Ultrasound-Assisted Extraction, LC–MS/MS Analysis, Anticholinesterase, and Antioxidant Activities of Valuable Natural Metabolites from Astragalus armatus Willd.: In Silico Molecular Docking and In Vitro Enzymatic Studies

The Astragalus armatus Willd. plant’s phenolic constituent extraction and identification were optimized using the ultrasound-assisted extraction (UAE) method and the LC–MS/MS analysis, respectively. Additionally, cupric reducing antioxidant capacity (CUPRAC), beta carotene, reducing power, DMSO alcalin, silver nanoparticle (SNP)-based method, phenanthroline, and hydroxyl radical tests were utilized to assess the extract’s antioxidant capacity, while the neuroprotective effect was examined in vitro against acetylcholinesterase enzyme. This study accurately estimated the chemical bonding between the identified phenolic molecules derived from LC–MS/MS and the AChE. The extract was found to contain sixteen phenolic substances, and rosmarinic, protocatechuic, and chlorogenic acids, as well as 4-hydroxybenzoic, hyperoside, and hesperidin, were the most abundant substances in the extract. In all antioxidant experiments, the plant extract demonstrated strong antioxidant activity and a significant inhibitory impact against AChE (40.25 ± 1.41 μg/mL). According to molecular docking affinity to the enzyme AChE, the top-five molecules were found to be luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol. Furthermore, these tested polyphenols satisfy the essential requirements for drug-like characteristics and Lipinski’s rule of five. These results highlight the significance of the A. armatus plant in cosmetics, as food additives, and in the pharmaceutical industry due to its rosmarinic and chlorogenic acid content.


Introduction
Synthetic compounds, notably tert-butylhydroquinone (TBHQ), propyl gallate (PG), and butylated hydroxytoluene (BHT), have been widely employed in the nutraceutical industries as the off-flavor development of foods as well as in pharmaceuticals to fight lipid oxidation [1].
Due to the negative impacts of these synthetic molecules, numerous research studies have concentrated on bioactive molecules from plants, which are used as safe antioxidant compounds [2,3]. These biomolecules have significant importance in reducing oxidative stress, which could destroy the biological mechanisms in the human body [3]. In reality, the preference for natural substances is also a consequence of the lack of adverse effects where numerous studies have shown that natural compounds counteract the negative effects of pharmaceutical treatments, implying that these natural molecules have greater medicinal value [4].
Various illnesses have been treated using plants [5] because of the existence of polyphenols, which have many biological activities [5]. Furthermore, the studies on enzyme inhibitors are regarded as the most effective in vitro strategies for a variety of diseases [6]. However, a complete understanding of the underlying physiological systems is required for the creation of therapeutic approaches [6]. Alzheimer's disease (AD), or neurodegenerative disease, is currently affecting older adults more than ever before. AD patients' brains were shown to have hyperactivity of acetylcholinesterase (AChE) [7]. The depletion of the acetylcholine (ACh) neurotransmitter by cholinesterase is linked to Alzheimer's etiology [7]. Currently, the most successful therapeutic strategy is the application of ChE inhibitors [8], particularly natural anticholinesterases and antioxidants. As most of these medicines have numerous adverse effects, it is desirable to use and manage them effectively.
Legumes are typically a significant source of high-quality protein, vitamins, minerals, and bioactive compounds [9]. The growing demand for leguminous plants as a result of the scarcity of agricultural land and water resources is turning into a severe problem. Thus, food plants with substantial levels of bioactive substances are becoming more difficult to acquire and much more expensive. As a result, an important research tendency in recent years has been the discovery of additional raw and environmentally safe sources of essential nutrients, particularly from non-conventional plants [10], while still able of offering excellent nutrients for industrial use under salinity and dry situations [10].
The plants from the Astragalus genus, members of the Fabaceae family, are often utilized in food and medicine as well as feed for livestock [9,10]. Among these herbs, we are interested in Astragalus armatus Willd., which belongs to the Leguminosae plant and is prevalent in Mediterranean climate zones in Europe and North Africa [9]. This species is also found in Algeria's Sahara Desert, Morocco, and Tunisia [10]. Aromatic compounds and essential oils are found in several Astragalus species, with a variety of medicinal and economic applications [11]. The capacity of this species to inhibit the enzymes associated with a variety of chronic illnesses has been demonstrated by several studies [10][11][12]. It has been demonstrated that several bioactive substances from this genus are effective in cancer treatment [12], including saponins, which had a substantial anticancer impact on hamsters [12]. Several polysaccharides and phenolic compounds with immunostimulatory properties are also found in this species [12].
However, a variety of solvents and methods have been employed to extract bioactive compounds from plants [12]. For this purpose, the current study aimed to extract the phenolic compound using an ultrasound-assisted extraction method, investigates the metabolite profiling of A. armatus using LC-MS/MS analyses, and assesses both its in vitro antioxidant potential and anticholinesterase effects. This study also reports a molecular docking analysis, where the identified polyphenols were used as ligands to test their inhibitory activity against AChE receptors to learn more about their mechanisms of action.

