1. Introduction

Figure 1. Oxidative stress and its mechanism in osteoarthritis; The primarily formed radicals, superoxide radical (O^2-•), can be converted to more harmful species such as hydroxyl radicals (OH^•) and hydrogen peroxide (H₂O₂). Imidazolium RvD1 analogues as antioxidant molecules scavenge ROS via their active sites to protect against the degradation of high molecular weight hyaluronic acid (HMW HA) to low molecular weight hyaluronic acid (LMW HA).

Figure 1. Oxidative stress and its mechanism in osteoarthritis; The primarily formed radicals, superoxide radical (O^2-•), can be converted to more harmful species such as hydroxyl radicals (OH^•) and hydrogen peroxide (H₂O₂). Imidazolium RvD1 analogues as antioxidant molecules scavenge ROS via their active sites to protect against the degradation of high molecular weight hyaluronic acid (HMW HA) to low molecular weight hyaluronic acid (LMW HA).

2. Materials and Methods

2.2. Analogues synthesis

2.2.1. Block A synthesis

The block A synthesis sequence was adapted from Gaetano et al. [37].

(i) 2-deoxy-3,4-O-(1-methylethylidene)-D-erythro-pentopyranose (1)

To a solution of 2-desoxy-D-ribose (18.7 mmol, 2.5 g) in anhydrous DMF (25 mL) under an argon atmosphere was added dierite dessicant (1.0 g), dimethoxy-propane (37 mmol, 3.8 g, 4.5 mL) and p-TSA (0.55 mmol, 0.1 g). The solution was stirred 3 h at room temperature. Triethylamine (TEA) (0.1 mL) was then added before to be filtered over celite pad. The filtrate was evaporated under pressure. The crude compound was purified by flash chromatography with EtOAc/Hexanes (7:3 + 1% TEA) to give 1.7 g (53 %) of compound 1. ¹H NMR spectra was in full agreement with literature data.

(ii) (Methoxycarbonyl propyl)triphenylphosphonium bromide (2)

To a solution of methyl-4-bromo-butanoate (25.0 g, 140 mmol) in toluene (250 mL) was added triphenylphosphine (33.5 g, 170 mmol) and stirred at 100°C for 24 h. The resulting solid was then filtered and washed with cold toluene to give compound 2 as a white solid (12.0 g, 20%).

(iii) Methyl (4Z)-6-[(4S,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl]hex-4-enoate (3)

To a solution of phosphonium salt 2 (9.6 g, 21 mmol) in anhydrous THF (60 mL) at -78 °C under an argon atmosphere was dropwise added KHMDS (1.0 M in THF, 20 mmol, 20.0 mL) over 10 min. The solution was then allowed to return at 0°C and was stirred for 1 h. To the later solution cooled down at -78°C was added compound 1 (1.5 g, 8.7 mmol) and stirred 20 min at this temperature before to be warmed at room temperature and stirred for an additional 30 min. The resulting solution was poured into a saturated ammonium chloride solution and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The resulting crude compound was purified by flash chromatography with EtOAc/Hexanes (1:1 + 1% TEA) to give 0.67 g (32%) of compound 3. ¹H NMR spectra was in full agreement with literature data.

(iv) Methyl (4Z)-6-[(4S,5S)-5-formyl-2,2-dimethyl-1,3-dioxolan-4-yl]hex-4-enoate (4)

To a solution of compound 3 (2.8 mmol, 0.67 g) in dichloromethane (DCM) (10 mL) was dropwise added a solution of Dess-Martin periodinane (5.6 mol, 2.4 g) in 10 mL of DCM. The solution was stirred for 2 h and then poured into a saturated solution of sodium bisulfite. The solution was then extracted with EtOAc. The organic layer was washed with a saturated solution of sodium bicarbonate, and then with brine, dried with sodium sulfate, filtered and evaporated under reduce pressure. The resulting crude product 4 was used as such toward next step. ¹H NMR spectra was in full agreement with literature data.

(v) methyl (4Z)-6-[(4S,5R)-5-ethynyl-2,2-dimethyl-1,3-dioxolan-4-yl]hex-4-enoate (5)

To K₂CO₃ (7.9 mmol, 1.1 g) under an argon atmosphere was dropwise added a solution of phosphonate (3.9 mmol, 0.67 g) in MeOH (10 mL). To the later solution was dropwise added a solution of aldehyde 4 (2.8 mmol, 0.67 g) in MeOH (45 mL) at room temperature and the solution was stirred for 3 h at room temperature. The resulting solution was poured into water, extracted with EtOAc, washed with brine, dried over sodium sulfate, filtered and evaporated under reduce pressure. The crude compound was purified by flash chromatography with EtOAc/hexanes (3:7 + 1% TEA) to give 330 mg (50%) of compound 5. ¹H NMR (400 MHz, Acetone- d₆) 5.57 – 5.36 (m, 2H), 4.77 (dd, J = 5.5, 2.2 Hz, 1H), 4.10 (ddd, J = 7.2, 6.5, 5.5 Hz, 1H), 3.59 (s, 3H), 3.05 (d, J = 2.2 Hz, 1H), 2.56 – 2.42 (m, 2H), 2.42 – 2.26 (m, 2H), 1.43 (s, 3H), 1.26 (s, 3H).¹³C NMR (100.6 MHz, Acetone-d₆) 172.7, 130.1, 125.8, 110.0, 109.3, 77.4, 76.3, 68.8, 50.7, 33.3, 29.4, 27.2, 25.6, 22.8 ppm. LRMS (APCI pos) m/z 253.1 [M + H].

(vi) Methyl (4Z)-6-{(4S,5R)-2,2-dimethyl-5-[(E)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethenyl]-1,3-dioxolan-4-yl}hex-4-enoate (Block-A)

In a microwave vial under an argon atmosphere was added compound 5 (1.3 mmol, 330 mg), TEA (0.4 mmol, 0.04 mL) and pinacolborane (2.5 mmol, 0.38 mL). At this mixture was added the Schwartz reagent (0.13 mmol, 34 mg) and the solution was heated at 60°C for 36 h. The resulting solution was poured into water, extracted with EtOAc, washed with brine, dried over sodium sulfate, filtered and evaporated under reduce pressure. The crude compound was purified by flash chromatography with EtOAc/hexanes (2:8 + 1% TEA) to give 105 mg (21%) of compound Block-A. ¹H NMR (400 MHz, Acetone- d₆) ) δ 6.60 – 6.39 (m, 1H), 5.64 (dd, J = 18.0, 1.4 Hz, 1H), 5.44 – 5.33 (m, 2H), 4.58 (td, J = 6.4, 1.4 Hz, 1H), 4.23 (dt, J = 7.7, 6.3 Hz, 1H), 3.58 (s, 3H), 2.41 – 2.24 (m, 4H), 2.18 (td, J = 7.4, 6.7, 4.5 Hz, 2H), 1.41 (d, J = 0.7 Hz, 3H), 1.28 (d, J = 0.7 Hz, 3H), 1.22 (s, 12H).¹³C NMR (100.6 MHz, Acetone-d₆) 172.6, 148.8, 129.6, 126.5, 108.0, 83.0, 79.8, 77.9, 50.7, 33.3, 27.5, 24.8, 24.2, 22.9 ppm. LRMS (APCI pos) m/z 381.2 [M + H].

2.2.2. Block B synthesis

The block B was prepared following a literature procedure [37], as shown in Figure 2. Briefly, 1,2-dimethylimidazole was treated with N-bromosuccinimide (2.2 eq) in chloroform and reacted for 14 h at room temperature to give the dibromo-imidazole compound – Block B. Reaction was quenched and compound isolated and/or purified. ¹H NMR data were in full agreement with that reported in the literature.

Figure 2. Chemical synthesis of Block A and Block B. Reagents, conditions and reaction yields.

Figure 2. Chemical synthesis of Block A and Block B. Reagents, conditions and reaction yields.

2.2.3. RvD1 analogues 1 and 2 syntheses

(i) 1-(4-bromo-1,2-dimethyl-1H-imidazol-5-yl)hexan-1-one (6)

This compound was prepared following a literature procedure [37] (Figure 3). ¹H NMR data were in full agreement with that reported in the literature.

Figure 3. Chemical synthesis of RvD1 analogues 1 and 2. Reagents, conditions and reaction yields.

Figure 3. Chemical synthesis of RvD1 analogues 1 and 2. Reagents, conditions and reaction yields.

