Kinetics and Mechanism of the Antioxidant Activities of C. olitorius and V. amygdalina by Spectrophotometric and DFT Methods.

The kinetics and mechanism of the antioxidant activities of the methanolic extract of the leaves of two vegetables [Corchorus olitorius (C. olitorius) and Vernonia amygdalina (V. amygdalina)] have been studied using experimental and theoretical approaches. The kinetics (second order and pseudo-first order) of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of the leaf extracts at varying times (30-90 min) were determined using the UV-visible spectrophotometry method at λmax = 517 nm, whereas the mechanism was studied by density functional theory at two levels of functionals (B3LYP and LC-ωPBE) using bond dissociation enthalpy and adiabatic ionization potential values. Molecular properties such as the highest occupied molecular orbital, lowest unoccupied molecular orbital, electronegativity (χ), electrophilicity (ω), hardness (η), and softness (S) of the predominant phenolic antioxidants were also compared. The second-order kinetics is favored by both plants rather than pseudo-first order; however, V. amygdalina with a second-order rate constant k 2 of 0.0152 (mM)-1 min-1 is faster in scavenging DPPH radicals than C. olitorius with a k 2 value of 0.0093 (mM)-1min-1. Chlorogenic acid and luteolin-7-O-β-glucuronide, which are the most abundant phenolic acid antioxidant in C. olitorius and V. amygdalina, both preferably scavenge the DPPH radical via a hydrogen atom transfer mechanism. This is evident from their lower bond dissociation enthalpy values than the adiabatic ionization potential values. Successful molecular docking of these phenolic compounds indicates that both compounds form favorable interactions with the therapeutic target, xanthine oxidase.

Introduction

The roles played by free radicals and oxidative agents in the formation of cell toxins, which causes changes in the cell structure and the destruction of its vitality, cannot be overemphasized.[1] Human cell protection against oxidation damage caused by free radicals has attracted a lot of attention from medical scientists.[2] Thus, the development of antioxidants that can scavenge and enervate free radicals has been of significant importance. Recently, tremendous research efforts have been devoted to finding foods of plant origin that possess antioxidant activities.[2] Plants that are rich in phenolic acids have been extensively studied and reported to be active against oxidative agents and free radicals.[3] Phenolic acids are classified as secondary metabolites that possess health-promoting properties found in vegetables and fruits. They are widely spread throughout the plant kingdom, and the antioxidant quality of a plant is related to its phenolic content.[4,5] Phenolic acids are phenols that possess a carboxylic acid functionality. They contain two distinguishing constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures.[6]

A large number of species in the Vernonia genus have been widely used as food and in medicine.[7]Vernonia amygdalina is one of the most common species used for these purposes. It is a shrub that is common to both Africa and Asia, and the plant’s extracts have been reported to be effective against amebic dysentery and gastrointestinal disorders.[8,9] Phytochemical screening of the methanolic extracts of the plant’s leaves reveals the presence of bioactive compounds such as flavonoids, alkaloids, anthraquinones, terpenes, saponins, coumarins, steroids, and phenolic acids.[10−12]V. amygdalina leaves are widely consumed because of their good source of antioxidants.[13] Phenolic acid species in V. amygdalina play a major role in the antioxidant activity of the plant, and the most abundant phenolic antioxidant in the methanolic extract of the plant’s leaves is luteolin-7-O-glucuronide,[14]Figure a.

Figure 1

(a) Luteolin-7-O-β-glucuronide and (b) chlorogenic acid.

Corchurus olitorius (also called Jute mallow) belongs to the genus Corchurus, and is an edible vegetable that either grows wildly or is cultivated in tropical Africa and in Asia.[15] The leaf extracts of the plant have been reported to exhibit a significantly high antioxidant activity among other consumable vegetables, and the role of phenolic acids in the relatively high antioxidant activity has been highlighted.[16] A quantitative study of the relative abundance of antioxidants in the methanolic extract of C. olitorius leaves reveals that 5-caffeoylquinic acid (chlorogenic acid) (Figure b) is the predominant phenolic compound contributing to the antioxidant activity of the plant.[17]

Many investigations[18−20] have been carried out to understand the kinetics and mechanisms of radical scavenging activities of phenolic antioxidants. According to the literature, the kinetics of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activities of phenolic antioxidants in the extracts of different plant species has been compared. Phenolics from red sweet potato scavenge DPPH radicals faster than phenolics from blue berries.[21] Different rates of reaction of ten tropical food extracts with free radicals have also been reported in another study.[22]

The activity of a phenolic compound against free radicals is majorly dependent on the structural properties of the phenolic compound.

