking results of the selected phytochemicals towardα-amylase enzyme were assembled in Table 7. The results showed that naringin, catechin, and kaempferol compounds had high affinities towards the active pocked of α-amylase and even better than the reference standard drug acarbose used in this study. The binding energy values were estimated to be −10.0 kcal/mol for naringin, −8.8 kcal/mol for catechin and −8.7 kcal/mol for kaempferol, while acarbose showed a binding energy of just of −8.2 kcal/mol. Chalcone was found to exhibit a good binding score equal to −7.8 kcal/mol, while gallic acid and p-coumaric acid showed the same moderate score of −6.4 kcal/mol.Table 7
Docking results against human pancreaticα-amylase enzyme.
Compoundα-amylase (2qv4)Binding affinity (kcal/mol)Involved residuesType of interactionsGallic acid−6.4Asp300, Glu233, Asp197, Tyr62, His299, His101, Arg195, Trp58, Leu165, Ala198HB, Pi-anion, Pi-Pi stacked, VDWp-Coumaric acid−6.4Asp197, Trp59, Gln63, Arg195, Tyr62, Asp300, Trp58, His299, Leu165, Glu60HB, Pi-Pi stacked, UDD, VDWKaempferol−8.7Asp300, His305, Trp59, Trp58, Glu233, Asp197, Tyr62, His299, His101, Arg195HB, Pi-anion, Pi-Pi stacked, VDWCatechin−8.8Asp300, Asp197, Gln63, Tyr62, Trp59, Glu233, His299, His101, Arg195, Trp58, Leu165, Ala198, His305HB, Pi-anion, Pi-Pi stacked, VDWNaringin−10.0Asp300, Glu233, His299, Arg195, Asp197, Tyr62, Trp59, His305, Lys200, Ala198, Ala307, Leu162, Trp58, Ser199, Val234, His201, Ile235, Tyr151HB, Pi-anion, Pi-Pi stacked, UAA, UDD, CHB, VDWChalcone−7.8Gln63, Trp59, Tyr62, Asp197, His305, Leu165, Trp58HB, Pi-Pi stacked VDWAcarbose−8.2Asp300, Asp353, Asp356, Glu233, Arg195, His101, His305, Tyr62, Asp197, His299, Ile235, Ala198, Leu162, Leu165, Trp58, Trp59, Trp357, Val354HB, UAA, CHB, VDWHB, Hydrogen bond; VDW, Van der waals forces; CHB, carbon hydrogen bond; UAA, unfavorable acceptor-Acceptor; UDD, unfavorable donor-donor.Further analysis of the interaction modes of the selected compounds was performed to explain the inhibition mechanism. These indicated that mainly hydrogen, hydrophobic, and electrostatic interactions were responsible for the binding of all molecules with the active siteα-amylase amino acid residues. As depicted in Figure 7, naringin, which exhibited the lowest binding energy and hence the most putative active inhibitor, interacted with α-amylase by several interactions with surrounding residues. It constructed conventional hydrogen bonds with four amino acids, namely Asp300, Glu233, His299, and Arg195; one salt bridge with Asp197,;one carbon hydrogen bond with Ala198 residue; two unfavorable donor-donor interactions with His299 and His305; and one unfavorable acceptor-acceptor contact with Lys200. This compound also formed two hydrophobic interactions as Pi-Pi stacked with Tyr62 and Trp59, and eight Van der Waals (VDW) contacts with Ala307, Leu162, Trp58, Ser199, Val234, His201, Ile235, and Tyr151 amino acids. Catechin, a flavanol compound in O. dillenii plants, was found to build three hydrogen bonds involving Asp300, Asp197 and Gln63 residues, in addition to numerous hydrophobic and electrostatic contacts: two Pi-Pi stacked (with Tyr62 and Trp59), a Pi-Anion (with Asp197) and eight VDW (Glu233, His299, His101, Arg195, Trp58, Leu165, Ala198 and His305) interactions. The complex kaempferol—α-amylase was stabilized by two hydrogen bonds, three hydrophobic interactions and six electrostatic contacts. His305 and Asp300 were involved in