Chemicals and Instruments
The chemical profile was performed by using LC-MS/MS (Shimadzu, Kyoto, Japan). The activity assays were evaluated by using a Shimadzu UV spectrophotometer and a BioTek Power Wave XS microplate reader (USA). All the standard chemicals utilized in the antioxidant activities and LC-MS/MS were purchased from Merck. Germany Sigma provided the AChE as well as the reactive products of antioxidant tests (Germany). All solvents were of analytical grade [13].

Plant Extract Preparation
The aerial parts of A. armatus were taken from Algeria's Sahara Desert (EL Oued). Therefore, the ultrasound-assisted extraction method was used in this investigation to perform the plant extraction. An ethanol-water mixture (70:30 v/v) was used to extract the plant. Following concentration with a rotary evaporator, the residual material was dissolved in water and then extracted using petroleum ether and ethanol, respectively [14]. The ethanolic fraction is the focus of our investigation.

Equipment and Chromatographic Parameters
The tandem MS system used in conjunction with the UHPLC (Nexera type Shimadzu, USA) was used for the LC-MS/MS technique [15]. Furthermore, The LC-MS analysis was also performed using LC-30AD binary pumps, a CTO-10ASvp column oven, a DGU-20A3R degasser, and a SIL-30AC autosampler, and the separation was carried out using a reversedphase C18 Inertsil ODS-4 analytical column of 150 mm × 4.6 mm × 3 m at 40 • C. Mobile phase A (H 2 O, ammonium formate (5 mM), and formic acid 0.1%) and mobile phase B (methanol, ammonium formate (5 mM), and formic acid 0.1%) were used in the elution gradient. The solvent flow rate was kept at 0.5 mL/min, and the injection was fixed at 4 µL. The ESI source was used in air pressure ionization, where the optimal ESI circumstances were determined to be a DL temperature of 250 • C, an interface temperature of 350 • C, a heat block temperature of 400 • C, a drying gas flow rate of 15 L/min, and a nebulizing gas flow rate of 3 L/min.

MS Instrumentation
The MS identification was performed using a Shimadzu LCMS 8040 triple quadrupole mass spectrometer equipped with an ESI source that could operate in both positive and negative ionization mechanisms. The data from LC-MS/MS was collected and processed using Lab Solutions software (Shimadzu, Kyoto, Japan). The multiple reaction monitoring (MRM) strategy was used to quantify the analyses. The assay of the explored substances was carried out after two or three shifts per substance. The first one was for quantification, whereas the second and/or third were for confirmation.

Method Validation Parameters for LC-MS/MS
The detailed analytical characteristics of LC-MS/MS reference compounds have already been documented in the literature [13][14][15], including the linearity ranges and rectilinear regression estimates for the standard chemicals examined. All the calibration curves for all chemical compounds were linear and reproducible, with a correlation coefficient of more than 0.991. Ertas and Yener [13][14][15] depict, furthermore, the disclosed analytical method's limit of detection (LOD) and limit of quantitation (LOQ). LOD varied from 0.05 to 25.8 g/L, while LOQ varied from 0.17 to 85.9 g/L. Furthermore, phenolic compound recoveries ranged from 96.9% to 106.2%.

Acetylcholinesterase Inhibitory Assay
The anti-AChE inhibitory assay of A. armatus extract was performed using the technique outlined in Ellman's work [23]. Briefly, 150 µL of a sodium phosphate buffer (100 Mm, pH 8.0), 10 µL of the extract, and a volume of 20 µL AChE were mixed. Then, 15 min after incubation at a suitable temperature for enzyme activation, 10 µL DTNB (0.5 mM) was introduced to the reactive mix. After that, 20 L of acetylthiocholine iodide was added to begin the reaction (0.71 mM). A 96-well microplate reader was used to measure the absorbance, and the percent inhibition was calculated as follows: where E is the enzyme's activity without the extracts, and S is the enzyme's activity with the extracts.
The experiments were carried out in triplicate. The standard compound used in the experiments was galantamine. A 50% inhibitory concentration was taken to represent the results (IC 50 ).