For good radical scavenging activity, the phenolic compounds should have the following structural characteristics:[23]

occurrence of multiple OH groups attached to the aromatic ring;

arrangement of these hydroxyls in the ortho-dihydroxy conformation, when possible;

planar structure of phenolics, which allows conjugation and electronic delocalization, as well as resonance effects; and

the presence of additional functional groups, such as the carbon–carbon double bond and the C=O carbonyl group.

An understanding of this relationship provides the information about the mechanism of radical scavenging activities of antioxidants.[24] Phenolics scavenge free radicals usually via any of the following three mechanisms:[23]The main mechanisms commonly employed for investigating the capability of phenolics to scavenge free radicals are the hydrogen atom transfer (HAT) and single electron transfer (SET).[19,25−28] The sequential proton loss-electron transfer (SPLET) mechanism has been less studied to date,[29−31] especially by computational methods, although it is also a relevant reaction path for investigating antioxidant activity.[32] In fact, the SPLET mechanism was reported to be favored experimentally in solvents that support X–H ionization when compared to the always present HAT process.[33−35] However, the computational aspect of this study focuses on the two main general mechanisms for investigating the radical scavenging capabilities of phenolics, the HAT and the SET.

Hydrogen atom transfer (HAT)

Single electron transfer (SET)

Sequential proton loss-electron transfer (SPLET)

The HAT and SET mechanisms have been associated with the calculation of two chemical properties: bond dissociation enthalpy (BDE) and adiabatic ionization potential (IP), respectively.[36] The density functional theory (DFT) method has been used in the evaluation of chemical properties because of its speed, ease, and accuracy.[37,38] Moreover, computationally determined BDE and IP values have been reported to correlate well with experimentally determined antioxidant parameters.[39,40] Research efforts in the use of the DFT method have revealed that the BDE and IP values are significantly influenced by levels of theory and the type of solvent, whereas basis sets do not play a significant role in BDE and IP calculations.[41] Among the DFT functionals, the B3LYP functional has been reported to show good performance in geometry optimization and its quite accurate prediction of X–H bond energetic and binding energies.[23,42] The LC-ωPBE functional has been successfully used in benchmarking the DFT method in probing antioxidant-related properties.[41] Although gas-phase calculations on antioxidant activities have been carried out on chlorogenic acid and luteolin-7-O-β-glucuronide,[24,38] little is still known about the calculations of the antioxidant activities of these compounds in the methanolic phase.

The intracellular production of reactive oxygen species is associated with many cellular events, including activation of enzymes such as xanthine oxidase (XO).[43] XO is a superoxide-producing enzyme with more than 1300 amino acid residues. It is generally known to have low specificity and can combine with other compounds and enzymes to create reactive oxidants, as well as oxidize other substrates. It has been implicated in the pathogenesis of chronic heart failure, cardiomyopathy in diabetes, and chronic wounds.[44−46]

This study was done to evaluate the kinetics (experimental approach) and mechanisms (theoretical approach) of radical scavenging activities of two vegetables, V. amygdalina and C. olitorius, and also to evaluate the inhibition potency (molecular docking) of the main phenolic compounds in these two vegetables; luteolin-7-O-glucuronide and chlorogenic acid against xanthine oxidase, an enzyme implicated in oxidative injury.

Experimental Section

Materials

The main apparatus and reagents used include a Life Science UV–visible spectrophotometer (Beckman Coulter DU 730), a rotary evaporator (Buchi rotavapor R-124), methanol, and 2,2-diphenyl-1-picrylhydrazyl (DPPH).