the formation of the hydrogen bonds. This latter also formed a Pi-Anion contact. Trp59 built two Pi-Pi stacked interactions with kaempferol. Moreover, Trp58, Glu233, Asp197, Tyr62, His299, His101, and Arg195 residues were stabilizing the complex by VDW contacts. As reported in Figure 4, eight conventional hydrogen bonds were observed between acarbose and binding site amino acids Asp300, Asp353, Asp356, Glu233, Arg195, His101, His305 and Tyr62. Meanwhile, Glu233 and Asp300 residues also formed a carbon hydrogen bond and unfavorable acceptor-acceptor contact with acarbose, respectively. Furthermore, ten electrostatic interactions, as VDW forces, were involved in the stabilization of acarbose- α-amylase complex. The residues were Asp197, His299, Ile235, Ala198, Leu162, Leu165, Trp58, Trp59, Trp357, and Val354. Surprisingly, chalcone, which exhibited a good binding energy, was found to have the smallest number of interactions with the receptor of all the compounds. These interactions included one hydrogen bond, two Pi-Pi stacked and four VDW interactions with the side chain amino acids Gln63, Trp59, Tyr62, Asp197, His305, Leu165, and Trp58, respectively. Gallic acid interacted with the residues Asp300, Glu233, Asp197, Tyr62, His299, His101, Arg195, Trp58, Leu165 and Ala198 via hydrogen bonds (two bonds), Pi-Anion (one interaction), Pi-Pi stacked (one interaction) and VDW (six interactions). In addition, p-coumaric acid was found to be surrounded by Asp197, Trp59, and Gln63 (via hydrogen bonds), Arg195 (unfavorable donor-donor interaction), Tyr62 (via Pi-Pi stacked interaction), and Asp300, Trp58, His299, Leu165, and Glu60 (via VDW forces) residues.Figure 7
2D-representations of the chemical bonding modes of the complexes formed between naringin, catechin, kaempferol, and acarbose compounds and the catalytic site residues of human pancreaticα-amylase.


## 7.1. Validation of Docking Protocol

The reliability of the docking protocol was checked by docking validation experiments. The native ligand (cocrystallized ligand) was redocked to the catalytic active site of the crystal structure ofα-amylase. The validation results showed that cocrystallized and redocked ligands were almost superimposed and had similar conformational poses in the binding pocket of α-amylase protein with a RMSD value of 1.172 Å, which is lower than 2.0 Å (the maximum allowable RMSD value). These findings clearly indicate the validity of the docking method used in this work. The validation results are displayed in Figure 6.Figure 6
Validation docking test results. Cocrystalized ligand (native ligand) is in green color, redocked ligand is in red color.


## 7.2. Docking Analysis for the Inhibitory Mechanism againstα-Amylase

The docking results of the selected phytochemicals towardα-amylase enzyme were assembled in Table 7. The results showed that naringin, catechin, and kaempferol compounds had high affinities towards the active pocked of α-amylase and even better than the reference standard drug acarbose used in this study. The binding energy values were estimated to be −10.0 kcal/mol for naringin, −8.8 kcal/mol for catechin and −8.7 kcal/mol for kaempferol, while acarbose showed a binding energy of just of −8.2 kcal/mol. Chalcone was found to exhibit a good binding score equal to −7.8 kcal/mol, while gallic acid and p-coumaric acid showed the same moderate score of −6.4 kcal/mol.Table 7
Docking results against human pancreaticα-amylase enzyme.
