The radical scavenging activity of muriolide in physiological environments: mechanistic and kinetic insights into double processes

Muriolide (MO) is a natural lactone that was isolated from Ranunculus muricatus. This compound exhibited good antioxidant activity in some experiments; however, the radical scavenging activity of MO in physiological environments has not been studied yet. In this study, the reaction between hydroperoxyl radical and MO was investigated in physiological environments by using density functional theory (DFT) calculations. It was found that MO exhibits excellent antiradical activity in water at physiological pH (k = 1.05 × 108 M−1 s−1) by the single electron transfer mechanism of the anion state. However, the activity in lipid media is moderate with k = 2.54 × 104 M−1 s−1 and is defined by the formal hydrogen transfer pathway. The antiradical reactions can occur in double processes; however, the first reaction may define the HOO˙ radical scavenging activity of MO. Compared with typical natural antioxidants, the antiradical activity of MO against HOO˙ radicals is slightly lower than Trolox in pentyl ethanoate. However, the activity of MO is approximately 808 times faster than that of the reference in aqueous solution. Thus, the data suggest that MO is a promising natural radical scavenger in the physiological environment.


Introduction
Ranunculus muricatus, which belongs to the genus Ranunculus (Ranunculaceae), is known as spiny fruit buttercup in Asia, Australia, South America, and Europe. 1 The plant has been used as a traditional drug to treat urinary infections, jaundice, diarrhea, dysentery, eczema, leprosy, and ringworm infection. [2][3][4] R. muricatus is also used as a remedy for coughs and asthma and a deworming agent for all types of livestock. 5,6 Studies showed that R. muricatus exhibited cytotoxic, antibacterial, antifungal, and particularly antioxidant properties. 7,8 The antioxidant activity of R. muricatus may be related to phenolic compounds such as avonoids, avonoid glycosides, and lactones found in the plant. 7,9,10 Muriolide (MO, Fig. 1) is a natural lactone that has been isolated from Ranunculus muricatus. 9 This compound exhibited good antioxidant activity against the DPPH radical scavenging activity (IC 50 ¼ 56.9 mM) and lipoxygenase enzyme testing (IC 50 ¼ 68.3 mM). Thus the radical scavenging activity of MO, particularly in the physiological systems, needs to be investigated; however, this issue has not been mentioned yet. Previous studies have shown that the computational method is one of the most convenient means of examining the relationship between structures and biological activity in the development of new medicines, such as antioxidants with increased activities. [11][12][13][14][15] Therefore, in this study, the antiradical activity of MO was thoroughly evaluated in double processes by using quantum calculations. In addition, the effects of solvents and molecule structure on the activity were also considered.

Computational details
Calculations were carried out with the Gaussian 09 suite of programs 16 by using the M06-2X/6-311++G(d,p) method. 17 The M06-2X functional is one of the most reliable methods to study thermodynamics and the kinetics of radical reactions, [18][19][20] and widely used to evaluate the antiradical scavenging activity in solvents (water for polar media and pentyl ethanoate for lipid environments) 15,21 with low errors compared to experimental data (k calc /k exp ratio ¼ 1-2.9). [21][22][23][24][25] The kinetic calculations were performed following the quantum mechanics-based test for the overall free radical scavenging activity (QM-ORSA) protocol, 12,21 with the SMD method for solvent effects, 26 by the Eyringpy code. 24,27 All of the species have been optimized directly in the specic environments, i.e. gas phase, pentyl ethanoate and water. The rate constant was calculated by using the conventional transition state theory (TST) and 1 M standard state at 298.15 K. 24,25,[27][28][29][30][31][32] where s is the reaction symmetry number, 33,34 k contains the tunneling corrections calculated using the Eckart barrier, 35 k B is the Boltzmann constant, h is the Planck constant, DG s is the Gibbs free energy of activation. The Marcus Theory was used to estimate the reaction barriers of single electron transfer (SET) reactions. [36][37][38] The free energy of reaction DG s for the SET pathway was computed following the eqn (2) and (3).
where DG SET is the Gibbs energy of reaction, DE SET is the nonadiabatic energy difference between reactants and vertical products for SET. 39,40 For rate constants that were close to the diffusion limit a correction was applied to yield realistic results. 21 The apparent rate constants (k app ) were calculated following the Collins-Kimball theory in the solvents at 298.15 K; 41 the steady-state Smoluchowski rate constant (k D ) for an irreversible bimolecular diffusion-controlled reaction was calculated following the literature as corroding to eqn (4) and (5). 21,42 where R AB is the reaction distance, N A is the Avogadro constant, and D AB ¼ D A + D B (D AB is the mutual diffusion coefficient of the reactants A and B), 41,43 where D A or D B is estimated using the Stokes-Einstein formulation (6). 44,45 h is the viscosity of the solvents (i.e. h(H 2 O) ¼ 8.91 Â 10 À4 Pa s, h(pentyl ethanoate) ¼ 8.62 Â 10 À4 Pa s) and a is the radius of the solute that was obtained in Gaussian calculations. The solvent cage effects were included following the corrections proposed by Okuno, 46 adjusted with the free volume theory according to the Benson correction 21,47-49 to reduce overpenalizing entropy losses in solution. All transition states were characterized by the existence of only one single imaginary frequency. Intrinsic coordinate calculations (IRCs) were performed to ensure that each transition state (TS) is connected correctly with the pre-complex (RC) and post-complex (PC).

