Part2: Bioactive Compounds From Ephedra Fragilis: Extraction Optimization, Chemical Characterization, Antioxidant And AntiGlycation Activities

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2.7.Antiglycation Activity 

2.7.1.UV-Visible Analysis

The UV-vis spectrum is a fast, consistent, and simple technique commonly used to detect protein conformational changes and complex formation. The absorption spectra of native and glycated BSA incubated for 15 days in the presence or absence of CEE/fractions as well as quercetin(positive control) are presented in Figure 3A.

It was clearly shown that the native BSA exhibits a characteristic peak at 入2s0 nm， which is mostly due to the aromatic amino acids, including tyrosine, tryptophan, and phenylalanine [47].

Upon modification with glucose， absorbance at λ280 nm was 60.57% more hyperchromic than native BSA. The increased absorption intensity at 入2s0 nm can be attributed to the glycation-induced unfolding of the protein helix, which can affect its normal physiological function.

Treatment with CEE/fractions reduced significantly the absorbance at入2so nm compared to glycated BSA, and this reduction varied markedly between fractions according to the solvent polarity. Overall, descending antiglycation activity was portrayed as EAF>WBF>DMF>CCE>WF>HE,which were 1.82,1.71,1.57,1.35,1.28,and 1.13-fold lower than glycated BSA. Nevertheless, this activity was markedly lower than that of quercetin used as a positive control (2.24-fold lower than glycated BSA). So, it can be clearly concluded from absorption studies that EAF from E.fragilis possesses a protective effect against BSA unfolding induced by protein glycation.

2.7.2. Inhibition of Protein Glycation in the BSA-Glu Model

AGEs are a heterogeneous group of compounds with fluorescence characteristics at λ440 nm when excited at入370 nm. The ability of CEE and its various fractions from E. fragilis to inhibit AGEs formation was evaluated using the BSA-glucose assay, and the results are presented in Figure 3B.

As evidenced from Figure 3B, the AGEs inhibition rate of all tested samples exhibited an upward trend with the increase of concentrations. At 1 mg/mL, CCE, HE, DME EAE, WBF, WF, and quercetin inhibited AGEs formation by 53.26,39.83,54.82,76.68,69.09,48.83, and 97.84%, respectively, after incubation for 15 days. The EAF(ICs =0.375±0.034 mg/mL)was the most effective AGEs inhibitor among all fractions, followed by WBF, DMF, CCE, and WF with IC50 values of 0.595 ± 0.047, 0.857 ± 0.018, 0.951 ± 0.099, and 1.044±0.032 mg/mL, respectively. HF showed the weakest activity with the ICso value as 1.212±0.063 mg/mL.

The high antiglycation potential of EAF could be due to the high amount of phenolic and flavonoid contents, which have been described as very good inhibitors of AGEs formation [48]. Higher antiglycation of the EAF of Liquidambar formosana Hance leaf extract was also reported by Zhang et al. [31]as compared with that of its dichloromethane, n-butanol, and water fractions.

As given in Table5, AGEs inhibition was strongly corelated in a positive manner to both TF(r=0.950;p<0.01)and TP(r=0.972;p<0.01)contents, which were in line with previously reported studies [2,29]. The AGEs inhibition was also correlated in a positive way to DPPH",ABTS,H2O2,reducing power, TAC,andβ-carotene assays withr= 0.930,

r=0.914,r=0.983, r=0.975,r=0.923,and r=0.963 (p<0.01),respectively. These reflected that AGEs inhibition is linked to the efficiency of primary antioxidants [6]. Kaewseejan and Siriamornpun [29] also reported that phenolic compounds prevented the formation of AGEs through its free radicals scavenging and antioxidant capacities.

2.7.3. Effects on Glycation-Induced Protein Oxidation

Glycation of proteins (Maillard reaction) is a reaction started by the covalent attachment of reducing sugar to an amino group of proteins (mainly lysine and arginine residues), which leads to producing an unstable and reversible product i.e., Schiff's base that further undergoes Amadori rearrangement to form more stable ketamine's named Amadori products. Subsequently, Amadori products undergo an enediol reaction to produce carbonylated proteins [48]. The degradation of this ketamine could generate free radicals such as superoxide radicals, which are further converted into HO·via Fenton reaction, causing oxidative and cellular damage [49].

