Theoretical Study on the Radical Scavenging Activity of Shikonin and its Ester Derivatives

Aim of study is to get a further insight on the contribution of the structural features to the radical scavenging potential of shikonin and its ester derivatives. The radical scavenging activity of shikonin and its ester derivatives have been studied by using density functional theory. The hydrogen bond property of the studied structures has been investigated by using the Atoms in Molecules (AIM) theory. The calculated results reveal that the hydrogen bond is important for good scavenging activity. The hydrogen atom transfer mechanism for shikonin and its ester derivatives are difficult to occur. However, shikonin and its ester derivatives appear to be good candidates for the one-electron-transfer mechanism. The shikonin ester derivatives investigated in this study are expected to be of high radical scavenging activity compared with parent compound shikonin. Taking this system as an example, we present an efficient method for the investigation of radical scavenging activity from theoretical point of view.


INTRODUCTION
Alkannin and shikonin are isohexenylnaphthazarin derivatives of hydroxynaphthoquinones.These two compounds are enantiomers (S/R) and mostly present as ester derivatives in the outer surface of the roots of at least 150 species belonging to the genera Alkanna, Lithospermum, Echium, Onosma, Anchusa and Cynoglossum of the Boraginaceae family (Akgun et al., 2009).Recently interest on these two compounds increased because of their pharmacological properties (Deng et al., 2010;Noula et al., 2010;Ordoudi et al., 2011).Phenolic compounds can act as antioxidants, by inhibiting biomolecules from undergoing oxidative damage through free radicals mediated reactions (Chang et al., 2007;Visioli et al., 1998).The antioxidant activity of the phenolic constituents is related to their structures.The intramolecular hydrogen bonds play important roles in stability of the radicals (Nenadis and Sigalas, 2008;Russo et al., 2000).However, to the best of our knowledge, no calculation about the antioxidant activity of shikonin and its ester derivatives has been reported so far.
Recently, (Albreht et al., 2009) reported a series of shikonin ester derivatives (Fig. 1).Herein the structural and electronic properties of shikonin and its ester derivatives and their radicals were investigated at Computational details: Phenoxyl radicals represent important intermediates in many biological applications (Halliwell and Gutteridge, 1989).Two main kinds of mechanism are reported for the radical scavenging processes of chain-breaking antioxidants ArOH (Leopoldini et al., 2004): H-atom transfer, Eq. 1, and one-electron transfer, Eq. 2.
In the H-atom transfer mechanism, the Bond Dissociation Energy (BDE) of the O-H bonds is an important parameter to evaluate the antioxidant action, because the weaker the O-H bond the easier the reaction of free radical inactivation will be.In the oneelectron transfer mechanism, the radical cation arising from the electron transfer must be stable, so it does not react with substrate molecules.In this case, the Ionization Potential (IP) is the most significant energetic factor for the scavenging activity evaluation.Molecules with the low IP and BDE values are expected to have high activity.
All calculations were performed by Gaussian 09 code (Frisch, 2009).The geometries of shikonin and 1-7 were optimized at the B3LYP/6-31+G (d) level without symmetry constraints.Single-point electronic energies (SPEs) were obtained at the B3LYP/6-311++G (d,p) level.The BDE and IP values were evaluated by the B3LYP/6-311++G (d,p) single-point energy calculations with the thermal contributions to enthalpy and the zero-point energy corrections at the B3LYP/6-31+G(d) level, respectively.The BDE values were calculated according to the formula BDE = H r + H h -H p , where H r is the enthalpy of the radical generated by H-abstraction, H h is the enthalpy of the H-atom (-0.499897 hartree at this level of theory), and H p is the enthalpy of the parent molecule.The IP values were determined according to the equation IP = E cr -E p , where p and cr indicate the parent molecule and the corresponding cation radical generated after electron transfer.