The gas phase evaluation
Study of the structure of the MO showed that the molecule can adopt multiple conformational structures. Thus, as an initial step, the possibility of MO conformers was examined 50 and then the M06-2X/6-311++G(d,p) method was used to analyze the six Fig. 2 The typical MO conformers and the relative free energy DG (compared with MO, kcal mol À1 ). lowest electronic energy conformers (Fig. 2). It was found that, the lowest DG value was observed at MO, those for MO1-MO5 were higher than that of MO about 3.6-7.1 kcal mol À1 . With the Maxwell-Boltzmann distribution, 51,52 it was found that MO is the dominant conformer (99.59%) in the relative tautomer populations, and this conformer has therefore been used in further studies.
The radical scavenging activity of MO was evaluated against HOOc radical. This radical species is the simplest of the biologically most important of the ROOc, i.e. peroxy radicals, and effective scavenging of these is sufficient to reduce oxidative stress in biological systems. 53 The HOOc radical, which is moderately reactive and one of the main antioxidant objectives, 54 has been widely used as a reference radical for modeling antioxidant activity in lipid and polar environments. 12,21,48,55 To understand how MO scavenges free radicals, the antioxidant reactivity of MO was rst evaluated following the three typical antioxidant mechanisms, including formal hydrogen transfer (FHT), sequential proton loss electron transfer (SPLET), and single electron transfer proton transfer (SETPT). 11 Those are dened by the thermochemical parameters (bond dissociation enthalpy (BDE), proton affinity (PA), and ionization energy (IE), respectively). Previous studied showed that the radical adduct formation (RAF) of HOOc radical was not favored for the p system of aromatic rings 48,56-58 such as in MO and therefore this mechanism was not considered in this study. Therefore, the thermodynamic parameters in the gas phase of MO were computed and results are presented in Table 1.
The lowest BDE values are presented at the phenolic groups, including O6(O7)-H bonds with BDE ¼ 79.6 and 79.0 kcal mol À1 , respectively. The BDE of the alcohol group (O11-H) is highest at 104.0 kcal mol À1 , and this is about $6 and 15.6 kcal mol À1 higher than those of the C11(12)-H bonds and C2-H bond, respectively. Calculated thermodynamic parameters in studied solvents (Table S1, ESI †) indicated that the lowest BDE values were also obtained at the O6 (7)-H bonds (BDE(O6(7)-H) ¼ 79.7, 79.0 and 82.6, 82.0 in pentyl ethanoate and water, respectively). Thus the results suggest that the O6(O7)-H bonds will dene the H-abstraction of MO following the FHT mechanism. It is clear from Table 1 that the PA and IE values are much higher than the BDEs. The lowest PA (PA(O7-H) ¼ 332.2 kcal mol À1 ) and IE values are around 4.21 and 2.34 times greater than the lowest BDE. Thus the radical scavenging of MO in the gas phase may be followed the FHT pathway rather than the SETPT and SPLET mechanisms. This result was conrmed by investigating the Gibbs free energies of the reaction between MO and HOOc radicals (Table 1). 25,59 The HOOc trapping activity of MO is spontaneous for FHT at O6(7)-H bonds (DG ¼ À5.4 and À6.0 kcal mol À1 , respectively), whereas the other reactions are unspontaneous with high positive DG values. Based on the calculated data, the MO + HOOc reaction may only follow the FHT mechanism, and thus this pathway should be investigated in the kinetic study.
In the next step evaluation of the HOOc trapping activity of MO, the kinetics of the HOOc + MO reaction following the primary mechanism (FHT at O6(7)-H bonds) in the gas phase were computed according to the (QM-ORSA) protocol, 12,21 the results are shown in Table 2 and Fig. 3. It was found that MO exhibited moderate hydroperoxyl antiradical activity with k Eck ¼ 1.63 Â 10 6 and 2.71 Â 10 6 M À1 s À1 for the O6-H and O7-H bonds, respectively. These reactions contribute about 37.5 and 62.5% in the overall rate constant (k overall ¼ 4.34 Â 10 6 M À1 s À1 ). However, the k overall of the HOOc + MO reaction in the gas phase is $4.3 times lower than that of Trolox (k ¼ 1.87 Â 10 7 M À1  s À1 ). 60 Hence, it appears to suggest that the hydroperoxyl antiradical activity of MO in nonpolar media might be lower than that of Trolox.

