7-O-Galloyltricetifavan: a promising natural radical scavenger

7-O-Galloyltricetifavan (7OGT), a natural flavonoid, is isolated from the leaves of Pithecellobium clypearia. The compound exhibits a variety of biological activities. This study details the evaluation of the HOO• antiradical activity of 7OGT by quantum chemistry calculations. The HOO• trapping activity of 7OGT in the gas phase (reference state) was discovered to follow the formal hydrogen transfer mechanism with a rate constant of k = 4.58 × 108 M−1 s−1. In physiological environments, 7OGT is predicted to be an excellent HOO• radical scavenger with koverall = 2.65 × 108 and 1.40 × 104 M−1 s−1 in water and pentyl ethanoate solvents, respectively. The HOO• antiradical activity of 7OGT in water at physiological pH is approximately 2000 times that of Trolox and substantially higher than that of other well-known natural antioxidants such as trans-resveratrol or ascorbic acid. Thus, 7OGT is an excellent natural antioxidant in polar environments.

Oxidative stress is now thought to play a role in several chronic diseases [8][9][10]. The ability of natural products to scavenge free radicals is an essential aspect of their anti-inflammatory, antibacterial and cancer-preventive properties, and it is the driving force behind the investigation of the antioxidant properties [11][12][13]. There is currently no information about the kinetics and mechanism of the HOO • + 7OGT in physiological conditions; however, computer calculations offer a convenient way to predict the antioxidant activity of organic compounds in physiological media [11,14,15]. Since 7OGT has exhibited a broad range of potent biological activities, this study aims to delve into the underpinning antiradical activity of 7OGT by a quantum chemical approach, using HOO • as a model radical.
Using the transition state (TS) theory at 298.15 K, 1 M standard state, the rate constant (k) was computed as follows [31][32][33][34]39]: where σ is the reaction symmetry number [29,30], κ contains the tunnelling corrections calculated using the Eckart barrier [35], k B is the Boltzmann constant, h is the Planck constant and ΔG ≠ is the Gibbs free energy of activation. The reaction barriers of single electron transfer (SET) reactions in media were determined using the Marcus theory [40,41]. The equations used to calculate the Gibbs free energy change of reaction ΔG ≠ for the SET pathway are where ΔE SET is the non-adiabatic energy difference among reactants and vertical products for SET, and DG 0 SET is the standard Gibbs free energy change of the reaction [42,43]. A correction was applied to rate constants that were close to the diffusion limit [11]. The apparent rate constants (k app ) for an irreversible bimolecular diffusion-controlled reaction were computed using the royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 9: 211906 was estimated using the literature [11,45], [44,46], where D A or D B is determined using the Stokes-Einstein formulation (2.6) [47,48].
To avoid over-penalizing entropy losses in solution, the solvent cage effects were added using Okuno's adjustments [49], which were modified with the free volume theory according to the Benson correction [11,[50][51][52].
For species with numerous conformers, all of them were energy minimized, with the lowest electronic energy conformer being included in the study. The existence of only one single imaginary frequency was a defining feature of all transition stages. To verify that each TS is accurately related to the pre-complex and post-complex, intrinsic coordinate calculations were completed. The calculations were carried out using Gaussian 09 software [53].