(ii) Methyl (4Z)-6-{(4S,5R)-5-[(E)-2-(5-hexanoyl-1,2-dimethyl-1H-imidazol-4-yl)ethenyl]-2,2-dimethyl-1,3-dioxolan-4-yl}hex-4-enoate (7)

In a 20 mL microwave vial, compound 6 (54 mg, 0.20 mmol) and Block A (100 mg, 0.26 mmol) were solubilised in DMF (1.2 mL) and 2M K₂CO₃ (0.2 mL). Argon was bubbled through the mixture for 15 min before addition of Pd(PPh₃)₄ (20 mg. 0.016 mmol). After sealing, the tube was heated in a microwave apparatus at 115^oC for 1 h. After cooling at room temperature, water was added and the aqueous phase extracted with EtOAc. The combined extracts were washed with water, brine and dried over Na₂SO₄. The residue was purified on silica gel eluting with toluene-EtOAc-ether (90:10:10 to 70:30:10) affording compound 7 (61 mg, 70%). ¹H NMR shows contamination with triphenylphosphine oxide and (1,2-dimethyl-1H-imidazol-5-yl)hexan-1-one. ¹H NMR (400 MHz, Methanol-d₄) δ 6.86 (d, J = 15.3 Hz, 1H), 6.43 (dd, J = 15.3, 6.8 Hz, 1H), 5.33 (m, 2H), 4.68 (m, 1H), 4.21 (q, J = 6.6 Hz, 1H), 3.65 (s, 3H), 3.49 (s, 3H), 2.29 (s, 3H), 2.18 (s, 6H), 1.58 (m, 2H), 1.41 (s, 3H), 1.26 (m, 7H), 0.81 (bs, 3H).

(iii) Methyl (4Z)-6-[(4S,5R)-5-{(E)-2-[5-(1-hydroxyhexyl)-1,2-dimethyl-1H-imidazol-4-yl]ethenyl}-2,2-dimethyl-1,3-dioxolan-4-yl]hex-4-enoate (8)

To an ice-cooled solution of compound 7 (60 mg, 0.13 mmol) in MeOH (1 mL) was added NaBH₄ (10 mg, 0.26 mmol). The mixture was stirred at 5^oC for 1 h then quenched with a 10% aqueous solution of NaH₂PO₄. The aqueous phase was extracted twice with EtOAc. The combined extracts were washed with water, brine and dried over Na₂SO₄. Evaporation gave quantitatively compound 8 (60 mg). ¹H NMR (400 MHz, Methanol-d₄) δ 6.59 (d, J = 15.6 Hz, 1H), 6.21 (dd, J = 15.6, 8.1 Hz, 1H), 5.43 (m, 2H), 4.68 (m, 1H), 4.21 (m, 1H), 3.65 (s, 3H), 3.62 (s, 3H), 2.30 (s, 3H), 2.18 (s, 3H), 1.92 (m, 1H), 1.76 (m, 1H), 1.49-1.19 (m, 6H), 0.88 (bs, 3H).

(iv) Methyl (4Z,7S,8R,9E)-7,8-dihydroxy-10-[5-(1-hydroxyhexyl)-1,2-dimethyl-1H-imidazol-4-yl]deca-4,9-dienoate (RvD1 analogue 2)

To a solution of crude compound 8 (60 mg, 0.13 mmol) in MeOH (1 mL) was added p-TSA (39 mg, 0.21 mmol) at 5^oC and stirred for 4 h. Triethylamine was added and MeOH was evaporated under reduce pressure. The residue was poured into water and extracted with EtOAc. The combined extracts were washed with water, brine, dried over Na₂SO₄, filtered and evaporated under reduced pressure to give crude material. This later compound was purified by flash chromatography using DCM/MeOH (9:1) as eluent to give compound RvD1 analogue 2 (33 mg, 60%). ¹H NMR (400 MHz, Methanol- d₄) δ 6.58 (d, J = 15.7 Hz, 1H), 6.27 (dd, J = 15.1, 7.3 Hz, 1H), 5.55 (m, 2H), 5.45 (m, 1H), 4.58 (m, 1H), 4.08 (m, 1H), 3.64 (s, 3H), 3.59 (s, 3H), 2.37 (m, 3H), 1.93-1.71 (m, 2H), 1.49-1.17 (m, 6H), 0.88 (bs, 3H). ¹³C NMR (100.6 MHz, Methanol- d₄) 174.0, 146.0, 149.9, 133.5, 130.1, 128.9, 127.4, 127.2, 126.6, 126.5, 121.6, 75.5, 75.4, 74.6, 74.5, 64.9, 50.6, 36.1, 36.0, 33.4, 33.3, 31.4, 31.3, 30.7, 30.2, 25.5, 25.4, 22.6, 22.3, 12.9, 11.2. LRMS (APCI pos) m/z 409.3 [M + H]. HPLC purity = 99.6%.

(v) (4Z,7S,8R,9E)-7,8-dihydroxy-10-[5-(1-hydroxyhexyl)-1,2-dimethyl-1H-imidazol-4-yl]deca-4,9-dienoic acid (RvD1 analogue 1)

To a solution of compound RvD1 analogue 2 (22 mg, 0.06 mmol) in MeOH (0.5 mL) was added LiOH (16 mg, 0.38 mmol). The mixture was stirred at 4^oC overnight and then poured into aqueous solution (10%) of NaH₂PO₄. The aqueous solution was extracted with EtOAc, washed with brine, dried over sodium sulfate, filtered and evaporated under reduce pressure to give 17 mg (80%) of compound RvD1 analogue 1 as a yellow solid. ¹H NMR (400 MHz, Methanol- d₄) δ 6.65 (d, J = 15.7 Hz, 1H), 6.42 (dd, J = 15.1, 7.3 Hz, 1H), 5.51 (m, 2H), 4.94 (m, 1H), 4.18 (m, 1H), 3.75 (s, 3H), 3.65 (m, 1H), 2.51 (s, 3H), 2.47-2.38 (m, 2H), 2.38-2.23 (m, 4H), 1.93-1.77 (m, 2H), 1.49-1.19 (m, 6H), 0.90 (bs, 3H). ¹³C NMR (100.6 MHz, Methanol- d₄) 177.2, 145.6, 145.5, 130.9, 130.8, 130.2, 130.1, 129.9, 129.3, 126.4, 126.1, 117.5, 117.4, 74.43, 74.41, 74.4, 74.3, 64.7, 64.6, 35.96, 35.9, 34.8, 31.33, 31.32, 31.3, 30.4, 30.3, 29.3, 25.34, 25.33, 23.3, 22.2, 12.9, 10.1, 10.0. LRMS (APCI pos) m/z 395.3 [M + H]. HPLC purity = 100.0%.

All results of analogues synthesis, including ¹H NMR spectrum, ¹³C NMR spectrum, LCMS chromatogram, and mass spectrum, were described in Figures S1-9.

3. Results and Discussion

3.1. Antioxidants and Radical Scavenging Activities of Resolvin Analogues

3.1.1. ORAC Assay

Several in vitro chemical assays have been developed and reviewed for measuring antioxidant capacity. The complicated mechanism among free radicals, substrates, and antioxidants makes it impossible to present an equation to express the kinetic order. Accurate quantification of antioxidant potential mandates the consideration of both inhibition degree, denoting the extent of free radical scavenging, and inhibition time, reflecting the persistence of the antioxidant effect. In this regard, the oxygen radical absorbance capacity (ORAC) assay stands out as the sole technique currently available that effectively integrates these complementary parameters into a single, informative value. Thus, this method is widely used in the nutraceutical, pharmaceutical, food industries, and biomedical studies [38]. In this assay, a radical-generating compound, 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH), produces biologically relevant oxygen radical species (ROS) via thermal decomposition in the presence of oxygen. As the ROS oxidizes the fluorescein, the fluorescence of the solution decreases over time. In the presence of an antioxidant, ROS can be scavenged, and the oxidation of fluorescein can be delayed until the antioxidant is depleted. Indeed, antioxidants protect fluorescein from ROS attacking by the way of hydrogen atom transfer (HAT) process (Figure 4).

Figure 4. ORAC assay mechanism; (A) Generating ROS (alkoxy radical) by thermal decomposition of 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH) in the presence of oxygen. (B) Reacting ROS with fluorescein (fluorescent probe) without antioxidant compounds and production of non-fluorescent molecule. (C) Inhibition of ROS by antioxidants and protection of fluorescein from radical attack.