The plant materials (leaves of V. amygdalina and C. olitorius) were obtained from the local market in Ilorin-Nigeria at their commercially marketable maturity and verified in the Department of Plant Biology, University of Ilorin, with the following Voucher numbers UILH/002/1022 and UILH/003/154, respectively.

Methods

Extraction

The leave samples were prepared, air dried at room temperature, and pulverized.

Typically, 100 g of the pulverized leaf material was macerated with 1.0 L of methanol for 5 days. The methanolic extract was then filtered using Whatman filter paper and concentrated under vacuum at 40 °C using a rotary evaporator (Buchi rotavapor R-124). Finally, the crude extract obtained was stored in sample bottles at 4 °C prior to antioxidant examinations.

DPPH Calibration

A stock solution of 2.54 × 10–2 mM was prepared and serial dilutions were made to obtain six (6) solutions with concentrations of 1.27 × 10–2, 6.35 × 10–3, 3.18 × 10–3, 1.59 × 10–3, 7.95 × 10–4, and 3.98 × 10–4 mM. The absorbance of the various concentrations was measured at a wavelength of 517 nm, being the wavelength of maximum absorbance using a UV–vis spectrophotometer (Beckman coulter), subsequently generating a graph of absorbance against concentration (calibration curve), where the slope of the graph represents the molar absorptivity (ε) of DPPH according to the Beer–Lambert law.

DPPH Scavenging Activity

The antioxidant potential of the methanolic crude extracts was determined based on their ability to scavenge the stable 2, 2-diphenyl-1-picrylhydrazl (DPPH) free radical adopting the methods of Brand-Williams et al.,[47] with modifications. Stock solutions of the extracts of V. amygdalina and C. olitorius were prepared to obtain a concentration of 1000 mg mL–1. Dilutions were made to obtain 0.50, 0.25, 0.125, 6.25 × 10–2, 3.13 × 10–2, 1.56 × 10–2, 7.81 × 10–3, 3.91 × 10–3, and 1.95 × 10–3 mg mL–1 concentrations; 2 mL of each of the extracts at different concentrations were mixed with 2 mL of methanol solution of DPPH at a concentration of 0.0254 mM. The mixture was then vigorously shaken and allowed to stand in the dark at room temperature for 30, 50, 70, and 90 min. The absorbance of the mixture was measured at 517 nm with a mixture of methanol and DPPH solution used as control. The absorbance results were obtained in triplicate, and the average values were used as the actual absorbance. The scavenging activity of the DPPH radical can be expressed as inhibition percentage using the following equation according to Brand-Williams et al.[47]where AB is the absorbance of the control (containing all reagents except the test compound), and AS is the absorbance of the test compound.

The IC50 value of the samples, which is the concentration of a sample required to inhibit 50% of the DPPH free radical, was calculated from the graph of percentage inhibition against extract concentrations.

Kinetic Analysis

At different time intervals, the concentrations of the DPPH radical in the extracts were calculated using the Beer–Lambert lawHere, A is the absorbance of the mixtures at a certain concentration (c) in mol dm–3, with a path length l (dm). ε is the molar absorptivity of the DPPH radical.

The kinetics of scavenging the free radical (in this case, DPPH) may be achieved via two types of second-order rate equationsThe second-order rate constant (k2) was determined by having the concentration of the antioxidant, {antioxidant}, to be in large excess compared to the concentration of the radical compound {DPPH}. This forces the reaction to vary only with the concentration of DPPH.{antioxidant} is assumed to remain constant throughout the reaction and can be modified to obtain different k1 values. The change in {DPPH} with time gives k1. Determination of k1 was repeated at different antioxidant concentrations for each of the samples, and the mean gave the overall k1.

Integration methodThen an integrated form of this type of second-order reaction rate equation was applied to the experimental results for V. amygdalina and C. olitoriuswhere {DPPH}0 and {DPPH} are the DPPH concentrations at time zero and any time “t”, respectively. A plot of against time gives a slope k2.