Protein oxidation is accompanied by carbonyl protein formation and loss of protein thiols, which are often employed as protein oxidation indicators[50]. As given in Figure 3C, the level of carbonyl content in native BSA was 1.16±0.04 nmol/mg protein, which was increased to more than 3.62-fold (4.21 ± 0.10 nmol/mg protein) upon glycation. The treatment with CEE and its various fractions reduced the level of carbonyl content with the increase of samples concentrations ranging from 0.1 to 1 mg/mL. Furthermore, the inhibition effect of quercetin on the formation of carbonyl proteins was stronger than that of all fractions at every concentration point. When the concentration was 1 mg/mL, CEE, HF, DMF, EAF, WBF, WF, and quercetin decreased the level of carbonyl content by 42.29, 17.37,58.68,73.44,69.17,28.84,and 98.68%, respectively, compared to native BSA. Overall descending, the inhibition of carbonyl content formation was portrayed as Q>EAF>WBF >DMF > CCE> WF >HF.

The effects of CEE/fractions on glycation-induced protein thiol oxidation are presented in Figure 3D. In native BSA, the level of protein thiol was 1.06± 0.086nmol/mg protein, which was decreased by more than three-fold(0.34± 0.021 nmol/mg protein) in glycated protein. In the presence of CEE/fractions, the level of the thiol group was significantly increased in a dose-dependent manner ranging from 0.1 to 1 mg/mL. Moreover, the level of protein thiol increased in the following order: HF<WF< CEE<DMF< WBF<EAE, and its corresponding levels at 1 mg/mL were 0.52±0.05,0.56±0.048, 0.64± 0.01,0.69±0.045, 0.82 ±0.081, and 0.95±0.059 nmol/mg protein, respectively, which were less effective than quercetin (1.03± 0.033 nmol/mg protein).

Similar results were observed in the plant Teucrium polium since the EAF was more effective than the other fractions (diethyl ether and water fractions) against glycation-mediated protein oxidation[51]. In their study, Golshahi and Bahramikia [52] also reported similar results when using several solvents with increasing polarity(diethyl ether and water) in the splitting of a medicinal plant Trachyspermum copticum. According to these authors, the EAF has the most potent protective effect against glycation-mediated protein oxidation, distantly followed by the diethyl ether and water ones. This demonstrates the presence in EAF of such compounds that might possess a preventive effect against hyperglycemia-induced oxidative damages to protein, which is believed to occur under the glycoxidation processes by reducing protein carbonyl formation and protecting protein thiols from oxidation as suggested by data.

2.8.Identified Phenolic Compounds in the EAF

The EAF, which showed the highest biological activity from other fractions, was selected for the identification of its main bioactive compounds by RP-HPLC. A total number of six compounds were identified by comparing their retention time with those of reference standards. The identified phenolic compounds are presented in Figure 4. Gallic, vanillic, caffeic, and ferulic acids were identified as phenolic acids, whereas only two compounds, namely rutin and quercetin, were identified as flavonoids. According to a study by Soumaya et al. [53], ferulic acid, luteolin-7-O-glucoside, myricetin, and kaempferol 3-O-rutinoside were identified as present in the EAF of aerial parts of Tunisian E.fragilis, whereas the presence of rutin, quercetin, gallic acid, and caffeine acid was only detected for the first time in our study. The disparity in the chemical composition of the EAF obtained from the same plant species can differ in different parts of a plant, the stage of plant development, the growth conditions (e.g., soil, light, temperature, water, humidity, and fertilizers), harvesting time, the drying system, and the extraction procedure [54]. The obtained results from the phytochemical fingerprint profile showed a good wealth of E.fragilis that had several phenolic compounds that are considered major contributors to the free radicals scavenging and antioxidant activities [55]. In addition, these compounds are known for their powerful antiglycation capacities [48]. Several studies have revealed the direct connection between the antioxidant activities of phenolic compounds and their antiglycation capacities.