Figure 4. ORAC assay mechanism; (A) Generating ROS (alkoxy radical) by thermal decomposition of 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH) in the presence of oxygen. (B) Reacting ROS with fluorescein (fluorescent probe) without antioxidant compounds and production of non-fluorescent molecule. (C) Inhibition of ROS by antioxidants and protection of fluorescein from radical attack.

In Figure 5, the typical time-dependent decays of fluorescence intensity of fluorescein induced by AAPH alone, or in the presence of antioxidants (final concentration range between 0.625 to 20 μM) were illustrated. After pre-incubation of fluorescein with different concentrations of the antioxidants (including Analogue 1, Analogue 2, RvD1, Ascorbic acid, and Trolox), dose-dependent inhibitions of the fluorescence decay were monitored. As seen in Figure 5A–E, both Analogues 1 and 2 showed the ability of free radical quenching in the radical propagation cascade. According to their shape of fluorescence decay curves, there is no clear distinction between lag time and fluorescein decay periods by increasing the concentrations of the Analogues indicating more complex scavenging kinetics. On the other hand, a very low change was observed for RvD1 by increasing the concentrations which means low scavenging kinetics at the tested condition (Figure 5C). Ascorbic acid and Trolox as controls were also tested to compare the antioxidant capacity of the samples. Although Ascorbic acid is a very strong antioxidant molecule, as seen in Figure 5D, it has a relatively low decay of fluorescence intensity at the tested concentration ranges. Trolox as another control showed a clear lag time proving that it has antioxidant properties to protect fluorescein from ROS (Figure 5E). As a blank control, fluorescein was exposed to excitation light at 485 nm in the absence of AAPH and antioxidants during the assay. The results proved that there is no significant fluorescence intensity change, thus, fluorescein is a stable fluorescent probe according to the analysis.

Figure 5. Signal curves for different antioxidant concentrations (0.625 to 20 μM) at 37 °C (Normalized to initial fluorescence signals after AAPH addition).

Figure 5. Signal curves for different antioxidant concentrations (0.625 to 20 μM) at 37 °C (Normalized to initial fluorescence signals after AAPH addition).

The antioxidant capacity correlates to the fluorescence decay curve, which is usually represented as the area under the curve (AUC) and calculated according to Equation 1. ORAC calibration curves are depicted in Figure 6 where Net AUC values were plotted against antioxidant concentrations. Trolox equivalent (TE) is a factor that presents the antioxidant capacity compared to Trolox as a standard. Based on this factor, both analogues have better antioxidant capacity compared to other tested antioxidants. Table 1 summarizes the ORAC values of all the samples expressed as μM concentration of Trolox equivalent.

Analogue 1 > Analogue 2 > RvD1 > Trolox > Ascorbic acid

Figure 6. ORAC calibration curves for Analogue 1 (A), Analogue 2 (B), RvD1 (C), Ascorbic acid (D), and Trolox (E).

Figure 6. ORAC calibration curves for Analogue 1 (A), Analogue 2 (B), RvD1 (C), Ascorbic acid (D), and Trolox (E).

Table 1. The Trolox Equivalent (TE) values for antioxidant samples.

Table 1. The Trolox Equivalent (TE) values for antioxidant samples.

Antioxidant	Analogue 1	Analogue 2	RvD1	Ascorbic acid	Trolox
TE Value	2.17	1.87	1.60	0.40	1

3.1.2. DPPH Assay

DPPH assay is one of the well-known and routinely practiced assessments of the free radical scavenging potential of antioxidant molecules since it is simple, quick, and relatively inexpensive. It is considered one of the standard and easy colorimetric methods for the evaluation of the antioxidant properties of pure compounds. This assay is based on the reduction of DPPH^• free radicals by antioxidant species accepting either a hydrogen atom or an electron (Figure 7). DPPH reagent is considered a stable radical and has limited similarities with peroxyl radicals [44]. Its stability originates from the delocalization of unpaired electrons on a nitrogen atom in the aromatic ring (diphenylamino group as an electron donor, and picryl as an electron acceptor). In this way, the DPPH radical does not dimerize like many other free radicals. The limited space surrounding the nitrogen atom sterically hinders the addition of bulky radicals to this region. Thus, the complex molecules with hydrogen donating sites (like RvD1 and its analogues) cannot approach the nitrogen atom and release the hydrogens there to the formation of hydrazine [45,46]. Since this assay is a colorimetric method, the reduction form of DPPH^• free radical (DPPH-H) can be followed by turning purple to a yellow color.

Figure 7. Reaction mechanism of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with two different types of antioxidants (small molecules and complex molecules) via hydrogen atom transfer (HAT) or single electron transfer (SET). Low possibility of interaction between complex molecules and DPPH radical due to the steric hindrance around the radical.

Figure 7. Reaction mechanism of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with two different types of antioxidants (small molecules and complex molecules) via hydrogen atom transfer (HAT) or single electron transfer (SET). Low possibility of interaction between complex molecules and DPPH radical due to the steric hindrance around the radical.

As all samples showed antioxidant activity in the ORAC assay at the range of 0.625-20 μM, the same range concentration was also chosen for the DPPH assay. Both control samples, Ascorbic acid and Trolox showed low scavenging percentages of 4.31±2.74 and 3.53±1.88 µM at 20 μM, respectively, whereas RvD1, Analogue 1, and Analogue 2 did not show any radical scavenging properties under the same conditions. To find the half-maximal inhibitory concentration (IC₅₀) value of the samples, higher concentrations in the range of 20-80 µM were also tested. IC₅₀ refers to the antioxidant concentration that scavenges 50% of the initial DPPH radicals in a specific but arbitrary time interval. The color change was monitored spectrophotometrically and utilized for the determination of parameters for antioxidant properties. In the cases of Ascorbic acid and Trolox, the purple color was changed to yellow with a concomitant decrease in absorbance at 515 nm. The IC₅₀ value was obtained for Ascorbic acid and Trolox around 66.87 µM and 67.13 µM, respectively. On the other hand, no radical scavenging was observed for RvD1, Analogue 1, and Analogue 2 even at higher concentrations.

It has been proved that some chemicals easily react with DPPH radicals by electron transfer or by donating hydrogen atoms. Especially phenolic compounds are the most reactive and important ones that react easily with DPPH• [47]. For instance, Trolox has a hydroxyl group on the aromatic ring, which shows good antioxidant activity via donating hydrogen mechanism, because the formed radical on Trolox can be stabilized by the delocalization within the aromatic ring. As mentioned above, sterically hindrance around the nitrogen atom of the DPPH radical inhibits all reactions, particularly the hydrogen transfer, which is necessary for the formation of a hydrogen-bonded complex. Complex molecules get in the way of each other more easily which blocks the access to DPPH radicals at low concentrations. Additionally, methanol, which is generally used as a solvent for the DPPH method, strongly binds H atoms and inhibits HAT processes. It may be concluded that DPPH^• radical might react with each antioxidant via different kinetics or might not react at all due to its stability. Furthermore, the reversibility of the reaction may lead to low readings of the antioxidant capacity of many antioxidants, consequently, the antioxidant capacity of many antioxidants is likely to be wrongly estimated.

3.1.3. FRAP Assay

The FRAP assay is a rapid and automated assay that measures the electron-donating ability of an antioxidant to reduce the ferric 2,4,6-tripyridyl-s-triazine complex [Fe³⁺-(TPTZ)₂]³⁺ to the intense, blue-colored ferrous complex [Fe²⁺-(TPTZ)₂]²⁺ under acidic condition (pH=3.6) [48]. Trolox equivalent (TE) is a unit of measurement used to express the antioxidant capacity of a substance relative to Trolox. All measurements for both Trolox and the sample should be done under the same conditions and concentrations [49]. The TE of ascorbic acid, RvD1, Analogue 1, and Analogue 2 were calculated based on the Trolox calibration curve. The TE values were obtained 242.53±46.16, 8.65±1.19, 12.71±2.51, and 14.12±2.14 µM at highest treated concentration (200 µM), respectively. Additionally, the relative percentage of reducing power were also calculated at the same concentrations and it was around 122.03%±22.81 for Ascorbic acid at 200 . However, this value for RvD1, Analogue 1, and Analogue 2 was much less, 4.12%±0.62, 6.17%±1.28, and 6.88%± 1.09, respectively (Figure 8). All data confirmed that the RvD1 analogues showed almost 2 times higher antioxidant activity compared to RvD1. To figure out why the analogues with potential antioxidant abilities have very little electron-donating ability compared to controls (ascorbic acid and Trolox), the redox condition mechanism should be considered. Redox conditions refer to media dominated by oxidants (electron acceptors), the substances that can oxidize other substances (cause them to lose electrons), or reductants (electron donors), the substances that can reduce other substances.

