Theoretical Study on the Antioxidant Activity of Natural Depsidones.

Depsidones are secondary metabolites in lichens with a range of potential health benefits. Among others, these compounds are believed to exhibit high hydroxyl radical and superoxide scavenging abilities, warranting a detailed investigation of their antioxidant properties. In this study, the radical scavenging activity of natural depsidones from Ramalina lichenized fungi was investigated in silico. Calculations of the thermodynamic parameters suggested that the main radical scavenging pathway follows the formal hydrogen transfer (FHT) mechanism; however, unexpectedly low rate constants were found in the CH3OO• scavenging reaction. Establishing that the depsidones are mostly ionized in an aqueous environment suggested that the single-electron transfer (SET) mechanism should not be ruled out. Consistently, depsidones were revealed to be excellent HO• and O2 •- scavengers in aqueous solutions (k = 4.60 × 105 - 8.60 × 109 M-1 s-1 and k = 2.60 × 108 - 8.30 × 109 M-1 s-1, respectively) following the sequential proton loss electron transfer (SPLET) mechanism. These results suggest that natural fungal depsidones are potent hydroxyl and superoxide radical scavengers in aqueous solutions.

Introduction

pan class="Chemical">Depsidones are aromatic compounds, which are usually isolated from lichens.[1,2] The structure of class="Chemical">pan class="Chemical">depsidones is characterized by the 11H-dibenzo[b,e][1,4]dioxepin-11-one ring that is formed by two 2,4-dihydroxybenzoic acid rings linked together by an ether and an ester moiety.[1]

The pan class="Chemical">depsidones of class="Chemical">pan class="Chemical">Ramalina lichenized fungi[2,3] that include salazinic acid (1), norstictic acid (2), stictic acid (or scopuloric) (3), connorstictic acid (4), cryptostictic acid (5), peristictic acid (6), variolaric acid (7), hypoprotocetraric acid (8), protocetraric acid (9), conhypoprotocetraric acid (10), gangaleoidin (11), and physodic acid (12) have gained the most attention due to their purported health benefits. Reported bioactivities of depsidones include radical scavenging, antimalarial, antihypertensive, antitrypanosomal, antiproliferative, antibacterial, antileishmanial, herbicidal, larvicidal, aromatase and cholinesterase inhibitor, and antifungal and antioxidant activities.[4] They also have health benefits as a reducing factor in allergic reactions in humans.[5−7]

Several studies span class="Chemical">howed that extracts from lichens have potential antioxidant properties[2,4,5,8−12] that are related to specific components of the extracts, in which the class="Chemical">pan class="Chemical">depsidones may play a major role. However, the radical scavenging and antioxidant activities of depsidones were only addressed in a handful of studies.[2,13,14] It was shown that the most potent antioxidant compounds of the depsidone family were those without a butyrolactone ring.[13] In particular, depsidones may have higher superoxide scavenging activity compared with that of typical antioxidants such as quercetin,[15] despite low inhibition in DPPH testing.[13] However, as a weak oxidant, superoxide can decompose to more potent and reactive oxygen species such as hydroxyl radicals.[13] Thus, the depsidones in lichens are likely to contribute to the antioxidant activity in biological systems by their potent superoxide and hydroxyl radical scavenging activities. Although most of the studies focused on confirming and quantifying the antioxidant properties of the depsidones, studies on the mechanism and kinetics of the antioxidant activity have not been performed yet. Furthermore, the relationship between chemical structure and the antioxidant properties of the depsidones is still an open question.

This study is aimed at evaluating the antioxidant properties of 12 compounds of the pan class="Chemical">depsidone class of the genus of class="Chemical">pan class="Chemical">Ramalina (Figure )[2] focusing on the following issues: (1) calculating thermodynamic parameters (bond dissociation energy (BDE), ionization energy (IE), and proton affinity (PA)) to evaluate the antioxidant properties of these compounds following three typical mechanisms,[16−20] formal hydrogen transfer (FHT), single-electron transfer followed by proton transfer (SETPT), and sequential proton loss electron transfer (SPLET); (2) studying the cooperation between structures and the antioxidant activity of the studied compounds; and (3) evaluating the kinetics of the reactions of the most potential antioxidants with radicals in the gas phase (CH3OO•) and aqueous solution (HO•, HOO•, CH3OO•, and O2•–) following the favored mechanisms.