Compoundα-amylase (2qv4)Binding affinity (kcal/mol)Involved residuesType of interactionsGallic acid−6.4Asp300, Glu233, Asp197, Tyr62, His299, His101, Arg195, Trp58, Leu165, Ala198HB, Pi-anion, Pi-Pi stacked, VDWp-Coumaric acid−6.4Asp197, Trp59, Gln63, Arg195, Tyr62, Asp300, Trp58, His299, Leu165, Glu60HB, Pi-Pi stacked, UDD, VDWKaempferol−8.7Asp300, His305, Trp59, Trp58, Glu233, Asp197, Tyr62, His299, His101, Arg195HB, Pi-anion, Pi-Pi stacked, VDWCatechin−8.8Asp300, Asp197, Gln63, Tyr62, Trp59, Glu233, His299, His101, Arg195, Trp58, Leu165, Ala198, His305HB, Pi-anion, Pi-Pi stacked, VDWNaringin−10.0Asp300, Glu233, His299, Arg195, Asp197, Tyr62, Trp59, His305, Lys200, Ala198, Ala307, Leu162, Trp58, Ser199, Val234, His201, Ile235, Tyr151HB, Pi-anion, Pi-Pi stacked, UAA, UDD, CHB, VDWChalcone−7.8Gln63, Trp59, Tyr62, Asp197, His305, Leu165, Trp58HB, Pi-Pi stacked VDWAcarbose−8.2Asp300, Asp353, Asp356, Glu233, Arg195, His101, His305, Tyr62, Asp197, His299, Ile235, Ala198, Leu162, Leu165, Trp58, Trp59, Trp357, Val354HB, UAA, CHB, VDWHB, Hydrogen bond; VDW, Van der waals forces; CHB, carbon hydrogen bond; UAA, unfavorable acceptor-Acceptor; UDD, unfavorable donor-donor.Further analysis of the interaction modes of the selected compounds was performed to explain the inhibition mechanism. These indicated that mainly hydrogen, hydrophobic, and electrostatic interactions were responsible for the binding of all molecules with the active siteα-amylase amino acid residues. As depicted in Figure 7, naringin, which exhibited the lowest binding energy and hence the most putative active inhibitor, interacted with α-amylase by several interactions with surrounding residues. It constructed conventional hydrogen bonds with four amino acids, namely Asp300, Glu233, His299, and Arg195; one salt bridge with Asp197,;one carbon hydrogen bond with Ala198 residue; two unfavorable donor-donor interactions with His299 and His305; and one unfavorable acceptor-acceptor contact with Lys200. This compound also formed two hydrophobic interactions as Pi-Pi stacked with Tyr62 and Trp59, and eight Van der Waals (VDW) contacts with Ala307, Leu162, Trp58, Ser199, Val234, His201, Ile235, and Tyr151 amino acids. Catechin, a flavanol compound in O. dillenii plants, was found to build three hydrogen bonds involving Asp300, Asp197 and Gln63 residues, in addition to numerous hydrophobic and electrostatic contacts: two Pi-Pi stacked (with Tyr62 and Trp59), a Pi-Anion (with Asp197) and eight VDW (Glu233, His299, His101, Arg195, Trp58, Leu165, Ala198 and His305) interactions. The complex kaempferol—α-amylase was stabilized by two hydrogen bonds, three hydrophobic interactions and six electrostatic contacts. His305 and Asp300 were involved in the formation of the hydrogen bonds. This latter also formed a Pi-Anion contact. Trp59 built two Pi-Pi stacked interactions with kaempferol. Moreover, Trp58, Glu233, Asp197, Tyr62, His299, His101, and Arg195 residues were stabilizing the complex by VDW contacts. As reported in Figure 4, eight conventional hydrogen bonds were observed between acarbose and binding site amino acids Asp300, Asp353, Asp356, Glu233, Arg195, His101, His305 and Tyr62. Meanwhile, Glu233 and Asp300 residues also formed a carbon hydrogen bond and unfavorable acceptor-acceptor contact with acarbose, respectively. Furthermore, ten electrostatic interactions, as VDW forces, were involved in the stabilization of acarbose- α-amylase complex. The residues were Asp197, His299, Ile235, Ala198, Leu162, Leu165, Trp58, Trp59, Trp357, and Val354. Surprisingly, chalcone, which exhibited a good binding energy, was found to have the smallest number of interactions with the receptor of all the compounds. These interactions included one hydrogen bond, two Pi-Pi stacked and four VDW interactions with the side chain amino acids Gln63, Trp59, Tyr62, Asp197, His305, Leu165, and Trp58, respectively. Gallic acid interacted with the residues Asp300, Glu233, Asp197, Tyr62, His299, His101, Arg195, Trp58, Leu165 and Ala198 via hydrogen bonds (two bonds), Pi-Anion (one interaction), Pi-Pi stacked (one interaction) and VDW (six interactions). In addition, p-coumaric acid was found to be surrounded by Asp197, Trp59, and Gln63 (via hydrogen bonds), Arg195 (unfavorable donor-donor interaction), Tyr62 (via Pi-Pi stacked interaction), and Asp300, Trp58, His299, Leu165, and Glu60 (via VDW forces) residues.Figure 7
2D-representations of the chemical bonding modes of the complexes formed between naringin, catechin, kaempferol, and acarbose compounds and the catalytic site residues of human pancreaticα-amylase.