Figure 8. Relative percentage of reducing power of Ascorboc Acid, RvD1, Analogue 1, and Analogue 2 in compared to Trolox as a standard control using FRAP assay.

Figure 8. Relative percentage of reducing power of Ascorboc Acid, RvD1, Analogue 1, and Analogue 2 in compared to Trolox as a standard control using FRAP assay.

There are several possibilities that the RvD1 and its analogues are not able to donate electrons easily to the ferric ion (Fe³⁺) and reduce it to a ferrous ion (Fe²⁺). The first possible reason can be related to the standard reduction potentials (E° values) of the reactants which can vary depending on the specific reaction conditions and solvent properties. E° values represent the standard reduction potential under specific conditions of 25°C and 1 M concentration [50]. For example, the acidic condition of the FRAP reaction (pH=3.6) may cause the protonation of the antioxidant compounds and as a result, it prevents the transfer of electrons from the antioxidant to the ferric ion. In the case of Trolox, the specific E° value depends on the context of the reduction reaction. At neutral pH (7.4), the relevant E° value is around -0.48 V which indicates a stronger electron-donating tendency compared to the acidic condition (E° = -0.35 V). The E° value shifting at acidic pH is because of the protonation of the phenolic hydroxyl, affecting its electron-donating ability slightly. But in the case of ascorbic acid, the specific E° values at pH 7.4 and 4.5 are -0.28 V and -0.05 V, respectively. Although both Trolox and ascorbic acid are commonly used reference standards in the FRAP, Trolox generally has a more negative E°, indicating a stronger tendency to donate electrons and reduce ferric ions. This suggests it might show higher FRAP activity compared to ascorbic acid at neutral pH. On the other hand, from the mechanism point of view, Trolox donates a single electron via its phenolic hydroxyl group, but ascorbic acid can donate one or two electrons depending on pH and reaction conditions. Thus, the two-step electron-donating of ascorbic acid adds more complexity compared with Trolox. Regarding the RvD1 and its analogues, they showed much lower FRAP value compared to Trolox and ascorbic acid. It can be concluded that the E° value might be less negative to be able to reduce the Fe³⁺ with an E° value of +0.77V. To unravel the precise electron transfer processes involved in these antioxidants’ activity, a comprehensive electrochemical studies is necessary to be done.

3.1.4. Hydroxyl Radical Scavenging (HRS)

This experiment was designed to investigate the antioxidant properties of RvD1 and analogues using CuCl₂ + H₂O₂-induced ROS generation in the acellular environment. Trolox was used as a positive control. As illustrated in Figure 9, RvD1, Analogue 1, Analogue 2, and Trolox reduced significantly the production of ROS in a time-dependent manner. After 8 h of incubation, the generation of ROS was reduced by 30, 50, 77, and 86 by Analogue 2, RvD1, Trolox, and Analogue 1, respectively, as compared to CuCl₂+H₂O₂ alone. These findings confirm the antioxidant activity of RvD1 and our synthetic analogues.

Figure 9. RvD1 and its analogues attenuate CuCl₂ + H₂O_2-induced ROS production. ROS were induced in a black 96-well plate by the addition of CuCl₂ (10 μM) to the H₂O₂ (100 μM) in the presence or absence of RvD1, Analogue 1 (A1), Analogue 2 (A2), and Trolox (TRX) at 10 μM. DCFH-DA probe was added at a concentration of 10 μM. The results are expressed as relative fluorescence units (RFU) (n=3).

Figure 9. RvD1 and its analogues attenuate CuCl₂ + H₂O_2-induced ROS production. ROS were induced in a black 96-well plate by the addition of CuCl₂ (10 μM) to the H₂O₂ (100 μM) in the presence or absence of RvD1, Analogue 1 (A1), Analogue 2 (A2), and Trolox (TRX) at 10 μM. DCFH-DA probe was added at a concentration of 10 μM. The results are expressed as relative fluorescence units (RFU) (n=3).

3.2. Protection of Hyaluronic Acid against ROS

Hyaluronic acid (HA) is a linear polysaccharide consisting of two alternating units, β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine. HA, found in synovial fluid, acts as an elastoviscous component that creates a friction-free surface for joint movement. The molecular weight of HA is more variable and most commonly synthesized as a high-molecular-weight polymer in normal biological fluids and tissues, with an average molecular mass of approximately 1000–8000 kDa [51]. Studies have shown that HA concentration remains relatively stable with age or tends to decline between 28 and 40 years old and remains at a low level beyond that age. Its concentration in human synovial fluid ranges from 1.5 to 3.6 mg/mL. Hence, HA with an average molecular weight of 1500 kDa was dissolved in PBS buffer solution (pH 7.4, 150 mM) with a concentration of 2 mg/mL. Then, the HA solution was incubated with CuCl₂ + hydrogen peroxide (H₂O₂) without or in the presence of antioxidants (RvD1, Analogue 1, Analogue 2, and Trolox). To evaluate the radical scavenging capability of the antioxidants, the following studies were performed.

3.2.1. Agarose Gel Electrophoresis

Here, the capacity of RvD1 and analogues was investigated to block ROS-induced HA degradation. To do so, the polymer was treated with RvD1 and analogues and then after with CuCl₂ + H₂O₂ for 16 h. The degradation of HA was evaluated by gel agarose electrophoresis. As indicated in Figure 10, the addition of RvD1 and analogues protected CuCl₂ + H₂O_2-induced HA fragmentation and preserved its integrity of HA, as compared to the untreated HA.

Figure 10. RvD1, Analogue 1 (A1), Analogue 2 (A2), and Trolox (TRX) inhibit CuCl₂ + H₂O_2-induced hyaluronic acid (HA) degradation. A solution of commercial HA (1 mg/mL, MW: 1000-2000 kDa) was exposed to 10 μM CuCl₂ and 100 μM H₂O₂ for 16 h in the presence or absence of RvD1, analogues or TRX at 10 μM. Samples were then loaded in agarose gel/1X TBE and then hyaluronic acid was revealed using a stain-all solution (n=3).

Figure 10. RvD1, Analogue 1 (A1), Analogue 2 (A2), and Trolox (TRX) inhibit CuCl₂ + H₂O_2-induced hyaluronic acid (HA) degradation. A solution of commercial HA (1 mg/mL, MW: 1000-2000 kDa) was exposed to 10 μM CuCl₂ and 100 μM H₂O₂ for 16 h in the presence or absence of RvD1, analogues or TRX at 10 μM. Samples were then loaded in agarose gel/1X TBE and then hyaluronic acid was revealed using a stain-all solution (n=3).

3.2.2. Gel Permeation Chromatography

Based on GPC results (Figure 11), the average molecular weight of HA was about 1,160,000 Da with a dispersity (Mw/Mn) of 1.103 and intrinsic viscosity of 13.5227. These values were changed significantly after the incubation of HA with hydrogen peroxide. The GPC chromatogram showed a symmetric broad peak with an average molecular weight of 217,211 Da, a dispersity value of 1.651, and an intrinsic viscosity of 4.7605. The degradation is because the hydroxyl radical can abstract hydrogen atoms at all ring C-H bonds except C-2 of N-acetyl hexosamine. The abstraction of hydrogen atoms generates carbon-center radicals which undergo a β-scission reaction resulting in the breakdown of the HA chains. ROS-mediated HA degradation occurs randomly and generates not only glycosidic linkage cleavage but also the possibility of other structural changes, including ring opening and the occurrence of different conformational characteristics. However, the RvD1 analogues as antioxidants scavenged the formed free radical and protected the HA from degradation. The GPC results showed that the average molecular weights of HA were almost the same as the control (free HA) which obtained 1,031.000, 1,043,000, and 1,026,000 Da for RvD1, Analogue 1, and Analogue 2, respectively. It can be concluded that the analogues have very good potential to protect HA from ROS degradation. All data obtained are summarized in Table 2 below which includes individual injection results.

Figure 11. GPC/SEC Chromatogram. Molecular weight comparison of HA after incubation with CuCl₂+H₂O₂ without or in the presence of RvD1 and RvD1 analogues as radical scavengers.

Figure 11. GPC/SEC Chromatogram. Molecular weight comparison of HA after incubation with CuCl₂+H₂O₂ without or in the presence of RvD1 and RvD1 analogues as radical scavengers.

