1. Introduction
Oxidative stress and inflammation have been considered important factors linked to diabetes mellitus, chronic pulmonary, cardiovascular, and Alzheimer’s diseases, in addition to the physiopathology of cancer. The oxidative stress is known by the overproduction of ROS (reactive oxygen species) leading to the oxidation of macromolecules, notably lipids, proteins, and nucleic acids, which causes the impairment of cellular functions and apoptosis [
1,
2,
3]. Inflammation is a protective process that involves an arsenal of enzymatic reactions and cellular activation. The prolongation of this process causes the loss of cell imbalance and biological system damage [
4]. Thus, for better-multifaceted disease management, it is necessary to develop new drugs implying antioxidant and anti-inflammatory activities. In this context, medicinal plants and derivatives have been widely used for therapeutic purposes to heal and protect against several illnesses. Bioactive compounds extracted from natural products can offer specific properties that could act on specific targets and exhibit several biological properties for treating particular diseases [
5,
6,
7]. The use of medicinal plants has increased in recent years. Therefore, screening biological activities remains an important step in therapeutic virtues research. In vitro assays, including enzyme inhibition assays, are commonly used for biological properties, bioactive natural compounds screening, and also for drug synthesis [
8,
9].
J. regia is a well-known worldwide species of the
Juglans genus belonging to the family of
Junglandacae. Fruits are consumed for their nutritional value phytotherapy [
10,
11,
12] and have been widely used in folk medicine, especially for diabetes and inflammatory diseases [
13] like eczema [
14] and gout disease [
15], particularly in Morocco [
16], Turkey [
17], Iran [
18], Italy and Romania [
19]. Leaves are used to treat digestive disorders like stomatitis, oral ulcer, and diarrhea [
14,
20,
21,
22]. Elsewhere, bark’s preparation, also called souak, is used for teeth hygiene and to treat bucco-dentaire sphere problems such as gum diseases, halitosis, and dental stain due to its depurative and antiseptic properties [
10,
23,
24,
25].
The literature reported several biological activities exhibited by
J. regia extracts, including anti-inflammatory and antioxidant activities [
26,
27,
28]. Each part of the plant showed different properties with regard to the cultivars [
29], extraction method ([
30], and geographic conditions [
31]. Moreover, the
J. regia phytochemical composition varied according to those parameters but the main molecules found are polyphenols, including phenolic acids (gallic acid, vanillic acid, syringic acid, ellagic acid, caffeic acid, ferulic acid, sinapic acid, chlorogenic acid), flavonoids, and tannins [
32].
J. regia extracts act on different inflammatory mediators; husk extracts can inhibit the nitric oxide (NO) production in macrophages [
28], kernel extracts inhibit the activation and the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) [
33] and are involved in the diminution of edemas [
34], and bark extracts can inhibit membrane hemolysis. In addition, leaf extracts inhibit cyclooxygenase-2 (COX-2), one of the most important targets of anti-inflammatory drugs [
35]. On the other hand, other researchers have focused on the anti-diabetic properties of
J. regia in particular leaves, which showed excellent hypoglycemic effects [
36,
37].
To the best of our knowledge, this work is the first comparative study realized in kernels, leaves, husk, and bark of J. regia extracts based on the phytochemical composition and antioxidant, anti-inflammatory, and anti-diabetic activities. To date, no study reported the inhibitory effect of J. regia extracts on lipoxygenase (LOX).
In this work, we investigated the chemical screening of methanolic extracts of different J. regia parts: kernels (MWK), leaves (MWL), husk (MWH), and bark (MWB). In addition, enzymatic inhibitory activities of these part extracts were evaluated against enzymes involving inflammation and diabetes mellitus. Moreover, several methods were used to evaluate their antioxidant capacities (DPPH, FRAP, ABTS).
