Is Usnic Acid a Promising Radical Scavenger?

Usnic acid (UA) is a natural product found in the lichen genera. Because of the phenolic groups in its structure, UA is suspected to be an antioxidant. Therefore, in this study, the radical scavenging of UA was investigated in physiological environments in silico by using kinetic calculations. It was found that the overall rate constant for the hydroxyl radical scavenging activity was approximately 109 M-1 s-1 in all environments, whereas the HOO• and CH3OO• radical scavenging activities were only significant in the polar environments with k in the range of 103-104 M-1 s-1. The results also revealed that the HO• scavenging activity followed the single electron transfer (SET) and radical adduct formation mechanisms; however, the SET pathway (for the dianion HU2-) played a dominant role in the scavenging of other studied radicals, including CH3O•, CCl3O•, CCl3OO•, NO2, SO4 •-, and N3 •. The activity of UA against these radicals was as high as that of typical phenolic acids such as ferulic acid, p-coumaric acid, caffeic acid, dihydrocaffeic acid, and sinapinic acid (k f ∼ 108 M-1 s-1) in polar solvents. Thus, UA is a promising natural antioxidant in aqueous environments.

Introduction

Usnic acid (UA), a natural product that is isolated from lichens, has potential antibacterial effects as well as other biological activities such as anti-inflammatory, anticancer, and antioxidant properties.[1−4] The antioxidant activity of UA was assessed by in vivo and in vitro studies.[1,5−8] It was reported that UA could reduce oxidative damage by increasing glutathione peroxidase activity, constitutive nitric oxide synthase, superoxide dismutase activity, and total glutathione activities.[6,9,10] UA showed strong antioxidant capacity in oxygen radical absorbance capacity assay, indicating significantly reduced radical oxygen species production,[11,12] whereas it showed no antioxidant activity in 2,2-diphenylpicrylhydrazyl assay.[13,14] This ambiguous behavior warrants a theoretical investigation of the free-radical scavenging activity of UA including a detailed evaluation of the mechanism of its action to understand its biological role. UA exists in two enantiomers: (+)-UA and (−)-UA, depending on the configuration of the methyl group at the 1′ position (Figure ). It was shown that the (+) enantiomer was more stable and exhibited higher biological activity in, for example, its antiviral and antibacterial functions than the (−) enantiomer.[2,15,16] Thus, in this study, the (+)-UA (Figure ) structure was used to evaluate the radical scavenging of UA.

Figure 1

Structure of UA.

This study aims to investigate the radical scavenging activity of UA in aqueous and lipid media by (i) thermodynamic calculations to identify the most likely mechanism of action and (ii) kinetic calculations to evaluate the activity of UA in HO•, HOO•, CH3O•, CCl3O•, HOO•, CH3OO•, CCl3OO•, NO, NO2 (free radical), O2•–, SO4•–, Br2•–, and N3• radical scavenging reactions.

Results and Discussion

Thermodynamic Study

Acid–Base Equilibria

The pKa values for UA are pKa1 = 4.4 (O3–H), pKa2 = 8.8 (O7–H), and pKa3 = 10.7 (O9–H).[17,18] Thus, under physiological conditions (pH 7.40), UA exists in both monovalent anionic state (O3–H bond, H2U–, 96.1%), and dianionic state (03–H and O7–H bonds, HU2–, 3.9%), and therefore, the H2U– and H2U– forms were used to evaluate the radical scavenging of UA in aqueous solution. In the lipid environment, the neutral UA (H3U) was modeled.

In this study, the radical scavenging of UA was evaluated following three main antioxidant pathways: single electron transfer (SET), formal hydrogen transfer (FHT), and radical adduct formation (RAF) in the studied solvents according to the following reactions:[19]

In the aqueous solution

Also, for the divalent anionic form[20]

In the lipid mediumwhere R• = HO•, HOO•. To model FHT, the direct hydrogen atom transfer pathway was calculated.