Figure 1

Structures of the 12 depsidones studied here for their antioxidant properties.

Structures of the 12 pan class="Chemical">depsidones studied here for their antioxidant properties.

Results and Discussion

Radical Scavenging Activity of Depsidones in the Gas Phase

Evaluating the Most Likely Mechanism

Antioxidant activity follows either of three typical mechanisms including n class="Chemical">FHT, SETPT, and SPLET.[16−20] The thermochemical class="Chemical">parameters (class="Chemical">pan class="Chemical">BDEs, IEs, PAs, and ΔG° of the reaction between the studied compounds and the CH3OO• radicals following these mechanisms) were first calculated to identify the main antioxidant mechanism of each studied compound. To reduce the duration of the calculations, the B3LYP/3-21G method was used first to calculate the BDEs and PAs of all possible X–H (X = C, O) bonds in the studied compounds; the results are presented in Tables S1 and S2 (Supporting Information, SI). The lowest BDEs or PAs of X–H (X = C, O) bonds, IEs, and ΔG° values were then calculated with higher accuracy at the ROB3LYP/6-311++G(2df,2p)//B3LYP/6-311G(d,p)[16,21−25] level of theory in the gas phase.[16,23,26] The results are presented in Table .

Table 1

Lowest Calculated BDEs, IEs, and PAs of the Depsidones and ΔG° of Reactions between the Studied Compounds and CH3OO• Radicals in the Gas Phase Following the Three Mechanisms (in kcal·mol–1)

	FHT mechanism			SETPT mechanism		SPLET mechanism
comp.	position	BDE	ΔG°	IE	ΔG°	position	PA	ΔG°
1	C5′–H	76.9	–5.7	191.7	163.2	O1–H	310.1	134.7
2	C1–H	79.9	–3.2	189.1	161.3	O1–H	312.2	137.0
3	C1–H	80.4	–2.7	187.3	159.4	O1–H	314.6	139.2
4	C1–H	79.9	–3.2	190.6	162.4	O1–H	316.4	142.7
5	C11′–H	74.4	–8.8	181.5	153.1	O1–H	320.1	144.5
6	C1–H	75.2	–6.4	182.9	155.2	COO–H	315.8	141.8
7	C1–H	83.6	0.8	186.2	158.4	O10–H	324.7	149.9
8	O8–H	85.8	2.9	172.8	144.8	COO–H	319.9	144.9
9	C9′–H	82.4	–0.3	183.1	154.6	COO–H	310.5	136.1
10	C9′–H	78.3	–4.2	175.3	146.6	COO–H	315.1	140.7
11	O8–H	87.7	4.5	175.3	147.3	O3–H	320.6	145.7
12	O8–H	84.5	1.8	174.6	146.8	COO–H	316.4	141.7

The results in Table span class="Chemical">how that the IE values are in the range of 172.8–191.7 kcal·mol–1. The lowest IE value is determined for compound 9 at 172.8 kcal·mol–1, whereas this value is the highest for 1 with IE = 191.7 kcal·mol–1. The sequence of vertical IE values in the gas phase is

pan class="Chemical">BDE(X–H) (X = C, O) values were calculated for assessing the possibility of class="Chemical">pan class="Chemical">FHT mechanism; in the gas phase, the values are in the range of 74.4–87.7 kcal·mol–1. The lowest BDE value was observed for compound 5 with BDE(C11′–H) = 74.4 kcal·mol–1, while that for compound 11 is the highest at 87.7 kcal·mol–1. On the basis of the gas-phase BDE values, the ability of H-donation of the studied compounds follows the sequence

In terms of the SPLET mechanism, the pan class="Chemical">PA values are in the range of 310.1–324.7 kcal·mol–1. The lowest class="Chemical">pan class="Chemical">PAs were observed at the O1–H position in compounds 1–5, while those of compounds 6–10 and 12 were at the COOH groups.