2.9. Molecular Docking Study of Identified Compounds

To clearly visualize the detailed mechanism by which the identified compounds in the EAF of E. fragilis bind with BSA and RAGE, we performed a molecular docking study. Docking results are presented in Table6, while interactions between the most active compound and targets are shown in Figure 5. Results showed that quercetin snugly fitted into the binding site, located in the hydrophobic cavity of subdomain IB of BSA with the lowest binding energy of-7.7 kcal/mol (Figure 5A).In contrast, lesser binding energy was obtained with ferulic acid, vanillic acid, caffeic acid, gallic acid, and rutin (-6.35,-6.05, -5.84,-5.25 and-4.41 kcal/mol, respectively). Usually, a high degree of negativity of binding energy is more effective and the compound would be used for controlling the glycation processes. From Figure 5B, it is clear that quercetin forms eight hydrogen bonds with SER109, ASP111, LEU112, LEU115, ARG144, ARG185, and ARG458of BSA, and four hydrophobic interactions mediated by the aliphatic amino acids(PRO110, PRO113, LYS114, and ARG144). Also, four amino acids(ASP108, HIS145, LEU189, and LEU462)surrounding quercetin interacted via van der Waal's forces. It has been reported that lysine and arginine are the main amino acid residues involved in the glycation process [56]. Therefore, the masking of quercetin to lysine and arginine residues could be one of the possible mechanisms of E. fragilis to inhibit protein glycation at an initial stage.

Engagement of AGEs products with RAGE is known to trigger, through ROS formation via NADPH oxidase and mitochondria [57], the activation of multiple intracellular signaling pathways (including JAK/STAT, phosphoinositol-3 kinase, rho GTPases, SAPK/INK MAP kinases, p38 and erk1/2 (p44/p42) MAP kinases), and culminating in the activation of the NF-kB transcription factors [58], leading to the pathogenesis of diabetes and aging-associated disorders [5]. Therefore, blocking the AGEs-RAGE interactions can repress stress-provoking signals transduction, which is considered a therapeutic strategy of inhibiting glycation at a later stage. Docking results with RAGE, as shown in Table 6, proved that gallic acid has the highest docking score(G=-6.8 kcal/mol) in comparison to those of vanillic acid, ferulic acid, caffeic acid, quercetin, and rutin(-6.68,-5.94,-5.89,-5.58, and -4.89 kcal/mol, respectively). Moreover, 2D modeling of gallic acid and RAGE showed that gallic acid formed five conventional hydrogen bonds with CYS38, GLY40, ALA41, LYS43, and SER83 of RAGE(Figure 5C, D). Also, LYS37 and LYS43 were responsible for the hydrophobic interactions of gallic acid with RAGE. Five amino acids surrounding gallic acid (GLU32, LYS39, PRO42, ASN81, and GLY82) were attached by van der Waal's forces, thereby stabilizing the gallic acid-RAGE complex by providing a strong cohesive environment. In summary, the current study has shown the efficient interaction of certain bioactive compounds in the EAF of E. fragilis with the target proteins of BSA and RAGE.E.fragilis could be a source of potential competitors to glucose and AGEs, which might resist their binding towards BSA and RAGE, respectively, and therefore reducing the subsequent development of oxidative stress and inflammation(Figure 6).