Docking Molecular Analysis
This study used docking molecular analysis to measure the binding degree affinity between AChE and the polyphenols of LC-MS/MS results. First, the crystal structure of the AChE enzyme with ID 4EY6 was loaded from the PDB database (https://www.rcsb.org, accessed on 23 August 2022). The 3D structure of this receptor was prepared using the Chimera 1.15 program by removing the ligands and the co-crystallized solvents, as well as the water molecules. The global atomic charge of the protein was also corrected, and the H-atoms were added. On the other hand, the structures of the polyphenols of LC-MS/MS serving as ligands were obtained from the PubChem database (https://pubchem.ncbi. nlm.nih.gov/, accessed on 23 August 2022) in their SMILES formats, where they were converted into 3D structures using the Chimera package, which was also used to assign them Gasteiger charges and hydrogen atoms. The program AutoDock Vina was used to perform molecular docking. The outcomes were expressed in terms of binding affinity, where the ligands with the highest binding affinity were selected for the investigation of their interactions with the AChE active site.

Drug Likeness and ADMET Profiling
The drug-like qualities of the chosen phenolic substances were measured with the Swiss ADME Web Service and using the rules of Lipinski and Veber [24,25], the different biochemical attributes were determined. In addition, the two web applications ProTox-II (https://tox-new.charite.de, accessed on 26 August 2022) [26] and ADMETlab 2.0 (https: //admetmesh.scbdd.com/, accessed on 26 August 2022) [27] were used to predict the toxicity of the selected ligands.

Statistical Analysis
The measurements were carried out in triplicate for each sample. The findings are expressed as mean ±standard deviation (SD). A one-way analysis of variance (ANOVA) of the bioassays was conducted using the PRISM GraphPad V: 5.00 (Trial), followed by the Tukey test at p 0.05.

Chemical Identification and Measurement of Phenolic Compounds
Several plants ingested as food have attracted increased interest due to the preventive impact of their phenolic compounds on cellular oxidative damage [13,14] and their availability in the human diet [28].

Statistical Analysis
The measurements were carried out in triplicate for each sample. The findings are expressed as mean ±standard deviation (SD). A one-way analysis of variance (ANOVA) of the bioassays was conducted using the PRISM GraphPad V: 5.00 (Trial), followed by the Tukey test at p0.05.

Chemical Identification and Measurement of Phenolic Compounds
Several plants ingested as food have attracted increased interest due to the preventive impact of their phenolic compounds on cellular oxidative damage [13,14] and their availability in the human diet [28].
Similar to the present work, and by using LC-ESI-MS, it has been documented that the A. armatus ethanolic fraction contains quercetin and kaempferol in low concentrations [12]. It was also mentioned that the A. armatus ethanolic fraction produced through sample extraction contained significant levels of cirsiliol (37.1 ± 1 µg/g DM).
The high concentration of phenolic content in the Astragalus genus has been observed previously [29]. Their structures were elucidated by the nuclear magnetic resonance (NMR) technical analysis in the form of 4 -dimethoxy isoflavane, 7,2 -dihydroxy-3 , isoliquiritigenin, formononetin, quercetin, ononin, kaempferol, vanillic, and p-hydroxybenzoic acids [29]. In addition, luteolin and its glucoside have been also isolated and described from A. galegiformis L., which confirms the richness of this genus in phenolic compounds. These advantageous compounds found in the extract explain the effectiveness of the ultrasoundassisted extraction technique on the determination of phenolic concentrations [1].
Additionally, this study provided the first significant findings on the high concentration of rosmarinic and chlorogenic acids in A. armatus. It may be suggested that this plant is a potential source of these two major substances. Several plant species have reportedly been tested to lengthen the shelf life of cheese and increase its quality, as well as for potential application in bread and cupcakes because of their high rosmarinic acid concentration [30]. Our results reinforce the possibility of also using this plant in this commercial context.