Table 2. GPC/SEC data of hyaluronic acid degradation with H₂O₂ without or in the presence of RvD1 and its analogues (Triple detection GPC results of the samples were calculated using Refractive Index Increment (dn/dc) value of 0.158 mL/g for Hyaluronic Acid in water).

Table 2. GPC/SEC data of hyaluronic acid degradation with H₂O₂ without or in the presence of RvD1 and its analogues (Triple detection GPC results of the samples were calculated using Refractive Index Increment (dn/dc) value of 0.158 mL/g for Hyaluronic Acid in water).

Sample	Vp1 (mL)	Mw (Da)	Mn (Da)	Mw/Mn2 (-)	IV3 (mg/mL)
Hyaluronic Acid	10.76	1,160,000	1,052,000	1.103	13.5227
Hyaluronic Acid/H2O2	11.97	217,211	131,579	1.651	4.7605
Hyaluronic Acid/H2O2 + RvD1	10.75	1,113,000	1,031,000	1.079	13.9877
Hyaluronic Acid/H2O2 + A1	10.71	1,126,000	1,043,000	1.079	13.9109
Hyaluronic Acid/H2O2 + A2	10.70	1,112,000	1,026,000	1.084	13.6087

¹ Peak Retention Volume. ² Polydispersity Index ₃ Intrinsic Viscosity.

3.3. Computational Study

Experimental methods have been used to determine the total antioxidant activity of samples. However, for a comprehensive evaluation of the radical scavenging activity of samples, computational methods are required. In this study, two concepts were used to analyze the antioxidant activity of samples: hydrogen atom transfer (HAT, which is based on the electronic and thermal enthalpy), and single-electron transfer (which is based on the frontier orbital energies of neutral molecules). To attain this, some parameters were calculated, such as energy gap (∆E), ionization potential (IP), bond dissociation enthalpy (BDE), hardness, softness, and electronegativity.

3.3.1. Geometry Optimization of samples

Geometry optimization is one of the most essential tasks in theoretical chemistry. It is a method to predict the three-dimensional arrangement of the atoms in a molecule employing the minimization of model energy. The molecular structures of all tested antioxidant samples are depicted in Figure 12 (designed by ChemDarw software version 12.0.0). As shown, the molecules have several functional groups such as aromatic ring, hydroxyl (O-H), ester (O=C-OR), carboxylic acid (O=C-OH), and carbon-carbon conjugated double (C=C) bonds. Among all functional groups, there are only four main structural motifs that contribute to the antioxidant activity of small molecules. These functional motifs include hydroxyl groups (-OH), amino groups (-NH), thiol groups (-SH), and isoprenoid groups. In this study, all tested antioxidants are in common with hydroxyl functional groups, thus comparing the bond strength of their O-H can be a way to estimate their antioxidant potential. It was found that the weaker O-H bond shows a higher antioxidant potential. Therefore, to get more data about geometrical molecular structure and their bonds, the DFT method with B3LY/6-31G (d, p) function was performed for optimization and analysis of the antioxidant samples, including Trolox, Ascorbic acid, RvD1, Analogue 1, and Analogue 2 (Figure 13). The bond lengths obtained through the optimized molecular structure of different samples are useful data to explain the HAT.

Within the Trolox structure, there are two hydroxyls (O-H) bonds, and the bond lengths related to phenol (O7-H8) and carboxylic acid (O31-H32) are 0.9741 and 0.9821 Å, respectively. Based on the shorter the bond the stronger the bond, the hydroxyl bond in carboxylic acid should be weaker because of the higher bond length. Since the bond length difference is very low around >1%, the determination of the better hydrogen donating cannot be very precise. In the case of Ascorbic acid, its structure includes four hydroxyl groups and the shortest O-H bonds are O12-H13 and O8-H20 with a length equal to 0.9772 Å and 0.9791 Å. Whereas the longest value belongs to O9-H19 and O17-H18 with a length equal to 0.9797 Å. As a result, O9 and O17 in Ascorbic acid and O7 in Trolox can be supposed as the active sites for hydrogen donating. As seen in Table 3, the longer O-H bond lengths in RvD1, Analogue 1, and Analogue 2 were related to O56-H58, O60-H62, and O26-H27 with the bond lengths of 0.9996, 0.9821, and 0.9981 Å, respectively. Interestingly, the O-H bond related to the carboxylic bond showed a higher bond length compared to another hydroxyl bond in RvD1 and analogue 1. As the number and type of hydroxyl group in the tested molecules are different, comparing the hydrogen-donating properties of the molecules is not possible. Therefore, the average bond length of the hydroxyl group was calculated to show the order of the antioxidant capacity for Trolox, Ascorbic acid, RvD1, Analogue 1, and Analogue 2 (with average bond length of 0.9781, 0.9789, 0.9856, 0.9801, and 0.9856 Å, respectively).

Analogue 2 = RvD1 > Analogue 1 > Ascorbic acid > Trolox

Figure 12. Molecular structure of Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D) and Analogue 2 (E).

Figure 12. Molecular structure of Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D) and Analogue 2 (E).

Figure 13. Geometrical molecular structure of Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D), and Analogue 2 (E) using Gaussian 09 - Ground states optimized at the B3LY/6-31G (d, p) function (all structures were also provided separately in the supporting information).

Figure 13. Geometrical molecular structure of Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D), and Analogue 2 (E) using Gaussian 09 - Ground states optimized at the B3LY/6-31G (d, p) function (all structures were also provided separately in the supporting information).

Table 3. Structural parameters of Trolox, Ascorbic acid, RvD1, Analogue 1, and Analogue 2: The bond length of the hydroxyl group (O-H).

Table 3. Structural parameters of Trolox, Ascorbic acid, RvD1, Analogue 1, and Analogue 2: The bond length of the hydroxyl group (O-H).

Antioxidants	Bond	Bond Length (Å)*
Trolox	O7-H8	0.9741
	O31-H32	0.9821
Ascorbic acid	O8-H20	0.9791
	O9-H19	0.9797
	O12-H13	0.9772
	O17-H18	0.9797
RvD1	O19-H20	0.9786
	O42-H44	0.9821
	O43-H45	0.9824
	O56-H58	0.9996
Analogue 1	O22-H23	0.9795
	O25-H27	0.9780
	O29-H30	0.9808
	O60-H62	0.9821
Analogue 2	O20-H21	0.9795
	O26-H27	0.9981
	O43-H44	0.9793

* (1 Å = 10^-10 m).

3.3.2. Bond Dissociation Enthalpy

Hydrogen Atom Transfer (HAT) is a crucial mechanism related to the antiradical activity of antioxidants. It is based on transferring hydrogen atoms with homolytic dissociation of O-H bond to free radicals. The antioxidant capacity of the HAT mechanism can be described by the Bond Dissociation Enthalpy (BDE) parameter.

BDE = H (ArO^•) + H (H^•) – H (ArOH)

(7)

Where H (ArOH) is the enthalpy of the neutral molecule, H (ArO^•) is the enthalpy of the radical generated via H atom abstraction, and H (H^•) is the enthalpy of H atom. The BDE values represent the energy necessary for cleaving the O-H bonds. The BDE values of all O-H bonds of samples are listed in Table 4. The antioxidant with the lowest BDE shows better antiradical properties. Table 4 shows clearly that the BDE value of O7-H8 of Trolox is the lowest in comparison with other hydroxyl groups in the gas phase. This means that the abstraction of the H atom from O7-H8 of Trolox is easier than other H atoms, which indicates that the radical created from this abstraction is more stable than other radicals. Moreover, among RvD1 and its analogues, the BDE value of the O22-H23 bond in the structure of Analogue 1 is the lowest, which indicates its higher antioxidant activity. According to the BDE values, the antioxidant activity of antioxidant samples decreases in the following order:

Trolox > Ascorbic acid > Analogue 1 > Analogue 2 > RvD1

All calculations of the neutral compounds and their radicals were calculated using Gaussian 09 software.

Table 4. O-H Bond Dissociation Enthalpy (BDE) of samples (kJ/mol).

Table 4. O-H Bond Dissociation Enthalpy (BDE) of samples (kJ/mol).

Antioxidants	Bond	BDE
Trolox	O7-H8	280.9
	O31-H32	346.9
Ascorbic acid	O8-H20	290.3
	O9-H19	293.9
	O12-H13	383.6
	O17-H18	380.5
RvD1	O19-H20	471.4
	O42-H44	421.6
	O43-H45	385.4
	O56-H58	415.0
Analogue 1	O22-H23	375.4
	O25-H27	380.5
	O29-H30	401.9
	O60-H62	411.2
Analogue 2	O20-H21	386.2
	O26-H27	403.0
	O43-H44	378.5

3.3.3. Ionization Potential

Single electron transfer (SET) is another main mechanism of antioxidants, in which a single electron transfers from the antioxidant to the free radical. The antioxidant capacity of the SET mechanism can be described by the Ionization Potential (IP) parameter.