The HOOc radical trapping activity of MO in physiological environments
3.2.1 The rst process. In aqueous environments, the radical scavenging activity of acidic species is typically dominated by the activity of the ionic forms. 18,59 Therefore, the protonation state of MO was rst evaluated at physiological pH to nd the most likely radical scavenging reactions. The thermodynamic section and the calculations for water medium (Table S1, ESI, † (PA(O6-H) ¼ 43.1 kcal mol À1 ), PA(O7-H) ¼ 42.9 kcal mol À1 ) showed that deprotonation was the easiest at the O7-H bond; thus, the pK a value of MO was computed for the O7-H bond based on the literature 59,61 and shown in Fig. 4.
The calculated pK a value was pK a ¼ 8.79. Therefore, in pH ¼ 7.40 aqueous solutions, MO exists in two states, including the neutral (HA, 96.1%) and anion (A À , 3.9%) states. Hence, these states were used in the kinetic study of the HOOc trapping activity of MO in water at pH ¼ 7.4. The overall rate constant (k overall ) of HOOc + MO reaction in the rst antiradical process were calculated according to eqn (7) and (8); the results are presented in Table 3 and Fig. 3, while the potential energy surfaces of the HOOc + MO reaction following the FHT pathway is shown in Fig. 5.
In the aqueous medium: As shown in Fig. 5, in the rst antiradical process, the reaction proceeds via the RCs that are more stable in terms of energy than the reactants about 3.4-5.8 kcal mol À1 . Then, the reaction can proceed to TSs from the RCs (the energy barriers around 12.0-17.0 kcal mol À1 ) before bottoming the lowest energy points (PCs) and forming the products. The energy barriers for the MO-O6-H + HOOc reaction in water and pentyl ethanoate are higher (from 0.3 to 2.1 kcal mol À1 , respectively) than those of the MO-O7-H + HOOc reaction. This suggests that the H-abstraction of the O7-H bond against HOOc radicals should be faster than that of the O6-H bond.
It is clear from the Table 3 that the hydroperoxyl radical trapping activity of MO in pentyl ethanoate is moderate with the k overall ¼ 2.54 Â 10 4 M À1 s À1 by the H-abstractions of the O6-H (G ¼ 25.2%) and O7-H bonds (G ¼ 74.8%). In contrast, MO exhibits an excellent HOOc trapping activity in the polar medium with the k overall ¼ 1.05 Â 10 8 M À1 s À1 . This process was dened by the SET reaction of the anion state (MO-O7 À , G $ 100%). The rate constants for the FHT reaction of the O6(7)-H bonds against HOOc radical in water are k f ¼ 8.17 Â 10 3 (2.02 Â 10 4 ) M À1 s À1 , whereas these reactions make negligible contributions ($0%) to the overall HOOc antiradical activity of MO. However, the reaction at the O7-H bond is faster than that of the O6-H bond in all of the studied media. This is in good agreement with the PES analysis results. Compared with the reference antioxidant Trolox (k ¼ 1.00 Â 10 5 and 1.30 Â 10 5 M À1 s À1 in pentyl ethanoate and water, respectively), 60 the HOOc trapping activity of MO is fairly lower in the lipid medium but about 808 times higher in water at physiological pH.
3.2.2 The second process of the radical scavenging. To gain further insights into the antioxidant of MO in the physiological environments, the hydroperoxyl radical scavenging activity of MO intermediates (the second antiradical process of MO) in pentyl ethanoate and water was investigated. As shown in the rst reaction step, the primary intermediates of MO + HOOc reaction in pentyl ethanoate were MO-O6c (25.2%) and MO-O7c (74.8%) radicals, while that for the aqueous solution was MO-O7c (100.0%). Thus these intermediates were used as reactants for the second reaction against HOOc radicals. The thermodynamic parameters (BDEs, IE) were computed for the most active positions and are shown in Table 4.