Mechanism Valuation

The ΔGo values of the UA + HO•/HOO• reactions were used for evaluating the preferred antioxidant mechanism. ΔGo values were calculated following each mechanism, and the results are presented in Table . It was found that the hydroxyl radical scavenging of UA is almost always spontaneous (ΔGo < 0) in the studied media, with the exception of the SET and RAF mechanisms at the C9′ position. However, the reaction of UA with HOO• radical was only spontaneous for the SET-2 mechanism. This suggests that the HOO• radical scavenging in the aqueous solution only occurs following the SET-2 mechanism. Thus, the overall rate constants can be calculated according to eqs –11.

Table 1

Calculated ΔGo Values of the Reactions of UA with HO• and HOO• Radicals in Water and Pentyl Ethanoate Solvents (in kcal/mol)

		OH		OOH
mechanism		water	pentyl ethanoate	water	pentyl ethanoate
set-1		7.6	56.0	31.1	77.0
set-2		–15.2		8.3
FHT	C10	–31.4	–28.3	1.5	5.1
	C12	–26.8	–22.8	6.1	10.6
	C14	–24.9	–22.5	8	10.8
	C15	–18.2	–15.6	14.7	17.8
	O3		–14.8		18.6
	O7	–26.6	–19.7	6.3	13.7
	O9	–22.3	–2.8	10.7	30.5
RAF	C2	–10.0	–7.4	20.2	30.0
	C3		–5.6		25.1
	C4	–20.9	–21.2	8.6	14.7
	C4′	–15.9	–16.1	9.0	15.6
	C6	–3.0	–3.2	24.1	29.4
	C6′	–13.8	–15.5	14.4	22.5
	C7	–17.5	–17.7	17.4	23.6
	C8	–22.8	–25.1	3.4	6.6
	C9	–6.3	–6.8	23.4	27.5
	C9′	1.3	0.7	30.4	37.2

In the aqueous solution

In the nonpolar solvent

Kinetic Study

HO• and HOO• Radical Scavenging Activity of UA

Calculations for the overall rate constant (koverall) of the HO• and HOO• radical scavenging reaction were carried out for all of the reactions by following eqs –11. The kinetic results and the optimized structures of typical transition states (TS) (Γ > 1%) are shown in Table and Figure , respectively.

Table 2

Gibbs Free Energies of Activation (ΔG⧧, kcal/mol), Tunneling Corrections (κ), Rate Constants (kapp, M–1 s–1), and Branching Ratios (Γ, %) at 298.15 K for the UA Oxidation by HO• and HOO• Radicals in the Studied Environmentsa

			water					pentyl ethanoate
	mechanism		ΔG⧧	κ	kapp	kf	Γ	ΔG⧧	κ	kapp	Γ
	set-1		7.7	6.9	1.50 × 107	1.44 × 107	0.2
	Set-2		3.9	5.7	4.10 × 109	1.60 × 108	1.7
OH•	HAT	O3						17.7	18.7	1.21 × 101	0.0
		O7	15.3	25.8	9.63 × 102	9.26 × 102	0.0	17.6	19.6	1.49 × 101	0.0
		O9	15.7	28.9	5.49 × 102	5.28 × 102	0.0	17.9	25.9	1.24 × 101	0.0
		C10	5.7	3.2	1.93 × 109	1.86 × 109	20.2	7.1	3.5	3.50 × 108	15.8
		C12	7.2	5.9	1.00 × 109	9.65 × 108	10.5	9.4	5.7	1.40 × 107	0.6
		C14	8	4.8	3.15 × 108	3.03 × 108	3.3	10.1	4.5	3.20 × 106	0.1
		C15	8.3	5.8	2.35 × 108	2.25 × 108	2.5	10.3	5.7	3.10 × 106	0.1
	RAF	C2	8.6	1.2	1.46 × 107	1.40 × 107	0.2	10.4	1.3	3.80 × 105	0.0
		C3					0.0	23.1	1.5	2.10 × 10–4	0.0
		C4	4	1.5	2.20 × 109	2.11 × 109	23	6.6	1.1	1.80 × 108	8.1
		C4′	6.6	1.1	3.22 × 108	3.10 × 108	3.4	7.1	0.6	4.50 × 107	2.0
		C6	7.3	1.2	1.25 × 108	1.20 × 108	1.3	8.5	0.8	5.78 × 106	0.3
		C6′	9.7	1.0	9.80 × 105	9.41 × 105	0.0	8.2	1.1	1.40 × 107	0.6
		C7	5.9	1.2	8.89 × 108	8.54 × 108	9.3	9.3	1.2	2.10 × 106	0.1
		C8	2.7	1.0	2.30 × 109	2.21 × 109	24.1	4.7	1.1	1.60 × 109	72.1
		C9	8	1.1	3.47 × 107	3.33 × 107	0.4	10.1	1.3	6.32 × 105	0.0
	koverall					9.18×109				2.22 × 109
OOH•	set-2		9.5	17.2	7.20 × 105	2.81×104	100