3. Materials and Methods 

3.1. Chemicals and Reagents

22-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS), butylated hydroxy-toluene (BHT), sodium azide, guanidine hydrochloride, tween-40,1,1-diphenyl-2-picrylhydrazyl (DPPH), Folin Ciocalteau reagent,β-carotene, linoleic acid, glucose, 2,4-dinitrophenylhydrazine(DNPH),5,5'-Dithiobis-(2-Nitrobenzoic Acid)(DTNB), ammonium molybdate, and polyphenols standards were purchased from Sigma-Aldrich(St. Louis, MO, USA). Hydrogen peroxide (H2O2)was purchased from Fluka (Basel, Switzerland). Aluminum chloride (AICl3), sodium carbonate (NazCO3), ferric chloride(FeCl3), potassium persulphate (K2S2Os), potassium ferricyanide (K3Fe(CN)6), sulfuric acid, and all solvents were obtained from Merck Life Science (Darmstadt, Germany). Ascorbic acid and trichloracetic acid (TCA) were obtained from Scharlau (Barcelona, Spain).

3.2.Plant Materials

E. fragilis (aerial parts) was collected from Dour Lagfifat, Oulad Teima, Taroudant, Morocco(latitude, 30°24'0"N; longitude,9°12'36"W) during May 2019. It was identified by Professor Najat ELKHIATI, a botanist from our institute, where a collection of voucher specimens was deposited. The plant was rinsed with distilled water, air-dried, powdered in a blender, and stored at 49C until use.

3.3.Experimental Design 

3.3.1.Selection of Variables

Many parameters are known to have significant effects on phenolic compounds extraction, such as the type solvent, solvent concentration, extraction time, and extraction temperature [59]. Different solvents such as acetone, methanol, and ethanol are suitable for the extraction of different phenolic compounds [16], but ethanol was selected as the solvent in this study, due to its edible safety and green manufacturing [60]. Therefore, all factors, including the ethanol concentration (X), extraction temperature (X2), and extraction time (X3) were selected as variables.

3.3.2. BBD for Extraction Optimization

An optimization procedure was developed using RSM to determine the effects of extraction factors and choose the optimum experimental extraction conditions of E. fragilis phenolic compound. A three-level, three-factor BBD was undertaken to investigate the impact of three independent factors including X1(ethanol concentration, %), X,(extraction temperature,°C), and X3(extraction time, h) on TP and TF contents of E, fragilis extracts [17]. For optimization purposes, a total number of 15 trials including three center points were carried out randomly(Table 1). Based on our preliminary single factor experiment (data not shown), all variables were set at three levels(-1,0 and+1),with X,(40,60 and 80%), X,(25,42.5 and 60°C), and X3(6, 15 and 24 h)(Table 1). The following second-order polynomial equation (Equation (1))was used to fit the response variables:

where Y is the predicted response;βo, β; βi, and β; are the regression coefficients for intercept, linear, quadratic, and interaction terms, respectively; and X;, and X; are the independent variables (i ≠ j).

3.3.3. Extraction Procedure

The powdered sample(10 g) was extracted by maceration method in a designed ethanol concentration(40-80%;1:10, w/v),at varying temperatures(25-60°C) for various periods (6-24 h) on an orbital shaker incubator(160 rpm). Gauze and Whatman filter paper no.1 were used to remove the insoluble mass. The filtrate was then dried at 40°C under low pressure using R-3 Rotavapor(Büchi) to yield the crude ethanolic extract (CEE).

3.4. Fractionation of the CEE Obtained under Optimum Condition

The CEE obtained under optimum condition was solubilized in distilled water (100 mL), and liquid-liquid extraction was performed with various solvents of increasing polarity to yield hexane fraction (HF,3×100 mL), dichloromethane fraction (DMF, 3×100 mL), ethyl acetate fraction(EAF.3×100 mL), a water-saturated n-butanol fraction (WBF, 3× 100 mL),

and the remaining water fraction (WF). These fractions were then filtered and dried as described above, and the extraction yield was recorded according to Equation (2):

Were Wo and W are the weight of dried CEE/fractions and initial weight of E.fragilis powder; respectively.

3.5. Phytochemical Analysis

The spectrophotometric techniques used to evaluate the phytochemical contents of E. fragilis extracts are detailed in the Supplementary Materials [61,62].

3.6. Biological Activities

Details of the antioxidant [43,63-67] and antiglycation activities [68-70] tests in vitro were given in the Supplementary Materials.