Isolation method: This may be applied to the kinetics of scavenging the free radical, DPPH, by a pseudo-first-order means. This method of obtaining the kinetics of antioxidant activity was adapted from Espín et al.[4] using DPPH and antioxidant concentrations of the extracts

We can, therefore, infer that DPPH was depleted from the medium under pseudo-first-order conditions according to the following equation:where {DPPH} is the radical concentration at any time (t), {DPPH}0 is the radical concentration at time zero, and k1 is the pseudo-first-order rate constant. This rate constant (k1) is linearly dependent (according to eq ) on the concentration of the antioxidant, and from the slope of these plots, k2 was determined.

DFT Studies

The structures of chlorogenic acid and luteolin-7-O-β-glucuronide used for the DFT studies were downloaded from the repository of PubChem.[48] In the ground state, the parent molecules were optimized at the B3LYP level of theory and with the 6-31G(d) basis set. Optimizations of the radicals (neutral and cation) were done at B3LYP/6-31G(d), starting from the optimized geometries of the parent molecules. A restricted approach was used for the optimization of the parent molecules, whereas an unrestricted open shell approach was adopted for the radicals. An implicit method was used for the solvation, with IEFPCM as the solvation model and methanol as the solvent. To characterize the geometries of the parent molecules and radicals as true minima, the harmonic vibrational frequency was calculated using both B3LYP/6-31G(d) and LC-ωPBE/6-31G. BDE and IP in methanol were determined using eqs and 15, respectively. These equations were adopted from eqs and 2.where Hrad is the enthalpy of the radical generated by hydrogen atom abstraction; HH is the solvation enthalpy of the hydrogen atom (taken from Bizarro et al.[49]). Ecr and Ep are the electronic energies of the cation radical generated after electron transfer and of the parent molecules, respectively. Note that the zero point energy corrections have been included in the enthalpy (H) and electronic energy (E) calculations. Furthermore, molecular descriptors such as the highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), electrophilicity (ω), electronegativity (χ), hardness (η), and softness (S) were calculated at this level of theory and with basis set (B3LYP/6-31G (d)/IEFPCM) to support the results. All calculations were done using the Gaussian 09 program suite[50] on the Lengau cluster machine, a high-performance computing resource of the Council for Scientific and Industrial Research (CSIR), Cape Town, South Africa.

Molecular Docking Studies

The X-ray crystal structure of the hypoxanthine–xanthine oxidase complex (PDB ID: 3NRZ)[43] was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb), and that of the standard ligand, febuxostat, a known non-purine selective inhibitor of xanthine oxidase,[51] was downloaded from the PubChem repository (Pubchem CID: 134018).[48] Prior to the docking experiments, the PDB crystal structure of the hypoxanthine–xanthine oxidase complex was edited to remove water molecules and all cocrystallized initiators to obtain the free xanthine oxidase used as the target molecule for the docking experiment. Following this initial preparation of the target and the compounds under study using the Discovery studio program,[52] molecular docking analyses were successfully performed with the AutoDock tools version 1.5.6 and AutoDock version 4.2.5.1 docking program.[53] The targeted docking of the two antioxidant compounds to the XO receptor as a target using the reported[43] binding sites (the amino acid residues within and around the binding pockets of the hypoxanthine and molybdopterin cofactor) in the hypoxanthine–xanthine oxidase complex was performed to detect the native ligand-receptor conformations in accordance with the AutoDock4 docking protocol.[53] The LGA stochastic search method[54] was implemented with the semi-empirical free-energy force field scoring function used to represent the potential energy surface of the ligand–protein interaction. A bounding simulation box was defined around the binding site of the protein by considering a distance of 3.0 Å away from the outermost atoms of the bound ligand in each of the x-, y-, and z-directions. To validate the docking method, the docking of hypoxanthine and febuxostat with the xanthine oxidase target was performed until a best docked ligand conformation with root mean square distance (RMSD) less than 1.50 Å was obtained.

Results and Discussion

Experimental Results

DPPH Calibration

The calibration of the DPPH solution is performed to generate a calibration curve, in which absorbance at 517 nm is plotted against varying concentrations of DPPH, and the slope, thereby, gives the molar absorptivity of DPPH (52.497). A linear regression (r2 value) of 0.997 was obtained from the curve.