IP = H (ArOH^•+) + H (e^-) – H (ArOH)

(6)

Where H (ArOH) is the enthalpy of the neutral molecule, H (ArOH^•+) is the enthalpy of the radical cation, and H (e^-) is the enthalpy of a single electron. Ionization Potential (IP) shows the ease of electron removal from antioxidant samples. An antioxidant sample with a higher IP value shows a lower electron donation tendency, therefore, an electron is hardly removed from the HOMO orbital of the neutral form of the compound. On the other hand, a compound with a lower IP value has a higher electron transfer capacity. The IP values of samples are listed in Table 5. According to the data in Table 5, Trolox has the lowest Ionization potential, so an electron can be easily removed from the HOMO orbital of neutral Trolox. In contrast, Ascorbic acid has the highest IP, which indicates its low antioxidant activity via the SET mechanism in comparison to other samples. Concerning RvD1 and its analogues, Analogue 2 represents better electron donating properties. According to the IP values, the antioxidant activity of antioxidant samples decreases in the following order:

Trolox > Analogue 2 > Analogue 1 > RvD1 > Ascorbic acid

Table 5. Ionization Potential (IP) of samples (kJ/mol).

Table 5. Ionization Potential (IP) of samples (kJ/mol).

Antioxidants	IP
Trolox	778.9
Ascorbic Acid	932.2
RvD1	875.3
Analogue 1	825.6
Analogue 2	806.1

3.3.4. Frontier Molecular Orbitals

Electronic properties of structures such as HOMO and LUMO energies and energy gaps (∆E) of compounds can be evaluated using frontier molecular orbital theory. The HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) are significant parameters that correlate with the antiradical activity of the samples. HOMO has electron donating ability; therefore, it is easy to remove electrons from this orbital. However, LUMO has electron accepting ability and it is easy for this orbital to accept electrons. To be more precise, in a compound with higher HOMO value, the donation of electrons is easier and as a result it may show better antioxidant activity. The electronic density distribution in the orbital permit’s prediction of the most probable sites in a molecule that can be easily attacked by free radicals and other reactive agents.

The HOMO and LUMO energies and the ∆E (LUMO-HOMO) of all antioxidant samples with B3LYP/6-31 + G (d, p) basis set are shown in Figure 14. The energy of HOMO is related to the electron-donating ability of an antioxidant; however, the shape of HOMO shows the sites for free radical attack. As seen in Figure 14A, the HOMO of Trolox is localized over the whole molecule except for the carboxylic acid functional group. The results of HOMO and LUMO energies for Ascorbic acid (Figure 14B) showed that the HOMO is localized on O8-H20, O9-H19, and the lactone ring, while the LUMO is not localized on the lactone ring but is very close to it. In the case of RvD1 (Figure 14C) and its two analogues (Figure 14D,E), the HOMOs are localized on conjugated double bond and imidazolium ring, respectively. Theoretically, the energies of HOMO orbitals are a great indicator of the scavenging activity of samples, relating to their electron-donating ability. Thus, it can be concluded that these regions are the most probable sites for free radical attack. Based on all the above-mentioned results, it can be perceived that the HOMO energy of Trolox (-5.294 eV) is the highest and the lowest HOMO is related to Ascorbic acid (-6.554 eV), which means that the radical scavenging ability of Trolox should be much better than Ascorbic acid as controls. Additionally, the HOMO energy values for the RvD1, Analogue 1, and Analogue 2 are -6.045, -5.914, and -5.690 eV, respectively. Based on these results, Analogue 2 with the highest HOMO energy is more probable to donate electrons than RvD1 and Analogue 1. According to the HOMO energy values in all samples, it can be assumed that radical scavenging ability based on HOMO energy arrangement is as follows. This evidence confirms the IP results which are shown above.

Trolox > Analogue 2 > Analogue 1 > RvD1 > Ascorbic acid

Figure 14. The energies and distributions of the HOMO and LUMO orbitals; Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D), and Analogue 2 (E).

Figure 14. The energies and distributions of the HOMO and LUMO orbitals; Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D), and Analogue 2 (E).

3.3.5. Quantum chemical parameters

The chemical activity of compounds can be calculated by the frontier molecular orbitals energies (HOMO and LUMO). Hardness (η), softness (σ), electronegativity ($\chi$), chemical potential (µ), and electrophilicity (ꞷ) are the quantum chemical parameters to evaluate the chemical behavior of compounds. The polarizability of molecules is an important factor which is associated with the band gap energy. A molecule with high band gap energy shows less polarizability. Generally, soft molecules are more strongly polarizable than hard molecules, so the softness value shows the ability to transfer electrons. On the other hand, the hardness value is related to the resistance of the molecule to lose electrons. Therefore, the hard molecules have a high HOMO-LUMO gap (∆E _gap) which tends to have high stability and low chemical reactivity with radicals. Overall, it may be said that the soft molecules have a low ∆E _gap, low stability, and high reactivity leading to higher antioxidant activity [52].

Electronegativity (χ) is the tendency of a molecule to attract electrons which is a factor to compare the antioxidant activity. According to the results, lower electronegativity is related to Trolox, which means that it has more ability to donate electrons rather than obtain them. Electrophilicity (ꞷ) is the ability of a molecule to take up electrons from the surroundings and depicts the electron-accepting ability of species. Compounds with lower electrophilicity indexes are more likely to donate electrons and exhibit higher antioxidant activity. The lowest electrophilicity is related to Analogue 1 (the strongest antioxidant among under-studied samples), and the highest electrophilicity is related to Ascorbic acid (the weakest antioxidant among under-studied samples). The formula of all parameters is based on the Koopmans theorem’s and is shown below. Table 6 and Table 7 show the equation for quantum chemical parameters for the studied molecules and the calculated value for quantum chemical parameters, respectively.

Table 6. The equations for calculation of quantum chemical parameters of antioxidants.

Table 6. The equations for calculation of quantum chemical parameters of antioxidants.

Hardness (eV)	Softness (eV)	Electronegativity (eV)	Electrophilicity (eV)	Chemical Potential (eV)
$\mathsf{\eta }=\frac{\mathbf{E}\mathbf{L}-\mathbf{E}\mathbf{H}}{2}$	$\mathsf{\sigma }=\frac{1}{\mathsf{\eta }}$	$\chi =\frac{-(\mathrm{E}\mathrm{H}+\mathrm{E}\mathrm{L})}{2}$	$\text{ꞷ}=\frac{\chi 2}{2\mathsf{\eta }}$	$\text{µ}=-\chi$

Table 7. The calculation of quantum chemical parameters for antioxidants; hardness (η), softness (σ), electronegativity (χ), electrophilicity (ꞷ), and chemical potential (µ).

Table 7. The calculation of quantum chemical parameters for antioxidants; hardness (η), softness (σ), electronegativity (χ), electrophilicity (ꞷ), and chemical potential (µ).

Antioxidants	EHOMO	ELUMO	η	σ	χ	ꞷ	µ
Trolox	-5.294	-0.655	2.31	0.432	2.974	1.914	-2.974
Ascorbic acid	-6.554	-1.324	2.61	0.383	3.938	2.970	-3.938
RvD1	-6.045	-0.959	2.54	0.393	3.500	2.411	-3.500
Analogue 1	-5.914	-0.490	2.71	0.369	3.201	1.890	-3.201
Analogue 2	-5.690	-0.553	2.56	0.390	3.121	1.902	-3.121

3.3.6. Molecular electrostatic potential (MEP)

The electrostatic potential map provides useful information related to the charge distribution of a molecule and information about the active sites of a molecule towards nucleophilic and electrophilic attack [53]. The MEP maps of antioxidant samples are shown in Figure 15. The electrostatic potential values of different sites are specified by different colors. The area with red color represents the most negative electrostatic potential (ESP, signifying electron-rich region for electrophilic attack), the blue color displays the most positive electrostatic potential (signifying electron deficient region for nucleophilic attack, such as radicals and charged molecules), and the green color illustrates the neutral electrostatic potential. Therefore, the negative ESP spots are significantly concentrated over oxygen atoms in all samples and nitrogen atoms in Analogues 1 and 2 (abundance of electrons in this region, active sites for electrophilic attack), and the positive ESP spots are mainly concentrated over some hydrogen atoms (absence of electrons in this region, active sites for nucleophilic attack) [54]. The most electronegative area (red region) is responsible for donating an electron and the most electropositive area (blue region) is responsible for donating proton.