To evaluate the effects of dispersion interactions on thermochemical parameters (pan class="Chemical">BDE, class="Chemical">pan class="Chemical">PA, and IE), the GD3 dispersion correction[27] was included by the (RO)B3LYP-GD3/6-311++G(2df,2p)//B3LYP-GD3/6-311G(d,p) level (Table S7, SI). It was found that the variations in thermochemical parameters between the B3LYP-GD3 and B3LYP levels are in the range of −0.3–1.7 kcal/mol (ΔBDE = 0.4–1.7 kcal/mol; ΔPA = −0.3–0.7 kcal/mol; ΔIE = 0.4–0.6 kcal/mol). Thus, the effects of dispersion interactions on thermochemical parameters are minor.

To evaluate the most likely mechanism for the studied pan class="Chemical">depsidones in the gas phase, the free energy change of the first step was calculated for each mechanism in a reaction with class="Chemical">pan class="Chemical">CH3OO• radicals, shown in Table . It was found that only the FHT pathway yielded exothermic and spontaneous reactions, apart from the reactions of compounds 7, 8, 11, and 12. The ΔG° values for the SETPT and SPLET mechanisms are in the ranges of 146.6–163.2 and 134.7–149.9 kcal·mol–1, respectively. Thus, the ROO• radical scavenging of studied depsidones is not supported in the gas phase. On the basis of the calculated data, the radical scavenging of the depsidones in the gas phase mainly follows the FHT mechanism and this mechanism will be further investigated in thermodynamic and kinetic calculations.

Reaction of CH3OO• Radicals with Depsidones

Potential Energy Surfaces (PES)

According to the thermochemical data (Table ), the main radical scavenging pathway in the gas phase is the pan class="Chemical">FHT mechanism; thus, the potential energy surface analysis focuses on the class="Chemical">pan class="Chemical">FHT mechanism shown in Figure and the corresponding transition states (TSs) are drawn in Figure . The ΔG° values of the reactions with the CH3OO• radical via the FHT mechanism were positive for compounds 7, 8, 11, and 12 (Table ); therefore, the potential energy surfaces were calculated only for compounds 1–6, 9, and 10 that have negative ΔG°. The results have the energies of transition states (TS) in the range of 10.4–13.0 kcal·mol–1 (Figure ). Among the studied compounds, the TS energy of compound 5 is the lowest at 10.4 kcal·mol–1. This is consistent with the lowest value of BDE that was determined for 5-C11′–H (74.4 kcal·mol–1). Nevertheless, the high values of TS energies suggest that these compounds are not suitable CH3OO• radical scavengers in the gas phase and thus very low activities are expected in all environments.

Figure 2

PES of the reaction between the selected compounds and CH3OO•.

Figure 3

Optimized geometries of TS in the reaction between the selected compounds with CH3OO•.

PES of the reaction between the selected compounds and pan class="Chemical">CH3OO•.

Optimized geometries of TS in the reaction between the selected compounds with pan class="Chemical">CH3OO•.

Kinetic Study

To confirm the results above, kinetic calculations were performed for the reactions between selected n class="Chemical">depsidones and class="Chemical">pan class="Chemical">CH3OO• radicals. The Gibbs free energy of activation (ΔG≠), tunneling corrections (κ), and rate constants (keck) were calculated at the M05-2X/6-311++G(d,p) level of theory[28] at 298.15 K in the gas phase, and the results are shown in Table . The rate constants for all of the reactions are in the range of 4.81 × 10–2 to 1.37 M–1 s–1. It is also worth noting that the FHT H-abstraction rate of 5 is the highest with k = 1.37 M–1 s–1, followed by that of compound 6 with 1.29 M–1 s–1, whereas the lowest rate constant is observed for compound 3 with k = 4.81 × 10–2 M–1 s–1. The rate constant of 5 is about 2–8 times faster than that of 1, 4, 9, and 10, and more than 30 times higher than that of compound 3. By including dispersion interactions (the dispersion correction GD3,[27]Table S8, SI), the ΔG≠ values were reduced by about 0.4–1.7 kcal/mol and thus the rate constants increased by around 1.1–6.7 times. Based on these results, compound 5 is expected to have the highest antioxidant scavenging activity. This result is in good agreement with the results of the thermodynamic study. However, compared with typical antioxidants, i.e., Trolox or ascorbic acid, these compounds have very low antioxidant activities, at least in apolar solvents.