2. Materials and Methods
2.1. Chemicals
Methanol, Folin–Ciocalteau reagent, (7.5%) Na2CO3, gallic acid, (5%) sodium nitrite solution (NaNO2), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), acetonitrile, (10%) aluminum trichloride (AlCl3), colchicine, Dimethyl sulfoxide (DMSO), ferric chloride (FeCl3), (0.1%) formic acid, nitrogen, iodine solution, PBS buffer, p-nitro-phenyl-α-D-glucopyranoside (p-NPG), (1%) Potassium ferricyanide, potassium persulfate, potassium phosphate buffer, quercetin, rutin, sodium hydroxide, sodium phosphate buffer, Kojic acid, (10%) trichloroacetic acid, Trolox, (4%) vanillin, hydrochloric acid (HCl) were analytical grade. L-Dopa, linoleic acid, tyrosinase, α-amylase, and α-glucosidase are purchased from Sigma.
2.2. Plant Material and Extraction
Leaves, husks, kernels, and bark of the plant J. regia were collected from the Taza region, Morocco. After the drying process, walnut parts were powdered and conserved in the dark at 4 °C. The extractions of leaves, husk, kernels, and bark powders (12.5 g) were realized by methanolic maceration (250 mL) for 24 h under agitation. Then, the mixture was filtered, the solvent was eliminated using a rotary evaporator at 60 °C, and the extracts were conserved at 4 °C.
2.3. Determination of Phenolic Contents
2.3.1. Total Phenolic Content
The determination of total phenolic content in
J. regia extracts was performed using the Folin–Ciocalteu procedure [
38]. 0.5 mL of Folin–Ciocalteau reagent was added to 0.5 mL of each extract and agitated. Then, 4 mL of 7.5 % Na
2CO
3 (
w/
v) was added, and the reaction was incubated at 45 °C for 30 min. Calibration curves were established using gallic acid. The absorbance was measured at 765 nm, and the total phenolic contents were expressed as mg gallic acid equivalents per g of the dry weight of extract (mg GAE/g of extract).
2.3.2. Total Flavonoid Content
The determination of the flavonoid content was carried out according to the aluminum trichloride method developed by Brighente et al. [
39]. In test tubes, 1 mL of each of the extracts (1mg / mL) and 6.4 mL of distilled water were successively introduced, then 0.3 mL of the sodium nitrite solution (NaNO
2 5%) was added. After 5 min, 0.3 mL of aluminum trichloride (AlCl
3 10%) was added. After 6 min, 2 mL of sodium hydroxide (1 M) was added, and the solution was agitated and allowed to stand for 30 min. The absorbances were measured at 510 nm. Rutin was used as a standard under the same analytical conditions. The flavonoid content is expressed in mg of rutin equivalent per g of dried extract (mg RE/g of the extract).
2.3.3. Total Tannin Content
The determination of total tannin contents was effectuated using the Julkunen–Tiitto [
40] method. 50 μL of each extract was mixed with 1.5 mL of 4% vanillin, then 750 μL of hydrochloric acid HCl was added. After, the mixture was incubated for 20 min at room temperature in the dark. Colchicine was used as standard. The absorbances were measured at 500 nm. The results are expressed in milligrams equivalent to catechin per gram of the extract (mg CE/g of extract).