Figure 15. The molecular electrostatic potential surface (ESP) of Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D), and Analogue 2 (E).

Figure 15. The molecular electrostatic potential surface (ESP) of Trolox (A), Ascorbic acid (B), RvD1 (C), Analogue 1 (D), and Analogue 2 (E).

3.3.7. Mulliken Charge Distribution

The Mulliken charge distribution of all samples is calculated on B3LYP at 6-31 G (d, p) level theory. Mulliken charge values for the atoms of all antioxidant samples are shown in Figure S9-13. In all samples, hydrogen atoms are positively charged, however, nitrogen and oxygen atoms acquire negative Mulliken atomic charge due to their electronegativity. Regarding carbon atoms, they are influenced by their substituents. Hence, all carbon atoms are negatively charged except for those which are boned with nitrogen and oxygen atoms. This is because electronegative atoms (N and O) pull out the partial charge from carbon atoms and make carbon atoms acquire a positive atomic charge.

5. Conclusions

References

1. Di Nicola, V. Degenerative osteoarthritis a reversible chronic disease. Regenerative Therapy 2020, 15, 149–160. [Google Scholar] [CrossRef]
2. Rahimi, M.; Charmi, G.; Matyjaszewski, K.; Banquy, X.; Pietrasik, J. Recent developments in natural and synthetic polymeric drug delivery systems used for the treatment of osteoarthritis. Acta Biomaterialia 2021, 123, 31–50. [Google Scholar] [CrossRef]
3. McClurg, O.; Tinson, R.; Troeberg, L. Targeting Cartilage Degradation in Osteoarthritis. Pharmaceuticals 2021, 14. [Google Scholar] [CrossRef]
4. Mehana, E.-S.E.; Khafaga, A.F.; El-Blehi, S.S. The role of matrix metalloproteinases in osteoarthritis pathogenesis: An updated review. Life Sciences 2019, 234, 116786. [Google Scholar] [CrossRef]
5. Panahi, Y.; Alishiri, G.H.; Parvin, S.; Sahebkar, A. Mitigation of Systemic Oxidative Stress by Curcuminoids in Osteoarthritis: Results of a Randomized Controlled Trial. Journal of Dietary Supplements 2016, 13, 209–220. [Google Scholar] [CrossRef]
6. Shi, Q.; Abusarah, J.; Zaouter, C.; Moldovan, F.; Fernandes, J.C.; Fahmi, H.; Benderdour, M. New evidence implicating 4-hydroxynonenal in the pathogenesis of osteoarthritis in vivo. Arthritis Rheumatol 2014, 66, 2461–2471. [Google Scholar] [CrossRef] [PubMed]
7. Morquette, B.; Shi, Q.; Lavigne, P.; Ranger, P.; Fernandes, J.C.; Benderdour, M. Production of lipid peroxidation products in osteoarthritic tissues: New evidence linking 4-hydroxynonenal to cartilage degradation. Arthritis & Rheumatism 2006, 54, 271–281. [Google Scholar] [CrossRef]
8. Kohen, R.; Nyska, A. Invited review: oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicologic pathology 2002, 30, 620–650. [Google Scholar] [CrossRef] [PubMed]
9. Zhuang, C.; Wang, Y.; Zhang, Y.; Xu, N. Oxidative stress in osteoarthritis and antioxidant effect of polysaccharide from angelica sinensis. International Journal of Biological Macromolecules 2018, 115, 281–286. [Google Scholar] [CrossRef] [PubMed]
10. Tudorachi, N.B.; Totu, E.E.; Fifere, A.; Ardeleanu, V.; Mocanu, V.; Mircea, C.; Isildak, I.; Smilkov, K.; Cărăuşu, E.M. The Implication of Reactive Oxygen Species and Antioxidants in Knee Osteoarthritis. Antioxidants 2021, 10. [Google Scholar] [CrossRef]
11. Henrotin, Y.; Mobasheri, A. Curcumin And Curcuminoids For The Treatment Of Osteoarthritis: Proof Of Concept. Journal of Tropical Medicinal Plants 2009, 10. [Google Scholar]
12. Berdiaki, A.; Neagu, M.; Spyridaki, I.; Kuskov, A.; Perez, S.; Nikitovic, D. Hyaluronan and Reactive Oxygen Species Signaling—Novel Cues from the Matrix? Antioxidants 2023, 12–824. [Google Scholar] [CrossRef]
13. Žádníková, P.; Šínová, R.; Pavlík, V.; Šimek, M.; Šafránková, B.; Hermannová, M.; Nešporová, K.; Velebný, V. The Degradation of Hyaluronan in the Skin. Biomolecules 2022, 12. [Google Scholar] [CrossRef] [PubMed]
14. Marinho, A.; Nunes, C.; Reis, S. Hyaluronic Acid: A Key Ingredient in the Therapy of Inflammation. Biomolecules 2021, 11. [Google Scholar] [CrossRef]
15. Schiller, J.; Volpi, N.; Hrabárová, E.; Šoltés, L. Hyaluronic acid: a natural biopolymer. Biopolymers: biomedical and environmental applications 2011, 70. [Google Scholar]
16. Soltés, L.; Kogan, G.; Stankovska, M.; Mendichi, R.; Rychlý, J.; Schiller, J.; Gemeiner, P. Degradation of high-molar-mass hyaluronan and characterization of fragments. Biomacromolecules 2007, 8, 2697–2705. [Google Scholar] [CrossRef] [PubMed]
17. Baron, D.; Baron, H.; Baerer, C.; Bodere, C.; Conrozier, T. Predictors for patient satisfaction of a single intra-articular injection of crosslinked hyaluronic acid combined with mannitol (HANOX-M-XL) in patients with temporomandibular joint osteoarthritis. Results of a prospective open-label pilot study (HAPPYMINI-ARTEMIS trial). BMC Musculoskeletal Disorders 2022, 23, 392. [Google Scholar] [CrossRef]
18. Pegreffi, F.; Reginster, J.-Y.; Bruyère, O.; Veronese, N. Comment on: The Effect of Intra-articular Hyaluronic Acid Injections and Payer Coverage on Total Knee Arthroplasty Procedures: Evidence From Large US Claims Database. Arthroplasty Today 2023, 21. [Google Scholar] [CrossRef]
19. Bruyère, O.; Honvo, G.; Vidovic, E.; Cortet, B. Assessment of the Response Profile to Hyaluronic Acid Plus Sorbitol Injection in Patients with Knee Osteoarthritis: Post-Hoc Analysis of a 6-Month Randomized Controlled Trial. Biomolecules 2021, 11, 1498. [Google Scholar] [CrossRef]
20. Rinaudo, M.; Lardy, B.; Grange, L.; Conrozier, T. Effect of Mannitol on Hyaluronic Acid Stability in Two in Vitro Models of Oxidative Stress. Polymers 2014, 6, 1948–1957. [Google Scholar] [CrossRef]
21. Mendoza, G.; Alvarez, A.I.; Pulido, M.M.; Molina, A.J.; Merino, G.; Real, R.; Fernandes, P.; Prieto, J.G. Inhibitory effects of different antioxidants on hyaluronan depolymerization. Carbohydr Res 2007, 342, 96–102. [Google Scholar] [CrossRef]
22. Mongkhon, J.-M.; Thach, M.; Shi, Q.; Fernandes, J.C.; Fahmi, H.; Benderdour, M. Sorbitol-modified hyaluronic acid reduces oxidative stress, apoptosis and mediators of inflammation and catabolism in human osteoarthritic chondrocytes. Inflammation Research 2014, 63, 691–701. [Google Scholar] [CrossRef]
23. Setti, T.; Arab, M.G.L.; Santos, G.S.; Alkass, N.; Andrade, M.A.P.; Lana, J.F.S.D. The protective role of glutathione in osteoarthritis. Journal of Clinical Orthopaedics and Trauma 2021, 15, 145–151. [Google Scholar] [CrossRef]
24. Henrotin, Y.; Kurz, B. Antioxidant to treat osteoarthritis: dream or reality? Current drug targets 2007, 8, 347–357. [Google Scholar] [CrossRef]
25. Suantawee, T.; Tantavisut, S.; Adisakwattana, S.; Tanavalee, A.; Yuktanandana, P.; Anomasiri, W.; Deepaisarnsakul, B.; Honsawek, S. Oxidative stress, vitamin e, and antioxidant capacity in knee osteoarthritis. Journal of clinical and diagnostic research: JCDR 2013, 7, 1855–1859. [Google Scholar] [CrossRef]