Table 2

Calculated ΔG≠ (kcal/mol), κ, and keck (M–1 s–1) at 298.15 K of the Reaction between the Selective Depsidones and CH3OO• in the Gas Phase

comp.	ΔG≠	κ	keck
1	20.6	216.2	9.64 × 10–1
2	21.5	232.7	2.51 × 10–1
3	22.5	241.5	4.81 × 10–2
4	21.3	163.6	2.47 × 10–1
5	20.2	141.8	1.37
6	20.5	221.7	1.29
9	21.4	128.4	1.69 × 10–1
10	21.1	111.7	2.41 × 10–1

Radical Scavenging Activity of Depsidones in Aqueous Solutions

Evaluating Acid–Base Equilibria

While the intact molecules would only exhibit very weak antioxidant activity, for aqueous media the protonation states of the acidic moieties of the n class="Chemical">depsidones have to be also considered.[28] Ionic forms of acidic molecules can exhibit vastly different antioxidant properties and may follow different mechanistic class="Chemical">pathways.[20,28,29] It is generally acknowledged that there is a close correlation between pKa values and structures of compounds.[30−35] Thus, to reduce the required calculation time, compound 1 was used as pKa reference for compounds 1–5, whereas compound 8 was the reference for compounds 8–12. On the basis of the calculations (Table S2, SI), the lowest class="Chemical">pan class="Chemical">PA values are found at the COO–H and O–H bonds. These groups were targeted for the investigation of the acid–base equilibria of the depsidones (DEP). The pKa was calculated by the model shown in eq following the literature[31] for the COOH and O–H phenolic groups, while the model shown in eq , in which the [H2O] value is 55.55 mol·L–1, was used for the O1–H group of compounds 1 and 6.[31,36] The values of pKa were defined by eqs and 4.[19,31,37]where the HRef is phenol for O–H phenolic groups (the experimental pKa(O–H) = 10.09 according to ref[38]) and salicylic acid for carboxylic groups (the experimental pKa(COOH) = 2.97 according to ref[39]). The calculated pKa values are presented in Figure . The pKa values for compound 1 are 5.56, 8.17, 9.92, and 13.61 for the O–H bonds at positions of 1, 10, 4, and 5-CH2-OH, respectively. The results are in good agreement with previous data.[40] The calculated pKa values for compound 8 are 2.51 (COOH), 7.83 (O3–H), and 13.56 (O9–H); those for compound 6 are 3.00 (COOH), 6.87 (O1–H), and 10.12 (O4–H); those for compound 7 are 7.90 (O10–H) and 9.49 (O1–H) bonds, respectively, and that for compound 11 are 5.63 (Figure ). The pKa values for these compounds are reported for the first time. Thus, at physiological pH (7.40) in aqueous solutions, the main stable forms of compounds 1–5 are H2A– (>90%), those of compounds 8–12 are H2A– (73%) and HA2– (27%). The main stable forms of compounds 6 and 11 are HA2– and A– (∼100%), respectively, whereas those of compound 7 are H2A (76%) and H2A– (24%).

Figure 4

Calculated pKa values of the studied compounds.

Based on the above data, the mechanism of antioxidant activity has to be re-evaluated for aqueous environments. The gas-phase data suggested that the dominant n class="Chemical">FHT mechanism yields very low rate constants in the class="Chemical">pan class="Chemical">CH3OO• radical scavenging reaction, practically under all circumstances; thus, FHT is not a viable pathway for radical scavenging. SET mechanisms were ruled out by thermochemical data. The only alternative is the RAF mechanism; however, it was shown before that it is generally not supported for ROO• radical scavenging.[41] However, considering only ROO• scavenging might be misleading. It was shown that HO• radicals cause the majority of tissue damage as well as damage to DNA in biological systems.[42,43] When considering HO• scavenging, several studies have shown that for acidic compounds the SET mechanism is the principal pathway in aqueous solutions.[29,44] Furthermore, the depsidones were also known as potential superoxide anion scavengers.[15] In this progress, O2•– generally donates an electron to depsidones to form stable O2 (triplet).[45] Thus, in aqueous solutions, it is important to consider HO• and O2•– scavenging as well, following the SET mechanismwhere R• = HO•, HOO•, and CH3OO•