2.4. HPLC-DAD-ESI-MS/MS
The chemical composition of
J. regia methanolic extracts was determined by high-performance liquid chromatography (Hewlett-Packard 1100 Agilent Technologies) equipped with a DAD detector and an electrospray HP 1100 MSD API (Agilent-Technologies, Palo Alto, CA, USA) under analytical conditions reported by Pallaufa et al. [
41]. A negative ionization mode, a capillary voltage of 3000 to 3500 V, and a fragmented variable of the order of 80 to 150 V were used. The column was a Poroshell 120 EC-C1, C18 (150 × 2.1) mm × 5 µm. The mobile phase was (A) 0.1% formic acid in the water, (B) acetonitrile. The established elution gradient was isocratic 15% B for 5 min, 15% B to 20% B over 5 min, 20–25% B over 10 min, 25–35% B over 10 min, 35–50% for 10 min. The total analysis time was 47 min, the flow rate was 0.5 mL/min. Double in-line detection was performed in the DAD using 280 nm and 370 nm as wavelengths and in a mass spectrometer (MS) connected to the HPLC system through the output of the DAD cell. MS detection was performed in a Qtrap API 3200 (Applied Biosystems, Darmstadt, Germany) equipped with an ESI source and a triple quadrupole ion trap mass analyzer. Zero-quality air was used as nebulizer gas (30 psi) and turbo gas for solvent drying (400 °C, 40 psi). Nitrogen served as a curtain (20 psi) and collision gas. The resolution of the quadrupoles was adjusted and the ion sputtering voltage was tuned at −4500 V (in negative mode). The MS detector has been set up to operate in two modes: Enhanced MS Analysis (EMS) and Enhanced Product Ion Analysis (EPIA) (EPI). To acquire an overview of all the ions in the sample, the EMS was utilized to capture the entire scan spectra. The defusing potential (DP) was −450 V, the input potential (EP) was −6 V, and the impact energy (CE) was −10 V. The spectra were recorded between
m/
z 100 and 1000 (in negative ion mode). For detected parent ion (s) discovered, the fragmentation pattern was then determined using EPI analysis under DP −50 V, EP −6 V, CE −25 V, and collision energy propagation (CES) 0 V [
42].
2.5. Antioxidant Assay
2.5.1. DPPH Method
A concentration of 1 mg/mL of each extract was prepared and diluted in methanol to obtain a range concentration from 10 to 1000 µg/mL. 1 mL of each sample concentration or standard was mixed with 0.5 mL of 0.2 mM DPPH methanolic solution. Trolox was used as a standard under the same conditions. Absorbances were measured at 517 nm after 30 min of incubation at dark conditions [
43]. The radical scavenging ability (RSA) was expressed in % according to the following equation where the Abs control is the absorbance of the solution containing all reagents except sample (or standard). IC50 was calculated from the plot of RSA vs. extract concentration.
2.5.2. ABTS Method
The antioxidant activity was determined by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method [
44]. 2 mM of ABTS was mixed with 70 mM of potassium persulfate. After incubation in the dark during 12–16 h, the ABTS
+ solution was diluted with methanol to adjust absorbance to 0.700 ± 0.005 at 734 nm. Thus, 1 mL of each extract concentration (1 mg/mL) or standard was added to 2 mL of diluted ABTS solution to obtain a range concentration from 25 to 1000 µg/mL and incubated for 1 min, and the absorbance was measured at 734 nm. Trolox is used as a standard compound. Scavenging activity in this assay was expressed as the concentration of the extract required to inhibit 50% of the free radical scavenging activity.
2.5.3. FRAP Method
The extract’s ferric ion-reducing power was determined by the FRAP method [
45] with some modifications. A concentration of 1 mg/mL of each extract was prepared and diluted in methanol to obtain a range concentration from 5 to 50 µg/mL. Trolox was used as standard. 1 mL of each prepared extract or standard was mixed with 1.25 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 1.25 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. After cooling, 1.25 mL of 10% trichloroacetic acid was added and centrifuged at 3000 rpm for 10 min. Finally, 1.25 mL of the supernatant was mixed with 1.25 mL distilled water and 0.25 mL FeCl
3 solution (0.1%,
w/
v). Absorbances were measured at 700 nm, and results were expressed as the 50% effective concentration (EC
50), which is the antioxidant concentration in mg/mL necessary to achieve an absorbance of 0.5.
2.6. Anti-Inflammatory Assay
2.6.1. Lipooxidase Inhibition Assay
The lipooxidase inhibition assay was realized according to the procedure described by Debayo et al. [
46]. A concentration of 1 mg/mL of each extract was prepared in methanol and diluted in 2M borate buffer to obtain a range concentration from 12.5 to 50 µg/mL. 12.5 µL of each extract concentration was added to 487.5 µL of 15-LOX (200 Units/mL) and kept at room temperature. After 5 min, 500 µL of linoleic acid dissolved in ethanol and diluted in the borate buffer were added to the enzymatic mixture and incubated for 5 min at room temperature. The absorbance was measured at 234 nm. Quercetin was used as a positive control, and DMSO was used as a negative control. The enzyme inhibition percentages were determined by the following equation.