Antioxidant Activity

The % inhibition of DPPH by V. amygdalina and C. olitorius in Figure shows that both plants have good inhibition ability at the experimental concentrations, although the inhibition ability of V. amygdalina is stronger than that of C. olitorius. However, C. olitorius has a lower IC50 value (1.15 × 10–2 μg mL–1) than V. amygdalina (2.95 × 10–2 μg mL–1) (Table ); this lower IC50 value of C. olitorius points to its higher potency (Figure .

Table 1

IC50 for V. amygdalina and C. olitorius

	IC50 (×10–2 μg mL–1)
time (min)	V. amygdalina	C. olitorius
30	3.20 ± 0.06	1.18 ± 0.12
50	3.01 ± 0.04	1.15 ± 0.08
70	2.97 ± 0.15	1.13 ± 0.23
90	2.87 ± 0.05	1.19 ± 0.08

Figure 2

% Inhibition of DPPH by V. amygdalina (a) and C. olitorius (b) for the 0–0.5 mg mL–1 concentration range at different incubation times.

Kinetics Analysis

The result of the second-order rate constant (k2) of the free radical scavenging activities of V. amygdalina and C. olitorius by the integration method is summarized in Figure . This result was obtained from a plot of against time (t).

Figure 3

Variation of with time for V. amygdalina (a) and C. olitorius (b) for determination of the second-order rate constant.

The second-order rate constant, k2, which is the slopes of the plots, was averaged to obtain an overall second-order rate constant of the antioxidant activities of the two plant extracts. An average k2 of 6.4503 and 3.0390 (mM)−1 min–1 were obtained for V. amygdalina and C. olitorius, respectively. This observation of a higher k2 in V. amygdalina led to the inference that V. amygdalina’s extract scavenges the DPPH radical faster than C. olitorius’s extract at the same concentration. It is worth noting that the second-order rate constants decrease with the concentration in both plant extracts; this means that the DPPH radical scavenging ability of the plant extracts decreases as the concentration decreases and that the antioxidant activities of both plant extracts against the DPPH radical become insignificant at concentrations below 0.0625 mg mL–1.

The r2 values of the trend lines over all concentrations were averaged to obtain the overall r2 values in both cases. The values are 0.9836 and 0.9911 for V. amygdalina and C. olitorius, respectively.

Using the isolation method, different pseudo-first-order rate constants (k1) were obtained according to eq , whose integrated form leads toTherefore, a plot of against times (t) gave a slope equal to −k1. The linear relationship between k1 and the concentration of the extracts (eq ) allows us to obtain k2 from the slope of their graphs. The second-order rate constants (k2) obtained from the pseudo-first-order rate constants are 0.0152 and 0.0093 (mM)−1 min–1 for V. amygdalina and C. olitorius, respectively. The linear regression coefficient (r2) of 0.9325 for V. amygdalina revealed that it is more favored in the pseudo-first-order kinetics than C. olitorius with a regression coefficient of 0.7999. However, both plant extracts follow second-order kinetics in scavenging the free radical because their regression coefficients are higher (>0.95) using the second order.

DFT Studies

The skeletal structure of chlorogenic acid contains one aromatic and one aliphatic ring with five OH groups, whereas the luteolin-7-O-β-glucuronide backbone consists of two aromatic and one aliphatic ring with six OH groups (Figure ). It is worth noting that the O–H groups of carboxylic acids on both antioxidants are not considered because they are in constant resonance with the (C=O) group and may not be available for H abstraction.

The BDE and IP values of O–H groups in the antioxidant species may serve as parameters to predict the mechanism of scavenging free radicals by antioxidants.[55] The lowest value between these two parameters indicates the preferred thermodynamic pathway (mechanism) of scavenging the DPPH radical.

