Kinetics and Mechanism of the Antioxidant Activities of C. olitorius and

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Kinetics and Mechanism of the Antioxidant Activities of C. olitorius and V. amygdalina by Spectrophotometric and DFT Methods Olaniyi K. Yusuff,† Modinah Adenike O. Abdul Raheem,*,† Abdulrahman A. Mukadam,† and Ridwan Oladayo Sulaimon‡ †

Department of Chemistry, Faculty of Physical Sciences, University of Ilorin, PMB 1515 Ilorin, Nigeria Department of Chemistry, King Fahd University of Petroleum and Minerals, P.O. Box 5061, 31261 Dhahran, Saudi Arabia

‡

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ABSTRACT: 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 k2 of 0.0152 (mM)−1 min−1 is faster in scavenging DPPH radicals than C. olitorius with a k2 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.

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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 © XXXX American Chemical Society

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 1a. 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 Received: March 27, 2019 Accepted: July 31, 2019

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DOI: 10.1021/acsomega.9b00851 ACS Omega XXXX, XXX, XXX−XXX

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Figure 1. (a) Luteolin-7-O-β-glucuronide and (b) chlorogenic acid.

methanolic extract of C. olitorius leaves reveals that 5caffeoylquinic acid (chlorogenic acid) (Figure 1b) is the predominant phenolic compound contributing to the antioxidant activity of the plant.17 Many investigations18−20 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 (i) Hydrogen atom transfer (HAT) ArOH + R· → ArO· + RH

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. 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-βglucuronide,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 superoxideproducing 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

(1)

(ii) Single electron transfer (SET) ArOH + R· → ArOH+ + R− → ArO· + RH

(2)

(iii) Sequential proton loss-electron transfer (SPLET) ArOH → ArO− + H+ −

.

.

(3) −

ArO + R → ArO + R

(4)

R− + H+ → RH

(5)

The main mechanisms commonly employed for investigating the capability of phenolics to scavenge free radicals are the B


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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 law

against xanthine oxidase, an enzyme implicated in oxidative injury.

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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.

Here, 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 equations (1) Integration method

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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 % inhibition =

AB −A s × 100 AB

(7)

A = εcl

−d{DPPH} = k 2{DPPH}2 (8) dt Then an integrated form of this type of second-order reaction rate equation was applied to the experimental results for V. amygdalina and C. olitorius 1 1 = k 2t + {DPPH}t {DPPH}0

(9)

where {DPPH} 0 and {DPPH} t are the DPPH concentrations at time zero and any time “t”, 1 against time gives a respectively. A plot of {DPPH}t

slope k2. (2) Isolation method: This may be applied to the kinetics of scavenging the free radical, DPPH, by a pseudo-firstorder means. This method of obtaining the kinetics of antioxidant activity was adapted from Espiń et al.4 using DPPH and antioxidant concentrations of the extracts −d{DPPH} = k 2{DPPH}{antioxidant} dt

(10)

The 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. −d{DPPH} = k1{DPPH} dt

(11)

wherek1 = k 2{antioxidant}

(12)

{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. We can, therefore, infer that DPPH was depleted from the medium under pseudo-first-order conditions according to the following equation: {DPPH)t = {DPPH}0 e−k1t

(6)

(13)

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 11) on the concentration

where AB is the absorbance of the control (containing all reagents except the test compound), and AS is the absorbance of the test compound. C


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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/631G(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/631G(d) and LC-ωPBE/6-31G. BDE and IP in methanol were determined using eqs 14 and 15, respectively. These equations were adopted from eqs 1 and 2. BDE = Hrad + HH − Hp

(14)

IP = Ecr − Ep

(15)

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 zdirections. 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.