Kinetic Study

As span class="Chemical">hown in the thermodynamic section, the effect of the dispersion interactions on the IE values was minor. Thus, the dispersion correction was not included in the kinetic calculations following the SET mechanism. To save calculation time, the kinetics of the reactions between the radicals and the studied class="Chemical">pan class="Chemical">depsidones were first calculated for aqueous environments at the M05-2X/6-31+G(d) levels,[28] and the results are shown in Table S5, SI. It was found that the ROO• (i.e., HOO• and CH3OO•) scavenging activities of the studied depsidones are generally much lower than HO• radical scavenging activities, with the rate constants <101 M–1 s–1. Thus, ROO• radical scavenging is not likely to occur in aqueous solutions. This is in good agreement with the obtained results in the gas phase. Therefore, the accurate calculation (using the M05-2X/6-311++G(d,p) method) was only focused on HO• and O2•– scavenging, and the results are presented in Table .

Table 3

Calculated Rate Constants (kapp, M–1 s–1, 298.15 K) of the Reaction between the Depsidones and the Radicals in Water Following the SET Mechanism

	HO•			O2•–
comp.	main forms	%	kapp	reagents	kapp
1	HA–	>90	2.90 × 106	HA•	2.70 × 108
2	HA–	>90	3.30 × 107	HA•	2.20 × 109
3	HA–	>90	4.60 × 105	HA•	3.80 × 109
4	HA–	>90	8.60 × 109	HA•	1.60 × 109
5	HA–	>90	1.60 × 108	HA•	3.30 × 109
6	HA–	∼100	3.00 × 108	HA•	5.50 × 109
7	HA–	24	8.50 × 109	HA•	3.20 × 109
8	H2A–	73	7.30 × 109	H2A•	7.90 × 109
	HA2–	27	8.60 × 109	HA•–	8.10 × 109
9	H2A–	73	9.30 × 108	H2A•	5.30 × 109
	HA2–	27	5.40 × 109	HA•–	7.30 × 109
10	H2A–	73	3.50 × 109	H2A•	6.80 × 109
	HA2–	27	8.60 × 109	HA•–	7.90 × 109
11	A–	∼100	8.60 × 109	A•	2.60 × 109
12	H2A–	73	3.00 × 109	H2A•	6.30 × 109
	HA2–	27	4.90 × 109	HA•–	8.30 × 109

As span class="Chemical">hown in Table , the steady-state Smolucclass="Chemical">pan class="Chemical">howski[46] rate constants (kD) of the studied compounds with HO• are about 109 M–1 s–1. The rate constants of 1, 2, and 3 with HO• following the SET mechanism are in the range of 4.60 × 105–3.3 × 107 M–1 s–1; for the other depsidones, nearly reach the kD at about 1.60 × 108–8.60 × 109 M–1 s–1. Thus, the HO• radical scavenging activities of 1, 2, and 3 following the SET mechanism are slower than those of the rest of the compounds. Among the studied depsidones, compounds 4, 8, 10, and 11 have the highest rate constants for HO• radical scavenging following the SET mechanism of 8.60 × 109 M–1 s–1. Comparing with typical antioxidants such as caffeine (koverall(OH) = 2.15 × 109 M–1 s–1),[47] glutathione (koverall(OH) = 7.68 × 109 M–1 s–1),[31] and Trolox (koverall(OH) = 2.78 × 1010 M–1 s–1)[28] in aqueous media, HO• radical scavenging of compounds 4, 7, 8, 9, 10, 11, and 12 is as fast as that of the most of the natural antioxidants.