Comparing the BDE and IP values (Table ) reveals that the H-atom transfer (HAT) mechanism is the more preferred mechanism by the two antioxidants because BDE is lower than IP for both B3LYP/6-31G(d) and LC-ωPBE/6-31G(d) levels of theory. Also, worthy of note is the relative performance of the two DFT functionals employed (Table ); the BDE and the IP values from the B3LYP functional are lower than their corresponding values from the LC-ωPBE functional. The lower IC50 value of C. olitorius (chlorogenic acid) (Table ) indicates its better antioxidant property than V. amygdalina (luteolin-7-O-β-glucuronide). Furthermore, the aromatic O–H groups in both antioxidants have lower BDE values than the aliphatic O–H groups with the terminal aromatic O–H groups, 2bOH and 1cOH, having the lowest BDE value for chlorogenic acid and luteolin-7-O-glucuronide, respectively. The decreased values of BDE for aromatic O–Hs may be associated with the extra stability caused by both intramolecular hydrogen bonding and delocalization of electrons of the aromatic rings, as reflected in the HOMO and LUMO energy diagrams shown in Figure . Therefore, the antioxidant property of these two compounds could be attributed to the net effect of the dissociation of aromatic O–H groups with the terminal aromatic O–H group having a more pronounced effect in each compound.

Table 2

BDE and IP of Chlorogenic Acid and Luteolin-7-O-Glucuronide in Methanol

		chlorogenic acid					luteolin-7-O-β-glucuronide
	BDE		IP (kcal mol–1)			BDE (kcal mol–1)		IP (kcal mol–1)
bond	B3LYP	LC-ωPBE	B3LYP	LC-ωPBE	bond	B3LYP	LC-ωPBE	B3LYP	LC-ωPBE
2aOH	100	129	129	135	3aOH	98	102	131	109
4aOH	99	103			4aOH	98	103
5aOH	95	101			5aOH	97	103
1bOH	77	79			1bOH	90	96
2bOH	70	74			1cOH	77	78
					2cOH	73	77

Figure 4

Optimized structures of (a, b) parent molecules and (c, d) most active radicals used for the DFT studies.

Despite having the same intramolecular hydrogen bond and being terminal, it is observed from Table that the 2bOH radical in chlorogenic acid has a slightly lower BDE, and hence, it is more stable than the 2cOH radical in luteolin-7-O-β-glucuronide. This observation can be explained based on the stability of the OH group adjacent to both 2bOH and 2cOH radicals. The OH group adjacent to 2bOH in chlorogenic acid is more stable because it is conjugated with an alkene group “meta” to it. This conjugation gives an increased stability because electron delocalization does not result in a pronounced perturbation of the benzene ring consisting of the OHs. This is evident from Figure as the HOMO of chlorogenic acid is distributed on the terminal ring, whereas the HOMO of luteolin-7-O-β-glucuronide is distributed centrally, revealing its slightly lower antioxidant activity compared to chlorogenic acid.

Figure 5

Distribution pattern of the electron density of the molecular orbitals in (a) chlorogenic acid and (b) luteolin-7-O-β-glucuronide after the DFT studies.

Due to its lower BDE value in methanol, chlorogenic acid is more active toward scavenging the DPPH radical via the HAT mechanism than luteolin-7-glucuronide, regardless of the additional ring and hydroxyl groups in the latter. The molecular descriptor values support this claim (Table . The energy gap between the frontier molecular orbitals (HOMO and LUMO) is lower in chlorogenic acid than that in luteolin-7-O-β-glucuronide. This reveals that chlorogenic acid requires less energy to scavenge the DPPH radical compared to luteolin-7-O-β-glucuronide. Other descriptors such as electronegativity (χ), electrophilicity (ω), hardness (η), and softness (S) support this claim (Table ). Lower electronegativity, hardness, and electrophilicity values make chlorogenic acid a more favorable antioxidant. Furthermore, the higher HOMO and softness values support this.

Table 3

Molecular Descriptors of Chlorogenic Acid and Luteolin-7-O-β-Glucuronide

molecular descriptors	chlorogenic acid	luteolin-7-O-β-glucuronide
EHOMO	–5.89	–6.34
ELUMO	–1.71	–1.93
Egap	4.19	4.41
χ	3.80	4.13
η	2.09	2.20
ω	3.45	3.87
S	0.24	0.23