## 8. Discussion

In this research, extracts were prepared from different organs ofO. dillenii fruits. Results showed high yield variability depending on the organ. This agreed with previous studies performed in different countries showing high variations in the ethanolic extract yields of Opuntia species. Indeed, yields between 3.93% and 6.25% were obtained with O. dillenii peel and juice from Taiwan [8]. This yield was between 6.20% and 15.5% for different Opuntia species from Mexico using the same extraction method. It was therefore suggested that extraction yields depend on genetic factors, harvesting routine, growing conditions, and cultivars and that the amounts of chemical compounds found in the extracts differed depending on the cultivar, climatology, harvesting conditions, and developing environment [46].For each fruit organ, the mean quantity of total polyphenols and total flavonoids was determined. Results showed differences depending on both factors which could be explained by different characteristics, as it is known that they can be related to genotypic factors, biotic (physiological stage, organs and species) and abiotic (edaphic) [47, 48]. Results obtained in the present study agree with other works showing that the total polyphenol content of O. dillenii fruits is low [8, 49]. As an example, Betancourt et al. [50] found the highest total phenolic compound content in a crude O. dillenii fruit extract from Colombia (311.61 μg/100 mg), while Embaby et al. [51] showed that O. dillenii fruits from Egypt had a low phenolic content (179.30 μg/100 mg).The different ethanolic extracts were then analyzed by HPLC for the determination of the major phenolic molecules. The present research highlighted that seeds ofO. dillenii fruits constitute a great source of flavonoids and polyphenols, which are important plant secondary metabolites [9]. For example, Katanic et al. reported low amounts of quercetin and kaempferol (0.6 and 2.7 mg/g, respectively) in the peel of this fruit [17]. In the work of Yilmaz and Toledo, the seeds presented the highest amount of polyphenols, followed by the peel and juice [7]. Another work reported the detection of quercetin, rutin, naringin, catechin, caffeic acid, gallic acid, chlorogenic acid, cinnamic acid, and syringic acid in O. dillenii fruits [52]. Also, Qiu et al. identified many phenolic compounds in the fruit, with 3-O-methyl quercetin, kaempferol and quercetin as the main phenols [53].It is well known that most of these molecules display antimicrobial activities and that they can prevent chronic diseases such as cardiovascular disease, inflammation, and cancer [9]. Furthermore, some compounds such as kaempferol have antimicrobial and antioxidant properties [17, 54]. The variability of phenolic molecules in the extracts may influence their biological activities as the number and position of hydroxyl groups in the aromatic rings may influence their toxicity towards microorganisms. On the other hand, the antimicrobial activity is not only dependent on the presence of phenolic compounds but also on the presence of various other secondary metabolites.Results obtained in the present study highlighted the interesting antibacterial activity of the extracts, which may be due to the presence of catechin and gallic acid, the major molecules in the active extracts. Indeed, it has been previously reported that these compounds possess antimicrobial activities. Gallic acid notably exhibits antibacterial capacity against various food-borne pathogens and food-borne harmful bacteria such asHelicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, and Listeria monocytogenes [55–57]. However, the observed activities were lower than those of the positive standard, which may be due to the inability of the antimicrobial agent to diffuse uniformly into the agar, as has already been reported by many researchers [58, 59]. The low phenolic compound content in the studied extracts could also be responsible for the observed inefficiency of the peel and seeds extracts, as there is a positive correlation between the concentration of phenolic compounds and their antimicrobial activity [60], with polyphenols and flavonoids representing important antimicrobial substances. Therefore, we suggest that the ethanolic extracts obtained from