Antioxidant Activity
The search for bio-antioxidants with potential applications in the food, cosmetics, and medical sectors is now gaining more interest among researchers [5]. The antioxidant activity of plant extracts has an important impact on a variety of processes, including their ability to reduce and scavenge free radicals as well as their capacity to absorb oxygen radicals [5]. As a consequence, the antioxidant potential of the ethanolic extract of A. armatus was estimated using seven antioxidant tests ( Table 2). To our knowledge, there is no published research in the literature on A. armatus using these antioxidant techniques. As shown in Table 2, the terms "IC 50 " and "A0.50 levels" refer to the concentration at 0.50 absorbance and the 50% inhibitory percent quantity, respectively. These two values were calculated using a linear regression approach and are presented as mean SD (n = 3). The ethanolic extract was found to have a significant antioxidant activity, drawing more attention to this leguminous plant as a very promising natural antioxidant that could potentially be engaged in the treatment of various physiological pathologies caused by imbalances in oxidative systems. The observed variances in antioxidant effects that are influenced by the extracting solvent utilized have previously been examined [5].
For this reason, we can confirm that the method of extraction can significantly contributes to increasing the yield of polyphenolic content and antioxidant capacity.
Additionally, antioxidants are very essential for preventing lipid peroxidation and cellular damage caused by free radicals [28]. Lipid peroxidation is a chain reaction involving free radicals, which are associated with a wide range of biological diseases [31]. As indicated by the results, the A. armatus extract demonstrated a potent anti-lipid peroxidation activity with a high half-maximal inhibitory concentration of 5.12 ±1.2 µg/mL more than α-Tocopherol and BHT (11.43 ± 0.23 and 9.65 ± 1.1 µg/mL), which served as the standards for the β-Carotene bleaching method. As shown in Table 2, rosmarinic and chlorogenic acids are both present in very high concentrations in the A. armatus extract (80,695.32 and 76,635.0 µg/g dry extract, respectively). Therefore, it may be suggested that the observed activity is caused by rosmarinic and chlorogenic acids and their synergic effects with other phenolic compounds.

Acetylcholinesterase Inhibitory Assay
Plants are the most important source to create novel AChE inhibitor drugs for the treatment of degenerative disorders, including AD, which is considered the most common disease worldwide.
A variety of endogenous enzymes must be inhibited in order to reduce this neuropathology [32]. There have been reports that a variety of plant compounds in extracts are effective alternatives for treating Alzheimer's disease. These bioactive substances have potent antioxidant activity that combats free radicals and reduces brain cell destruction [33,34].
There are currently few studies on the Astragalus genus and the ability of its species to inhibit enzyme function. Through this research, we aimed to demonstrate this plant's capacity to obstruct AChE's catalytic site.
As seen in Table 3, the AChE inhibitory activity of A. armatus was compared with that of galantamine. The plant's IC 50 value for blocking AChE was 40.25 µg/mL. The analysis using statistics demonstrates that the values were significantly different (p < 0.05). Table 3. Galantamine standard and A. armatus fraction IC 50 values (g/mL) for inhibiting acetylcholinesterase activity.

Extract IC50 Values (g/mL) for Inhibiting Acetylcholinesterase Activity
A. armatus 40.25 ± 1.3 a Galantamine 34.75 ± 1.1 b These findings can be explained by the existence of chemicals in the extracts that are responsible for the inhibition of the AChE [4] and particularly by the ethanolic extract's flavonoids. Our results agree with those of the investigations conducted by Teyeb et al. [35]. Additionally, the A. setulosus plant exhibited a good inhibition against BChE, according to a previous study by Zengin [36], while A. leporinus Boiss. var. hirsutus extract demonstrated a potent inhibitory activity, with an IC 50 value of 66.15 ± 4.08 µg/mL.

Binding Mode Analysis Using a Molecular Docking Approach
Molecular docking has become a much more frequently used technique in the computeraided drug development process. This groundbreaking strategy can significantly reduce energy use, costs, and time in drug discovery by screening large pharmaceutical libraries for future drug substances [37]. In the current work, we used a molecular docking method to screen the inhibitory effects of the polyphenolic components of LC-MS/MS.
It is well-established from crystal studies that Ser203 and His447 are the two major amino acids important for AChE's catalytic properties. These two are located in the catalytic and oxyanion hole residues, which also include Glu202, Tyr33, and Trp86, located in the choline-binding pocket, and Trp236, Phe338, Phe297, and Phe295, composing the acyl-binding pocket of the enzyme [38,39]. The binding mode of the co-crystallized inhibitor galantamine, as shown in Table 4, clearly identified these key residues. The docked reference molecule (galantamine) gave a binding energy value of −10.3 Kcal/mol and showed four hydrogen bonds, including one with the key amino acid Glu202. In addition, this molecule interacts by forming a P-Stacked amide bond with Gly121 and six Pi-alkyl bonds with Tyr337, Phe338, Phe295, Phe297, His447, and Trp86. Based on the high scores given by the top-five compounds (Table 4), it is evident that they all had a very strong affinity with the catalytic site of the enzyme. In addition, they all had interactions with at least one of the key amino acids (Table 4). With a docked score of −10.8 Kcal/mol, luteolin was determined as the ligand with the greatest inhibition potential. This molecule interacts by forming five hydrogen bonds with Asn87, Tyr133, Gly448, and Ser125. The key amino acid Trp86 provides two Pi-Pi-stacked interactions with these ligands. Moreover, several Van der Waals attractions reinforce the stability of the luteolin-AChE complex (Figure 3).