Molecular Docking

Molecular docking results showed that the studied compounds interacted favorably with the target (xanthine oxidase) via several nonbonded interactions, which were mainly hydrogen-bonding and hydrophobic interactions. The docking results revealed that chlorogenic acid and luteolin-7-O-β-glucuronide formed stable complexes with XO (Figure ), which showed that they interacted well with the active sites of XO. The analyses of the binding affinities, which express the binding free energies of these potential antioxidants, febuxostat, and the control ligand, hypoxanthine, reported in Table , showed that chlorogenic acid and luteolin-7-O-β-glucuronide have higher binding free energies than the control ligand. Chlorogenic acid binds significantly with the target via conventional hydrogen-bonding interactions with ALA 1079, SER 1080, GLU 802, and MET 1038; carbon–hydrogen bonds with ARG 912; pi–alkyl interactions with ALA 1078; and pi–sigma interactions with PHE 914. On the other hand, luteolin-7-O-β-glucuronide showed conventional hydrogen bonds with GLY 799, ARG 912, THR 1083, GLN 1194, GLY 1260, MET 1038, and SER 1080; carbon–hydrogen bonds and pi–alkyl interactions with ALA 1078; and pi–sulfur interactions with MET 1038. The control ligand, hypoxanthine, interacted via hydrogen bonds with ARG 880, THR 1010, and GLU 802; pi–pi stacking with PHE 914; pi–alkyl interactions with ALA 1078, ALA 1079, VAL 1011, and LEU 1014; and pi–pi T-shaped stacking with PHE 914. Finally, the reference compound, febuxostat, which is a known xanthine oxidase inhibitor, displays conventional hydrogen-bonding interactions with PHE 798, ARG 912, ALA 1079, SER 1080, and GLN 1194; carbon–hydrogen bonds with GLU 1261; alkyl interactions with ALA 910, ALA 1078, and MET 1038; and pi–alkyl interactions with MET 1038 and PHE 914. These results regarding hypoxanthine and febuxostat are in agreement with those found in the literature.[56] An increase in hydrophobicity has been reportedly linked to an increase in stability.[57,58] We observed that the trio of chlorogenic acid, luteolin-7-O-β-glucuronide, and febuxostat, which have closely higher binding affinities than hypoxanthine, interacted with amino acids ALA 1078, SER 1080, and ARG 912. This shows that these three compounds all preferentially bind to XO at the binding sites of its molybdopterin cofactor, MTE, which acts as a catalyst for the enzymatic actions of XO. Thus, the compounds will successfully inhibit XO by disrupting the catalytic pathway for the activation of the XO enzymatic action of generating superoxides. This can be attributed to the presence of more phenolic rings in the trio, which further supports and explains the antioxidant activity of chlorogenic acid and luteolin-7-O-β-glucuronide against the therapeutic xanthine oxidase. The molecular interactions of the studied compounds are depicted in Figure a–d.

Figure 6

Molecular interactions between (a) chlorogenic acid, (b) luteolin-7-O-β-glucuronide, (c) febuxostat, and (d) hypoxanthine and the amino acid residues of therapeutic xanthine oxidase.

This result indeed further supports and explains the antioxidant activity of the methanolic extracts of C. olitorius and V. amygdalina.

Conclusions

The kinetics and mechanism of the antioxidant activities of two popular vegetables (V. amygdalina and C. olitorius) have been analyzed using spectrophotometric, DFT, and molecular docking methods. Methanolic extracts of the plant leaves contain phenolic antioxidants as the major agent responsible for their DPPH scavenging activities. Although both plants prefer second-order kinetics in scavenging the DPPH radical, V. amygdalina is faster than C. olitorius in this process, as 0.0152 and 0.0093 (mM)−1 min–1 were obtained for V. amygdalina and C. olitorius, respectively. DFT analysis revealed that the dominant phenolic acids in both plants prefer the HAT mechanism in scavenging DPPH as compared to the SET mechanism, based on a comparison of their BDE and IP values of the hydroxyls, OHs. It is also evident from the DFT analysis that delocalization of electrons plays a more important role in the stabilization of O–Hs than the number of O–H groups or number of rings. In addition, molecular docking studies showed that both chlorogenic acid and luteolin-7-O-β-glucuronide form favorable interactions with the therapeutic target, xanthine oxidase, with their antioxidant potency comparable to that of febuxostat, a potent antioxidant drug used as the control.