the juice of O. dillenii could be used to purify compounds with antibacterial activities or as a food ingredient.Thein vitro anti-diabetic activities of the O. dillenii extracts were analyzed. One therapeutic approach for diabetes consists of reducing postprandial hyperglycemia by delaying glucose absorption by inhibiting carbohydrate hydrolyzing enzymes (α-amylase and α-glucosidase), thereby preventing an increase in postprandial glucose [61, 62]. Pancreatic α-amylase is a digestive enzyme that catalyzes the initial step of starch hydrolysis to maltose and ultimately glucose [63]. Alpha-glucosidases are digestive enzymes located at the brush edge of enterocytes that catalyze the hydrolysis of α-(1,4)-glycosidic bonds of food disaccharides that reach the jejunum. Residues from the digestion of starch lead to the formation of absorbable glucose by intestinal cells [64, 65]. As a result, much research has focused on the identification of potential α-amylase and α-glucosidase inhibitor compounds for the prevention and treatment of diabetes.Results obtained here showed the interesting ability of all extracts to inhibit the activity ofα-glucosidase and α-amylase, with peel extracts displaying the highest activities. The differences in the activities reported for the different extracts may be related to differences in their chemical compositions. According to the HPLC data, the main compounds in the peel extracts were quercetin, kaempferol, and naringin, each of which is known for its ability to inhibit the activity of α-glucosidase and α-amylase. Those molecules, and possibly other active compounds may inhibit enzymes that hydrolyze polysaccharides and disaccharides. They may be nonspecific, in which case they would inhibit both α-amylase and α-glucosidase. It has been suggested that there may be a correlation between the content in phenolic compounds and the ability to inhibit the activity of α-glucosidase and α-amylase [66–69]. However, plant extracts with a high polyphenol content do not always exert a high inhibitory activity on α-amylase [70] which highlights the importance of the nature of different molecules and the interactions between them [71]. In addition, there is also a reported correlation between flavonoids and the inhibition of α-glucosidase and α-amylase. Some of the molecular mechanisms by which these compounds exert anti-diabetic effects have already been evaluated [72–74]. These phenolic compounds, which are characterized by antioxidant activities, are known to have anti-diabetic activities by regulating the disturbed oxidative medium under diabetic conditions [75]. Tadera et al. evaluated the inhibitory activity of six groups of flavonoids on yeast and rat intestinal α-glucosidase and porcine pancreatic α-amylase. For pancreatic α-amylase inhibition, luteolin, myricetin, and quercetin were the most potent compounds, showing inhibition percentages of 61%, 64%, and 50%, respectively. Quercetin, kaempferol, luteolin, naringin, and cyanidin showed inhibition percentages of 91%, 82%, 92%, 73%, and 99%, respectively, against the yeast α-glucosidase. Weak inhibitory activity was observed in the presence of intestinal α-glucosidase [61]. In addition, other authors have shown that catechins can inhibit the activity of pancreatic α-glucosidase and α-amylase [63, 76]. From those results, it can be concluded that the antihyperglycemic effect of O. dillenii is related to its richness in natural bioactive compounds that have an important antidiabetic property while reacting alone or in synergy.On the other hand, molecular docking simulations of the predominating metabolites identified inO. dillenii peel ethanolic extract were performed toward the human pancreatic α-amylase enzyme. Results clearly indicated the presence of bioactive molecules with high binding affinities to the α-amylase. Interestingly, naringin, catechin, and kaempferol exhibited lowest binding energies and therefore highest activities than the standard drug acarbose. Furthermore, all the studied compounds were found to form several hydrogen, hydrophobic, and electrostatic interactions with the same amino acid residues that interact with the cocrystallized ligand, which can explain the high stability of the complexes formed between these molecules and α-amylase. Investigation