26. Lee, C.H. Role of specialized pro-resolving lipid mediators and their receptors in virus infection: a promising therapeutic strategy for SARS-CoV-2 cytokine storm. Archives of Pharmacal Research 2021, 44, 84–98. [Google Scholar] [CrossRef]
27. Yaribeygi, H.; Atkin, S.L.; Simental-Mendía, L.E.; Barreto, G.E.; Sahebkar, A. Anti-inflammatory effects of resolvins in diabetic nephropathy: Mechanistic pathways. Journal of Cellular Physiology 2019, 234, 14873–14882. [Google Scholar] [CrossRef] [PubMed]
28. Molaei, E.; Molaei, A.; Hayes, A.W.; Karimi, G. Resolvin D1, therapeutic target in acute respiratory distress syndrome. European Journal of Pharmacology 2021, 911, 174527. [Google Scholar] [CrossRef] [PubMed]
29. Benabdoune, H.; Rondon, E.-P.; Shi, Q.; Fernandes, J.; Ranger, P.; Fahmi, H.; Benderdour, M. The role of resolvin D1 in the regulation of inflammatory and catabolic mediators in osteoarthritis. Inflammation Research 2016, 65, 635–645. [Google Scholar] [CrossRef] [PubMed]
30. Serhan, C.N.; Petasis, N.A. Resolvins and protectins in inflammation resolution. Chemical reviews 2011, 111, 5922–5943. [Google Scholar] [CrossRef] [PubMed]
31. Benabdoun, H.A.; Kulbay, M.; Rondon, E.P.; Vallieres, F.; Shi, Q.; Fernandes, J.; Fahmi, H.; Benderdour, M. In vitro and in vivo assessment of the proresolutive and antiresorptive actions of resolvin D1: relevance to arthritis. Arthritis Res Ther 2019, 21, 72. [Google Scholar] [CrossRef]
32. Norling, L.V.; Headland, S.E.; Dalli, J.; Arnardottir, H.H.; Haworth, O.; Jones, H.R.; Irimia, D.; Serhan, C.N.; Perretti, M. Proresolving and cartilage-protective actions of resolvin D1 in inflammatory arthritis. JCI insight 2016, 1. [Google Scholar] [CrossRef]
33. Dravid, A.A.; Dhanabalan, K.M.; Naskar, S.; Vashistha, A.; Agarwal, S.; Padhan, B.; Dewani, M.; Agarwal, R. Sustained release resolvin D1 liposomes are effective in the treatment of osteoarthritis in obese mice. Journal of Biomedical Materials Research Part A 2023, 111, 765–777. [Google Scholar] [CrossRef]
34. Inactivation, E. Resolvin D1 and Its Aspirin-triggered 17R Epimer. THE JOURNAL OF BIOLOGICAL CHEMISTRY 2007, 282, 9323–9334. [Google Scholar] [CrossRef]
35. Orr, S.K.; Colas, R.A.; Dalli, J.; Chiang, N.; Serhan, C.N. Proresolving actions of a new resolvin D1 analog mimetic qualifies as an immunoresolvent. Am J Physiol Lung Cell Mol Physiol 2015, 308, L904–L911. [Google Scholar] [CrossRef]
36. Benderdour M, Fernandes J, Maltais R, Sanceau JY. Resolvin Analogs Compounds, Methods and Used Thereof. In: WO2023137554A1. vol. WO2023137554A1. Canada; 2023.
37. de Gaetano, M.; Butler, E.; Gahan, K.; Zanetti, A.; Marai, M.; Chen, J.; Cacace, A.; Hams, E.; Maingot, C.; McLoughlin, A.; et al. Asymmetric synthesis and biological evaluation of imidazole- and oxazole-containing synthetic lipoxin A4 mimetics (sLXms). European Journal of Medicinal Chemistry 2019, 162, 80–108. [Google Scholar] [CrossRef]
38. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.A.; Prior, R.L. High-Throughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. Journal of Agricultural and Food Chemistry 2002, 50, 4437–4444. [Google Scholar] [CrossRef] [PubMed]
39. Goldschmidt, S.; Renn, K. Zweiwertiger Stickstoff: Über das α, α-Diphenyl-β-trinitrophenyl-hydrazyl.(IV. Mitteilung über Amin-Oxydation). Berichte der deutschen chemischen Gesellschaft (A and B Series) 1922, 55, 628–643. [Google Scholar] [CrossRef]
40. Blois, M.S. Antioxidant Determinations by the Use of a Stable Free Radical. Nature 1958, 181, 1199–1200. [Google Scholar] [CrossRef]
41. Benzie, I.F.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Analytical Biochemistry 1996, 239, 70–76. [Google Scholar] [CrossRef]
42. Govindappa, C.M.; Sadananda, T.S.; Chandrappa, C.P. Phytochemical Analysis of Loranthus micranthus Extracts and Their in Vitro Antioxidant and Antibacterial Activities. Journal of Biologically Active Products from Nature 2014, 4, 303–315. [Google Scholar] [CrossRef]
43. Chen, H.; Qin, J.; Hu, Y. Efficient Degradation of High-Molecular-Weight Hyaluronic Acid by a Combination of Ultrasound, Hydrogen Peroxide, and Copper Ion. Molecules 2019, 24. [Google Scholar] [CrossRef]
44. Yogesh, K.; Jha, S.N.; Ahmad, T. Antioxidant potential of aqueous extract of some food grain powder in meat model system. Journal of Food Science and Technology 2014, 51, 3446–3451. [Google Scholar] [CrossRef] [PubMed]
45. Foti, M.C. Use and Abuse of the DPPH• Radical. Journal of Agricultural and Food Chemistry 2015, 63, 8765–8776. [Google Scholar] [CrossRef] [PubMed]
46. Munteanu, I.G.; Apetrei, C. Analytical Methods Used in Determining Antioxidant Activity: A Review. International Journal of Molecular Sciences 2021, 22. [Google Scholar] [CrossRef]
47. Gulcin, İ.; Alwasel, S.H. DPPH radical scavenging assay. Processes 2023, 11, 2248. [Google Scholar] [CrossRef]
48. Lang, Y.; Gao, N.; Zang, Z.; Meng, X.; Lin, Y.; Yang, S.; Yang, Y.; Jin, Z.; Li, B. Classification and antioxidant assays of polyphenols: a review. Journal of Future Foods 2024, 4, 193–204. [Google Scholar] [CrossRef]
49. Gliszczyńska-Świgło, A. Antioxidant activity of water soluble vitamins in the TEAC (trolox equivalent antioxidant capacity) and the FRAP (ferric reducing antioxidant power) assays. Food chemistry 2006, 96, 131–136. [Google Scholar] [CrossRef]
50. Baranowska, M.; Suliborska, K.; Chrzanowski, W.; Kusznierewicz, B.; Namieśnik, J.; Bartoszek, A. The relationship between standard reduction potentials of catechins and biological activities involved in redox control. Redox Biology 2018, 17, 355–366. [Google Scholar] [CrossRef]
51. Cowman, M.K.; Lee, H.-G.; Schwertfeger, K.L.; McCarthy, J.B.; Turley, E.A. The Content and Size of Hyaluronan in Biological Fluids and Tissues. Frontiers in Immunology 2015, 6. [Google Scholar] [CrossRef]
52. Nasidi, I.I.; Kaygili, O.; Majid, A.; Bulut, N.; Alkhedher, M.; ElDin, S.M. Halogen Doping to Control the Band Gap of Ascorbic Acid: A Theoretical Study. ACS omega 2022, 7, 44390–44397. [Google Scholar] [CrossRef] [PubMed]
53. Tabrizi, L.; Nguyen, T.L.A.; Dao, D.Q. Experimental and theoretical investigation of cyclometalated phenylpyridine iridium(iii) complex based on flavonol and ibuprofen ligands as potent antioxidant. RSC Advances 2019, 9, 17220–17237. [Google Scholar] [CrossRef] [PubMed]
54. Farrokhnia, M. Density Functional Theory Studies on the Antioxidant Mechanism and Electronic Properties of Some Bioactive Marine Meroterpenoids: Sargahydroquionic Acid and Sargachromanol. ACS Omega 2020, 5, 20382–20390. [Google Scholar] [CrossRef]