Drug-Likeness and ADMET Profiling
In Table 5, the ADME results of the top-five ligands measured using the SWISS-ADME server are displayed. Luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol have molecular weights of 286. 24, 302.24, 272.25, 360.31, and 286.24 g/mol, respectively; the results of these phenolic substances suggested that they could be easily transported, distributed, and absorbed through the biological membranes [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. In addition, luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol were found to have LogP values of 1.73, 1.23, 1.84, 1.52, and 1.58, respectively, which are consistent with Lipinski's rule of five. Additionally, the hydrogen bond donor and acceptor numbers of these ligands were less than five and less than ten, respectively, which satisfies the AD-ME standards of H-bond donors and acceptors. The ADME results showed that luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol had topological polar surface (TPSA) values of 111.13, 131.36, 86.99, 144.52, and 111.13 Å 2, respectively. These values fall within the acceptable range not exceeding Å2, as outlined earlier by Cecchelli [41]. Furthermore, the atom molar refractivity (AMR) values of the selected components varied between 71.57 for naringenin and 91.40 for rosmarinic acid, both of which fall within the margins designated for this criterion between 40 and 130 [42]. Compound solubility is another important biochemical criterion for the choice of a biologically active molecule. LogS is the measurement that represents this parameter. For the five best polyphenolic compounds, this measurement was between −3.71 for luteolin and −3.16 for quercetin,

Drug-Likeness and ADMET Profiling
In Table 5, the ADME results of the top-five ligands measured usin SWISS-ADME server are displayed. Luteolin, quercetin, naringenin, rosmarinic aci kaempferol have molecular weights of 286.24, 302.24, 272.25, 360.31, and 286.24 respectively; the results of these phenolic substances suggested that they could be transported, distributed, and absorbed through the biological membranes [25-4 addition, luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol were fou have LogP values of 1.73, 1.23, 1.84, 1.52, and 1.58, respectively, which are consisten Lipinski's rule of five. Additionally, the hydrogen bond donor and acceptor numb these ligands were less than five and less than ten, respectively, which satisfies th ME standards of H-bond donors and acceptors. The ADME results showed that lu quercetin, naringenin, rosmarinic acid, and kaempferol had topological polar s (TPSA) values of 111.13, 131.36, 86.99, 144.52, and 111.13 Å 2, respectively. These fall within the acceptable range not exceeding Å2, as outlined earlier by Cecchel Furthermore, the atom molar refractivity (AMR) values of the selected componen

Drug-Likeness and ADMET Profiling
In Table 5, the ADME results of the top-five ligands measured using the SWISS-ADME server are displayed. Luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol have molecular weights of 286.24, 302.24, 272.25, 360.31, and 286.24 g/mol, respectively; the results of these phenolic substances suggested that they could be easily transported, distributed, and absorbed through the biological membranes [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. In addition, luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol were found to have LogP values of 1.73, 1.23, 1.84, 1.52, and 1.58, respectively, which are consistent with Lipinski's rule of five. Additionally, the hydrogen bond donor and acceptor numbers of these ligands were less than five and less than ten, respectively, which satisfies the ADME standards of H-bond donors and acceptors. The ADME results showed that luteolin, quercetin, naringenin, rosmarinic acid, and kaempferol had topological polar surface (TPSA) values of 111.13, 131.36, 86.99, 144.52, and 111.13 Å2, respectively. These values fall within the acceptable range not exceeding Å2, as outlined earlier by Cecchelli [41]. Furthermore, the atom molar refractivity (AMR) values of the selected components varied between 71.57 for naringenin and 91.40 for rosmarinic acid, both of which fall within the margins designated for this criterion between 40 and 130 [42]. Compound solubility is another important biochemical criterion for the choice of a biologically active molecule. LogS is the measurement that represents this parameter. For the five best polyphenolic compounds, this measurement was between −3.71 for luteolin and −3.16 for quercetin, reflecting good solubility, which corresponds to fairly good absorption and distribution. The drug-likeness analysis of the top-five ligands of LC-MS/MS revealed that these metabolites have an advantageous pharmacological profile and can thus be classified as drug-like substances. The toxic behavior of the five ligands was also studied, and its results are reported in Table 6. The results from this analysis are more or less reassuring of the safety of these molecules. None of the ligands are hepatotoxic or androgenic disruptors. However, quercetin showed potential for carcinogenic and mutagenic activity, and similar to galantamine, chlorogenic acid may pose an immunotoxicological risk.