3.7.RP-HPLC Analysis of EAF

Analysis of phenolic compounds in the EAF was performed with Agilent 1100(Agilent Technologies, Santa Clara, CA, USA)equipped with a ZORBAX Eclipse SB-C18 reversed-phase analytical column of 100×4 mm and 3.5 um particle size [71]. The column temperature was kept constant at 48°C. Isocratic elution with acetonitrile, 0.1%acetic acid in water (12:88, v/v) as mobile phase, and 1 mL/min flow rate ensured good separation of polyphenols in the EAF of E.fragilis. The injected volume was 10 μL and chromatograms were measured at 330 nm. The retention times of phenolic compounds in the EAF were compared to those of purely available standards to identify them.

3.8. Molecular Docking

AutoDock Tools(ADT) version 1.5.6 was used to perform a molecular docking study. SDF format of all compounds was obtained from PubChem database and then converted to 3Dpdb file using Open Babel GUI (version 2.4.1). The crystal structures of BSA(PDB ID:4ORO) and RAGE(PDB ID:4LP5)were collected from the RCSB Protein Data Bank (PDB). Briefly, the proteins were firstly prepared for docking by (i) removing all heteroatoms and water molecules,(i) adding polar hydrogen atoms, and (ii) assigning Kollman charges. The grid box dimension was set to x=126,y=126,z=126 and x=80,y=80.Z= 90 with grid center of×=8.415,y=21.626,z=106.57 andx=37.98,y=-43.581,z=9.371 with a grid spacing of 0.375A created around the binding site of BSA and RAGE, respectively. The docking software was run 100 times using the Lamarckian Genetic Algorithm(LGA)to find the best binding pose. The ligand with the lowest binding energy score was chosen for further investigation. Discovery Studio software version 2020 (BIOVIA, San Diego, CA, USA) was used for visualizing docking results.

3.9. Statistical Analysis

Design-Expert software version 119(Stat-Ease Inc., and Minneapolis, MN, USA) was used to perform RSM. Data analysis was performed by one-way analysis of variance (ANOVA)followed by Duncan's post-hoc test using SPSS 26.0(IBM Co., USA);p<0.05 was considered statistically significant. Pearson correlation analysis was conducted to investigate correlations between variables and their significance. All graphics were constructed using GraphPad Prism 7.0 software (San Diego, CA, USA). All experiments were conducted in triplicate and presented as mean values ±standard deviation (SD).

4. Conclusions

RSM with a BBD was employed to set the optimized parameters for extraction of the bioactive compounds from the Moroccan medicinal herb E. fragilis. The optimum ethanol concentration, extraction temperature, and extraction time were predicted for maximum

extraction yield of phenolic compounds and showed to be 61.93%, 44.43℃C, and 15.84 h, respectively. The CEE obtained under optimum extraction conditions and its various fractions were analyzed for their TP and TF contents, as well as their antioxidant and antiglycation activities. The EAF fraction shows the highest TP and TF contents and the strongest antioxidant activities compared to other fractions. Also, the evaluation of several biomarkers such as UV-vis absorption spectrum, specific AGEs fluorescence, carbonyl content, and free thiols group, showed the greatest protective effect of EAF against glycation mediated by glucose. Furthermore, a significant positive relationship was observed between the antioxidant capacities of phenolic compounds and their antiglycation activities. This indicates that phenolics compounds may be the main predominant components responsible for both antioxidant and antiglycation activities. The bioactive compounds in the EAF were characterized by RP-HPLC analysis and a total number of six compounds were identified. In silico molecular docking analysis also displayed an effective interaction between quercetin and gallic acid with BSA and RAGE as target proteins, respectively. Collectively, this study suggests that E.fragilis might be a potential source of natural bioactive compounds with powerful antioxidant and antiglycation activities and should be applied in the treatment and prevention of aging and glycation-associated complications. Further studies on bioactive compounds isolation and pharmacological screening (i.e., cytotoxicity study) need to be conducted to explore the phytochemistry and mechanisms of action of pharmacological properties of E. fragilis.

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