The gas phase evaluation
Following the established protocol [19,54], the antioxidant activity of 7OGT was first evaluated according to the three main radical scavenging mechanisms: sequential electron transfer followed by proton transfer (SETPT), formal hydrogen transfer (FHT) and sequential proton loss followed by electron transfer (SPLET). In the two-step reactions such as the SETPT and SPLET pathways, the first step reaction (i.e. SET and proton loss (PL) for the SETPT and SPLET pathways, respectively) normally has the higher activation energy, with the exception of the proton dissociation of acidic moieties in water that is considered separately. Therefore, the thermochemical parameters i.e. proton affinity (PA), bond dissociation energy (BDE) and ionization energies (IE) that characterize the PL, FHT and SET reactions, respectively, were computed for all relevant bonds of 7OGT with the M06-2X/6-311++G(d, p)//M06-2X/6-31+G(d) method in the gas phase [21]. The results are presented in table 1. The results showed that the BDE values of the C−H range from 84.3 to 99.0 kcal mol −1 , while the BDEs are 73.9-85.3 kcal mol −1 for O-H bonds. This suggests that the hydroxyl groups are the thermodynamically preferred sites of activity via the hydrogen transfer reaction. The lowest BDE values were presented at the O4' − H bond (73.9 kcal mol −1 ) and the O13−H bond (77.1 kcal mol −1 ). These sites are believed to play a key role in 7OGT's radical scavenging activity via the FHT mechanism. According to this data, 7OGT has lower BDE(O−H) values than e.g. vanillic acid (85.2 kcal mol −1 ), [16] puerarin (87.3 kcal mol −1 ), [54] resveratrol (83.9 kcal mol −1 ), [55] or viniferifuran (82.7 kcal mol −1 ) [56]. This suggests that 7OGT could exhibit faster antiradical activity (following the FHT mechanism) than these natural antioxidants.
As shown in table 1, the lowest IE and PA values are significantly higher (about 2.42 and 4.60 times, respectively) than those of the BDE. Therefore, the antiradical activity of 7OGT is expected to favour the FHT mechanism in lipid media. The calculated Gibbs free energy changes (ΔG°) of the 7OGT + HOO • reaction via the main mechanisms: FHT, PL, which is the first step of SPLET, and SET as the first step of the SETPT suggest that the FHT reaction is spontaneous (ΔG°< 0) for most of the sites, apart from the C3(4)H−bonds; however, the PL and SET reactions are not spontaneous (ΔG°> 0) in any cases. The Marcus theory was also used to estimate the reaction barriers of the SET reaction in the gas phase [40,41]; however, this reaction was negative in the studied conditions (ΔG ≠ (SET) = 155.6 and λ = 21.2 kcal mol −1 ). Thus kinetics of HOO • + 7OGT reaction were computed following the FHT mechanism at the positions that yielded negative ΔG°.
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 9: 211906 Kinetic studies of the HOO • + 7OGT reaction were performed following the QM-ORSA protocol with the M06-2X/6-311 ++ G(d,p) method [11,27,28], and results are presented in table 2 [22,27]. The calculated results suggest that the HOO • trapping activity of 7OGT is defined by the FHT reaction at the O4 0 position; therefore, this reaction will be further analysed in physiological media.

The radical scavenging activity of 7-O-Galloyltricetifavan in physiological environments
Previous research has shown that the antiradical activity of phenolic compounds in aqueous solutions is dominated by anion states [37,38]. The protonation states of 7OGT were investigated at physiological pH to discover potential radical scavenging mechanisms [16,38,57]. Based on the calculated data [58], pK a1 and pK a2 values were 6.87 and 8.29, respectively (figure 3). Therefore, in water at pH = 7.4, three states, including neutral (H 2 A: 20.7%), anion (HA − : 70.2%) and dianion (A 2− : 9.1%) will be used for studying the radical scavenging activity, whereas the neutral state will be considered in the lipid medium ( pentyl ethanoate solvent).
The calculated results in the vacuum suggest that the HOO • antiradical activity in non-polar media follows the hydrogen transfer mechanism at the O4 0 − H bond. Thermodynamic evaluation in pentyl  royalsocietypublishing.org/journal/rsos R. Soc. Open Sci.   royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 9: 211906 ethanoate and water (electronic supplementary material, table S2) did not differ at the most likely site of activity (BDE = 74.6 and 78.9 kcal mol −1 and ΔG°= −10.7 and −9.6 kcal mol −1 in the lipid and water media, respectively) for the neutral state (H 2 A), however, the PL and SET pathways of this state are not spontaneous in either media (ΔG°> 0). Previous studies showed that for a compound containing acidic moieties the SET reaction of the dissociated states should be also considered in the aqueous solution [16,18,21,37,54,57]. Thus, the kinetics of the radical scavenging activity of 7OGT against HOO • radical in physiological media were carried out following equations (3.1) and (3.2) below, and the results are presented in table 3.
Lipid medium, Water at physiological pH, According to the results (table 3)

Conclusion
The hydroperoxyl antiradical activity of 7OGT was successfully evaluated by computational chemistry. The results showed that the antiradical activity of the 7OGT in non-polar media such as gas phase and pentyl ethanoate follows the FHT reaction (k = 4.58 × 10 8 and 1.40 × 10 4 M −1 s −1 , respectively). 7OGT also presented excellent antiradical activity (k overall = 2.65 × 10 8 M −1 s −1 ) in the aqueous solution. The HOO • radical scavenging of 7OGT is faster than that of typical antioxidants such as trans-resveratrol, Trolox, deoxynimbidiol, ramalin, 8-hydroxyconiothyrinone B and ascorbic acid. Thus, 7OGT is one of the most potent natural antioxidants identified thus far in polar environments.  Table 3. ΔG ≠ (kcal mol −1 ), λ, κ, k app , k f , k overall (M −1 s −1 ) and Γ (%) of the 7OGT + HOO • reaction in the physiological media.