As shown in Table 4, the BDE values of the most active positions (O6-H and O7-H bonds) are in the range of 69.2 to 71.1 kcal mol À1 , much lower than those of MO in the rst step (Table 1)   however, the SET mechanism is not favored for the intermediates due to the large positive DG (DG ¼ 26.3-75.8 kcal mol À1 ). Thus the HOOc radical scavenging activity of intermediates was dened by the FHT pathway (DG < 0, Table 4); thus, these reactions were used for kinetic investigating.
Previous studies also showed that the reaction between antiradical intermediates with radical i.e., HOc and HOOc most probably proceeds through triplet transition states, 55,62 and thus, the result was used to evaluate the mechanism and kinetics of the second antiradical reaction of MO. The potential energy surfaces are shown in Fig. 5, whereas the possible mechanisms and kinetic data are presented in Fig. 5 and Table  4, respectively.
As shown in Fig. 5, the reactions of intermediates (MO-O6c and MO-O7c) and HOOc radicals proceed via RCs, TSs and PCs, while the PC species are less stable in terms of energy than the reactants. This is in line with previous studies in phenolic compounds. 55,62 The energy barriers of the reactions are about 20.1-22.7 kcal mol À1 , which is much higher than those of the rst step (the energy barriers around 12.0-17.0 kcal mol À1 , Table 5). The overall rate constants of the intermediates (MO-O6c or MO-O7c) + HOOc reactions in pentyl ethanoate is 1.07 Â 10 À4 M À1 s À1 , while that for the aqueous solution is 1.80 Â 10 À2 M À1 s À1 . Thus the HOOc radical scavenging activity in the second reaction step of MO following the FHT pathway is about 10 6 -10 8 times lower than those of the rst reaction step (Fig. 6),    Table 4) are lower than those of MO (Table 1) by about 10 kcal mol À1 , and the Gibbs free energies for the intermediates (MO-O6c or MO-O7c) + HOOc reactions are DG ¼ À16.0 to À18.8 kcal mol À1 (Table 4). These results suggest that the HOOc radical scavenging of MO at the second process is supported by the thermodynamic properties (the low BDE values and DG < 0); however, this reaction hardly occurs due to the low rate constant values. Thus the antiradical activity should be considered in both thermodynamic and kinetic data rather than based on thermodynamic considerations alone. Based on the calculated data, the HOOc trapping activity of MO in nonpolar and polar environments was mainly dened by the rst step.

Conclusion
The hydroperoxyl radical scavenging activity of muriolide in the physiological environment has been successfully investigated in silico. The result showed that MO exhibited moderate activity (k ¼ 2.54 Â 10 4 M À1 s À1 ) in the nonpolar media, whereas the activity was excellent with k ¼ 1.05 Â 10 8 M À1 s À1 in water under the physiological pH. The antiradical reactions could occur in two steps; however, the rst step reaction dened the HOOc radical scavenging activity of MO. In nonpolar conditions, the FHT mechanism via the O6-H and O7-H bonds determined the antiradical activity, whereas the SET mechanism of the anionic state dened the activity in the polar medium. The HOOc + MO reaction in pentyl ethanoate is slightly lower than Trolox, but it is approximately 808 times faster than that of the reference in the aqueous solution. Thus, MO is an effective radical scavenger in the physiological environment.

Conflicts of interest
There are no conicts to declare.