2.6.2. Tyrosinase Inhibition Assay
The anti-tyrosinase activity was determined according to Huang et al. [
47] procedure. A concentration of 1 mg/mL of each extract was prepared in methanol and diluted in 0.05 M PBS buffer (pH 6.5) to obtain a range concentration from 25 to 100 µg/mL. Tyrosinase enzymatic solution (333 U/mL) and L-Dopa (5 mM) were prepared in the PBS buffer. 50 µL of each extract concentration was mixed with 200 µL enzyme solution (3 U/mL), and the mixture was incubated at 37 °C. After 10 min, 500 µL of the substrate (L-Dopa) was added. The enzymatic reaction solution was then incubated for 30 min at 37 °C. The absorbance was measured at 510 nm. The percentage of inhibition was determined by the following formula, where Abs represents absorbance. Kojic acid was used as a standard.
2.7. Anti-Diabetic Activity
2.7.1. α-Amylase Inhibition Assay
The effect of
J. regia extracts on α-amylase activity was assessed according to Kusano et al. [
48] method with some modifications. A concentration of 1 mg/mL of each extract was prepared in methanol and diluted in phosphate buffer (pH 6.9) to obtain a range concentration from 25 to 100 µg/mL. 200 µL of starch solution (substrate) was added to 100 µL of the buffer, and 250 µL of α-amylase (30 U/mL) was then incubated at 37 °C for 15 min. For the sample test, the enzyme was incubated with 250 µL of each extract concentration for 15 min. After adding substrate, the enzymatic reaction was conducted for 15 min and then stopped using 400 µL HCl (0.1 M). Total and remaining starch were measured at 630 nm after adding 500 µL of iodine solution (25 mM). For the positive control, acarbose was used. The percentage of inhibition was calculated by the following formula.
2.7.2. α-Glucosidase Inhibition Assay
The α-Glucosidase inhibition activity was tested according to Li et al. described method with slight modifications [
49]. A concentration of 1 mg/mL of each extract was prepared in methanol and diluted in 1 M potassium phosphate buffer (pH 6.8) to obtain a range concentration from 250 to 1000 µg/mL. 100 µL of sample or acarbose (positive control), 380 µL of p-nitro-phenyl-α-D-glucopyranoside (p-NPG) (0.53 mM), and 250 µL of α-Glucosidase solution (0.015 Units/mL) were mixed in the buffer. After incubating at 37 °C for 20 min, 1mL of Na
2CO
3 (0.1 M) was added to quench the reaction. The IC
50 value is determined by the concentration of α-Glucosidase inhibitor necessary to inhibit 50% of activity under assay conditions. The absorbance was measured at 405 nm, and the inhibition percentages were determined using the following equation:
2.8. Statistical Analysis
Raw or log-transformed measured parameters were tested for normality and homogeneity of variance to meet the assumptions for parametric statistics. As the assumptions were violated for all parameters (extracts and assays), non-parametric analyses of variance, followed by a Dunn pairwise comparison test of means, were performed. The critical level of significance is set at 0.05.
4. Conclusions
J. regia is a well-known species widely used for its nutritional benefits and also for its therapeutic properties, as reported in numerous ethnobotanical studies conducted in many countries.
This study is the first to emphasize the variability in antioxidant and biological characteristics across the various parts of the walnut. This variability will be used to guide in vivo studies and, consequently, the uses by herbalists of those parts of plants exhibiting the most relevant activities.
Results highlighted the potential antidiabetic properties of kernels and husk extracts as well as the anti-inflammatory properties of bark extract. In fact, the phenolic profile determined by HPLC-DAD-ESI-MS/MS showed the richness of J. regia in bioactive compounds. However, further investigations concerning the isolation of main chemical compounds, as well as the evaluation of their antioxidant, antidiabetic, and anti-inflammatory effects, are required to determine the molecular mechanisms involved in these biological activities. Moreover, in vivo explorations and toxicological investigations are needed to determine the main pharmaceutical parameters of these compounds as well as to validate their safety.