The nuclear reorganization energy (λ); kf = f × kapp, (f(H2UA–) = 0.961, f(H2UA2–) = 0.039).

Figure 2

Optimized structure of typical TSs.

The overall rate constant for the HO• radical scavenging in the polar environment (koverall = 9.18 × 109 M–1 s–1) was slightly higher than that for the nonpolar media (koverall = 2.22 × 109 M–1 s–1). The SET mechanism played a minor role (<2%) in the koverall in either of the studied solvents, whereas the contributions of RAF mechanism in the hydroxyl radical scavenging of UA were about 60 and 80% in water and pentyl ethanoate, respectively. The FHT pathway contributed 36.5% of the koverall in the aqueous solution and 16.6% of the koverall in the lipid media. Thus, the results indicate that the RAF and FHT mechanisms decide the hydroxyl radical scavenging of UA in the polar solvent and the RAF mechanism in the nonpolar solvent. The hydroxyl radical scavenging of UA is somewhat lower than that of typical antioxidants such as melatonin,[21] dopamine,[22] indolinonic hydroxylamine,[23] ramalin,[24] indole-3-carbinol,[25] and Trolox[26] in all of the studied media. Thus, UA is a moderate hydroxyl radical scavenger.

It is important to note that the SET-2 mechanism played a deciding role in the HOO radical scavenging of UA with kapp = 7.20 × 105 M–1 s–1 (kf = 2.81 × 104 M–1 s–1) in the polar solvent; however, the mechanism did not occur with UA in lipid media. Based on these results, UA has a similar activity to Trolox (k = 8.96 × 104 M–1 s–1) in the HOO• scavenging reaction in polar environments.

Broad Spectrum Radical Scavenging Activity of UA in Aqueous Solution

As shown in the thermodynamic section, the HU2– state could eliminate radicals in the aqueous medium following the SET-2 mechanism. Thus, in this section, the radical scavenging activity of HU2– was investigated against a broad range of radicals including CH3O•, HOO•, CH3OO•, CCl3OO•, NO, NO2 (free radical), O2•–, SO4•–, Br2•–, and N3•, and the results are presented in Table .

Table 3

Calculated Kinetic Parameters of the Reaction between HU2– and Selected Radicals Following the SET-2 Mechanism in Aqueous Solution at pH 7.4a

radical	ΔG⧧	λ	kD	kapp	kf
HO•	3.9	5.7	8.40 × 109	4.10 × 109	1.60 × 108
CH3O•	2.8	7.7	8.10 × 109	7.10 × 109	2.77 × 108
CCl3O•	0.2	54.8	7.60 × 109	7.60 × 109	2.96 × 108
HOO•	9.5	17.2	8.20 × 109	7.20 × 105	2.81 × 104
CH3OO•	11.0	16.6	8.00 × 109	5.80 × 104	2.26 × 103
CCl3OO•	0.5	18.3	6.80 × 109	6.80 × 109	2.65 × 108
NO	96.4	16.2	8.40 × 109	1.30 × 10–58	5.07 × 10–60
NO2	2.0	30.0	8.20 × 109	7.80 × 109	3.04 × 108
O2•–	55.3	19.1	8.60 × 109	1.80 × 10–28	7.02 × 10–30
SO4•–	3.0	21.0	7.80 × 109	6.50 × 109	2.54 × 108
N3•	2.7	4.9	8.10 × 109	7.10 × 109	2.77 × 108