It is important to notice that O2•– scavenging of the pan class="Chemical">depsidones occurs significantly with the rate constants in the range of 2.60 × 108–8.30 × 109 M–1 s–1. class="Chemical">pan class="Chemical">Hydroxyl radical and superoxide anion scavenging of depsidones may occur as a regeneration cycle (Figure ), in which the depsidone anions donate an electron to the hydroxyl radicals to form depsidone radicals (k = 4.60 × 105 to 8.60 × 109 M–1 s–1) and in the half-revered cycle the radicals withdraw an electron from O2•– to convert the initial anions (k = 2.60 × 108 to 8.30 × 109 M–1 s–1, Table ). As a result, the radicals (HO• and O2•–) are killed to form the stable species of oxygen (HO– and O2 triplet). This process can increase the protective effects of depsidones against oxidative stress. Thus, these compounds are promising candidates for HO• and superoxide anion radical scavenging in polar environments.

Figure 5

Regeneration cycle of hydroxyl and superoxide radical scavenging of depsidones following the SET mechanism in the polar environment.

Regeneration cycle of pan class="Chemical">hydroxyl and class="Chemical">pan class="Chemical">superoxide radical scavenging of depsidones following the SET mechanism in the polar environment.

Conclusions

The radical scavenging activity of natural pan class="Chemical">depsidones from class="Chemical">pan class="Chemical">Ramalina lichenized fungi was investigated by thermodynamic and kinetic calculations in the gas phase as well as in an aqueous solution. It was found that the BDE values are in the range of 74.4–87.7 kcal·mol–1. The lowest BDE(C–H)s were observed for salazinic acid (1), cryptostictic acid (5), peristictic acid (6) at 76.9, 74.4, and 75.2 kcal·mol–1, respectively. However, the rate constants of CH3OO• scavenging of the studied compounds following the FHT mechanism in the gas phase are low in the range of 4.81 × 10–2 to 1.37 M–1 s–1. This suggests that depsidones are not good ROO• radical scavengers in either polar or apolar environments. However, these compounds are excellent HO• and O2•– radical scavengers in aqueous media following the SET mechanism with the rate constants in the ranges of k = 4.60 × 105 to 8.60 × 109 M–1 s–1 and k = 2.60 × 108 to 8.30 × 109 M–1 s–1, respectively. These results suggest that the natural fungal depsidones exhibit potential hydroxyl and superoxide anion radical scavenging activity in polar environments.

Computational Methods

In this study, the (RO)B3pan class="Gene">LYP/6-311++G(2df,2p)//B3class="Chemical">pan class="Gene">LYP/6-311G(d,p) calculation model, which is highly recommended for calculating thermochemical properties (BDE, PAs, and IEs) due to a demonstrated good agreement with experimental values (i.e., a BDE error of ∼2 kcal/mol compared with experimental values),[16,21−25] was used to compute the thermochemical properties of studied compounds, in which the reaction enthalpies of the individual steps (in the gas phase, at 298.15 K and 1 atm) were computed following the literature.[16,17,23] The enthalpies of the proton and electron were refereed from previous studies, while that of the hydrogen atom was computed at the same level of the studied compounds.[48−51]

The quantum mechanics-based test for the overall free radical scavenging activity (QM-ORSA) protocol[28] that is best suited to perform kinetic calculations in the gas phase as well as in the aqueous solution (the solvation model density (pan class="Disease">SMD)) was used (kcalc/kexp ratio, 1.0–2.9).[19,28,37] The rate constant was computed according to the transition state theory (at 298.15 K, 1M standard state) following the literature[18,52−57] and is presented in Table S6, SI.

For the species that have multiple conformers, all of these were investigated and the lowest electronic energy conformer was included in the analysis.[19,37] The correction of transition states was confirmed by calculating intrinsic n class="Chemical">coordinate calculations (IRCs) and the presence of only one single imaginary frequency. All calculations were performed with the Gaussian 09 suite of programs.[58] Kinetics were calculated by the M05-2X/6-311++G(d,p) calculating metclass="Chemical">pan class="Chemical">hod as it is highly recommended for calculating rate constants (kcalc/kexp ratio, 1.0–2.9)[20,28] using the Eyringpy code.[59,60]