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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 1 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 1); this lower IC50 value of C. olitorius points to its higher potency (Figure 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 suite50 on the Lengau cluster machine, a highperformance 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 reported43 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 ligandreceptor conformations in accordance with the AutoDock4 docking protocol.53 The LGA stochastic search method54 was implemented with the semi-empirical free-energy force field

Table 1. IC50 for V. amygdalina and C. olitorius IC50 (×10−2 μg mL−1) time (min) 30 50 70 90

V. amygdalina 3.20 3.01 2.97 2.87

± ± ± ±

0.06 0.04 0.15 0.05

C. olitorius 1.18 1.15 1.13 1.19

± ± ± ±

0.12 0.08 0.23 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 3. This result was obtained from a plot of 1 against time (t). {DPPH}t

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. D


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Figure 3. Variation of

1 {DPPH}

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 1). 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 2) 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 2); 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 1) 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 4. 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. Despite having the same intramolecular hydrogen bond and being terminal, it is observed from Table 1 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-βglucuronide. This observation can be explained based on the stability of the OH group adjacent to both 2bOH and 2cOH

with time for V. amygdalina (a) and C.

olitorius (b) for determination of the second-order rate constant.

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 11, whose integrated form leads to ij {DPPH}t yz zz = −k t lnjjj 1 j {DPPH}0 zz k {

(

Therefore, a plot of ln

(16)

{DPPH}t {DPPH}0

) against times (t) gave a slope

equal to −k1. The linear relationship between k1 and the concentration of the extracts (eq 12) 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

Table 2. BDE and IP of Chlorogenic Acid and Luteolin-7-O-Glucuronide in Methanol chlorogenic acid

luteolin-7-O-β-glucuronide −1

BDE

−1

IP (kcal mol )

IP (kcal mol−1)

BDE (kcal mol )

bond

B3LYP

LC-ωPBE

B3LYP

LC-ωPBE

bond

B3LYP

LC-ωPBE

B3LYP

LC-ωPBE

2aOH 4aOH 5aOH 1bOH 2bOH

100 99 95 77 70

129 103 101 79 74

129

135

3aOH 4aOH 5aOH 1bOH 1cOH 2cOH

98 98 97 90 77 73

102 103 103 96 78 77

131

109

E


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Figure 4. Optimized structures of (a, b) parent molecules and (c, d) most active radicals used for the DFT studies.

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.

the OHs. This is evident from Figure 5 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.

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 F


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reported in Table 3, showed that chlorogenic acid and luteolin7-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 hydrogenbonding 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

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 3). Table 3. Molecular Descriptors of Chlorogenic Acid and Luteolin-7-O-β-Glucuronide molecular descriptors

chlorogenic acid

luteolin-7-O-β-glucuronide

EHOMO ELUMO Egap χ η ω S

−5.89 −1.71 4.19 3.80 2.09 3.45 0.24

−6.34 −1.93 4.41 4.13 2.20 3.87 0.23

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 3). Lower electronegativity, hardness, and electrophilicity values make chlorogenic acid a more favorable antioxidant. Furthermore, the higher HOMO and softness values support this. 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 6), 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,

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. G


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Notes

acid and luteolin-7-O-β-glucuronide against the therapeutic xanthine oxidase. The molecular interactions of the studied compounds are depicted in Figure 6a−d. This result indeed further supports and explains the antioxidant activity of the methanolic extracts of C. olitorius and V. amygdalina.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge the Council for Scientific and Industrial Research (CSIR), Cape Town, South Africa, for granting them access to their High-Performance Computing resources for this work.

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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-βglucuronide 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.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00851. Data for the measured % Inhibition of DPPH by V. amygdalina and C. olitorius for the different DPPH concentrations at 30, 50, and 90 min experimental times (Tables S1 and S2, respectively), the calibration curve for DPPH from the measured absorbances at different DPPH concentrations (Figure S1), the plot of

(

ln

■

[DPPH]t [DPPH]0

REFERENCES

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) against experimental time for the determi-

nation of the pseudo-first-order rate constant for V. amygdalina and C. olitorius (Figure S2), and the Cartesian coordinates of the optimized structures of chlorogenic acid (Scheme S1) and luteolin-7-O-βglucuronide (Scheme S2) used for the DFT calculations (PDF)

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*E-mail: [email protected]., [email protected]. uk. Tel: +2348035952356. ORCID

Modinah Adenike O. Abdul Raheem: 0000-0002-9188-4002 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. H


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Kinetics and Mechanism of the Antioxidant Activities of C. olitorius and

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