kf = f × kapp, f(H2UA2–) = 0.039.

As shown in Table , UA is suggested to exhibit good activity against CH3O•, CCl3O•, CCl3OO•, NO2, SO4•–, and N3• radicals with the kapp in the range of 4.10 × 109–7.80 × 109 M–1 s–1, at a rate fairly similar to the diffusion-limited rate constants (kD). UA exhibited moderate alkylperoxy (i.e., HOO• and CH3OO•) antiradical activity with kapp = 103–104 M–1 s–1, whereas NO and O2•– scavenging could not occur under the studied conditions. Comparing with hydroxycinnamic acid derivatives such as ferulic acid, p-coumaric acid, caffeic acid, dihydrocaffeic acid,[20] and sinapinic acid,[27] the predicted activities for UA against CH3O•, CCl3O•, CCl3OO•, NO2, SO4•–, and N3• radicals were as high as those of these compounds (kf ∼ 108 M–1 s–1). Thus, UA is a promising natural antioxidant in aqueous solution.

Conclusions

The radical scavenging of UA was investigated in aqueous and lipid (pentyl ethanoate) media. It was found that UA had moderate HOO• and CH3OO• radical scavenging activities in aqueous solution (k = 103–104 M–1 s–1), whereas activity in lipid media was generally low. The overall rate constants for the hydroxyl radical scavenging were koverall = 9.18 × 109 and 2.22 × 109 M–1 s–1 in polar and nonpolar solvents, respectively. The HO• antiradical activity mainly followed the SET and RAF mechanisms; however, the SET-2 pathway played a deciding role in the radical scavenging activity of the other studied radicals. UA exhibited activities against CH3O•, CCl3O•, CCl3OO•, NO2, SO4•–, and N3•, which were as high as those of typical phenolic acids such as ferulic acid, p-coumaric acid, caffeic acid, dihydrocaffeic acid, and sinapinic acid (kf ∼ 108 M–1 s–1) in polar solvents. Thus, UA is a promising radical scavenger in aqueous solution.

Computational Methods

In this study, the quantum mechanics-based test for the overall free-radical scavenging activity protocol was applied to perform the kinetic calculations in water and pentyl ethanoate environments by using the M05-2X/6-311++G(d,p) method with the solvation model based on density that has been widely used for evaluating the radical scavenging activity of antioxidants because of low errors compared with experimental data (kcalc/kexp ratio = 1–2.9).[23,26,28−31] Rate constants were computed according to the TS theory (at 298.15 K, 1 M standard state) following the equation[31−37]where σ is the reaction symmetry number,[38,39] κ contains the tunneling corrections calculated using the Eckart barrier,[40]kB is the Boltzmann constant, h is the Planck constant, and ΔG⧧ is the Gibbs free energy of activation.

The SET reaction barriers were corrected following the Marcus theory,[41,42] whereas the apparent rate constants (kapp) in solvents were computed following the literature.[43,44] Okuno’s corrections[45] of the free volume theory following the Benson correction were also applied to minimize overpenalizing entropy in solution.[46] All of the studied species (molecules, radicals, anions, and TSs) were treated by the hindered internal rotation method to obtain the lowest energy conformers that were used in the further studies.[47] Intrinsic coordinate calculations were carried out to confirm that the TSs connect to the reactants and products. All of the calculations were performed by using the Gaussian 09 program package[48] and the Eyringpy program[49,50] depending on each individual task.