of the crystal structure of α-amylase demonstrated the presence of important residues in the active pocket, especially the three amino acids Asp300, Glu233, and Asp197 [77–79]. These three characteristic residues are the enzymes’ key residues that contribute to the overall catalytic activity of α-amylase enzyme [80, 81]. Brayer et al. [82] demonstrated by kinetic analyses that the substitution of Asp197 leads to a 106-fold decrease in catalytic activity of α-amylase enzyme, while the substitution of Glu233 and Asp300 residues decline the activity by 103-fold. As it can be noted from the docking results, most of the studied compounds showed interactions with these enzymes’ key residues and that may explain their strong binding at the active site, specifically naringin, catechin, and kaempferol compounds which have been already reported as potent antidiabetic agents [83–85]. Besides these observations, it is very exciting to note that the number of rings contained in the studied molecules increases the binding affinity to the receptor, hence the inhibitory potential. Overall, and based on the obtained observations, it can be concluded that the docking results are in accordance with the obtained in vitro experimental outcomes and demonstrate that O. dillenii ethanolic extract is rich in natural compounds that can be promising leads to developing highly active inhibitors against diabetes type 2, which would be better than the acarbose used currently. However, more theoretical and experimental validations are essential to determine and better understand the therapeutic effect of these compounds in the inhibition mechanism of diabetes.


## 9. Conclusions

The present study revealed that the extracts produced from seeds, juice, and peel ofO. illeniid fruits have an interesting chemical composition, including the presence of high polyphenol quantities (193.73 ± 81.44 to 341.12 ± 78.90 gallic acid eq [g/100 g dry weight]). Notably, gallic acid, vanillic acid, quercetin 3-O-β-D-glucoside, kaempferol, and syringic acid, which were the dominant constituents, are well known for their remarkable biological activities, including ant-diabetic, antibacterial, and antifungal properties [57, 60]. Results also showed that the yield, chemical composition, and biological activity of O. illeniid seeds, juice, and peel extracts varied according to the plant organ and extraction solvent, with the highest yields being obtained with ethanol extracts (8.41 ± 5.19%). In this study, a noteworthy anti-diabetic effect was highlighted for Opuntia extracts, in both in vitro and in silico assays, suggesting that they could potentially be used as a phytomedicine in healthy and diabetic subjects to prevent the incidence of diabetes and delay the development of complications in diabetics. The results of the in silico assays, which were consistent with the experimental outcomes, indicated the presence of naringin, catechin, and kaempferol as antidiabetic bioactive compounds. In addition, they provided a deeper insight into overall molecular interactions between the selected compounds and α-amylase enzyme, notably highlighting high-binding affinities. In conclusion, the study demonstrated that these natural metabolites in O. dillenii ethanolic extracts can be good alternatives to the standard approved drug acarbose.As a perspective, it would be interesting to study the mechanisms of action of those extracts and their optimal dosages as well as their method of administration for the patients. In addition, these extracts could be used in the agrifood industry as additives, dyes, and natural food preservatives thanks to their antibacterial properties. Based on the chemical profile ofO. dillenii fruits, bio-guided assays could be performed to isolate the main pharmacologically active constituents, which could then be used in the prevention and treatment of various diseases, representing one of the possible prospects for the food and nutraceutical industries. The high efficacy and decreased side effects of this plant highlight the possibility of using medicinal plants instead of chemical drugs. Moreover, this approach will provide great benefits for Moroccan farmers and increase their economic income.



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*Source: 9471239-2022-10-30.xml*