RESULTS AND DISCUSSION
Hydrogen atom transfer mechanism: Starting from the absolute minima of each species, two radicals from shikonin and 1-7 were obtained upon H-atom abstraction from every hydroxyl phenolic group, whereas a single radical cation for each parent molecule was obtained by removing one electron.Figure 2 presents the spin densities of 5-OH and 8-OH radicals for shikonin and 1-3 as representatives.Hydroxyanthraquinone derivatives are usually considered radical scavengers due to their excellent delocalization possibility.For both 5-OH and 8-OH radicals, the spin distribution indicates the oxygen atoms O 11 and O 13 from which the H atoms are removed as the most probable radical centers.
The calculated BDE and IP values of shikonin and 1-7 are presented in Table 1.The BDE values characterize the hydrogen atom donating ability of shikonin and 1-7.On the basis of BDE values, it is evident that the radical scavenging activity of 5-OH and 83.9 (0.0) 84.0 (-1.4) 175.3 (-5.7) a : The relative BDE and IP (in the parentheses) are referred to shikonin 8-OH radicals for 1-7 is predicted to be almost equal to that of shikonin radical.Such efficiency is related to the further stabilization of the derived formation of an intramolecular hydrogen in both 5-OH and 8-OH radicals.The trend of BDE values for the examined compounds suggests that an essential factor for a good radical scavenging activity is the intramolecular hydrogen bond type structure, and the introduction of ester groups for shikonin decreases (increases) slightly the radical scavenging activity of 5-OH (8-OH) radicals for 1-7.However, abstraction of the H atom from the hydroxyl in positions 5 and 8 implies the breaking of the hydrogen bonds formed with the 4 and 1-keto groups, respectively.Hence, the hydrogen atom transfer for shikonin and 1-7 are difficult to occur.
Single electron transfer mechanism: Figure 3 presents the spin densities for the radical cations arising from shikonin and 1-3 as representatives.The unpaired electron delocalized over the naphthoquinones and the substituent groups on 3-position, as shown in Fig. 3.The results displayed in Table 1 reveals that the lowest Table 2: Electronic density at BCP ρ(r) bcp , the laplacian∇ 2 ρ(r) bcp (in au) and the hydrogen bond energy E HB (in kcal/mol) of shikonin and 1-7 IP value is found for 1, followed by 7, 3, 4, 2, 5, and 6.The electron-donating ability of shikonin and 1-7 seems to be related to an extended electronic delocalization over all the molecules.Systems having a high degree of π-delocalization are the most active among the oneelectron-transfer mechanism.
Furthermore, the introduction of the ester groups for shikonin leads to a decrease the IP values compared with that of shikonin, particularly for 1.For conjugated system, the IP is raised in energy, so the abstraction of an electron becomes very easy.The trend of IP values for the examined compounds suggests that an essential factor for a good activity as antioxidant is the hydrogen bond type structure, and it can be further improved by the introduction of the ester groups for the molecules.The IP values of 1-7 are lower than that of shikonin, suggesting that 1-7 are expected to be of high radical scavenging activity compared with parent compound shikonin.
Atoms in molecules analysis: Atoms in molecules (AIM) (Bader, 1991) theory was applied at the B3LYP/6-311++G(d,p) level to investigate the topology of electron density and the OH⋅⋅⋅O hydrogen bond properties of the studied structures.The electron densities, ρ(r) bcp , their Laplacians, ∇ 2 ρ(r) bcp , and the potential electron energy densities V(r) bcp at bond critical points (BCPs) are frequently used as indicators of hydrogen bond.The hydrogen bond energy E HB (defined as-De, where De is the hydrogen bond dissociation energy) in molecules can be estimated within the framework of the AIM analysis using the relationship: Espinosa et al. (1998).Comparing the ρ(r) bcp , their Laplacians ∇ 2 ρ(r) bcp , and E HB in neutral forms with those in radical cations shown in Table 2 and SI, one can find that the strength of H 14 ···O 15 hydrogen bonds in radical cations is stronger, while the corresponding strength of H 12 ···O 16 hydrogen bonds is weaker than those in their neutral forms, respectively.An enhancement of the strength of hydrogen bond in radical cations confers high stability to radical cations of acylshikonin derivatives.

CONCLUSION
The introduction of ester groups for shikonin decreases/increases the radical scavenging activities of 5-OH/8-OH radicals slightly.Shikonin and its ester derivatives appear to be good candidates for the oneelectron-transfer mechanism, particularly for acetylshikonin (1).The introduction of the ester groups for shikonin decreases the IP values compared with that of shikonin.All the shikonin ester derivatives are expected to be of high radical scavenging activity compared with parent compound shikonin.

Fig. 1 :
Fig. 1: Geometries of shikonin and its ester derivatives, along with atom numbering

Fig. 3 :
Fig. 2: The spin densities of 5-OH and 8-OH radicals for shikonin and 1-3 Table 2 presents the topological parameters and the E HB values for shikonin and 1-7.As a general rule, hydrogen bonds are characterized by positive values of ∇ 2 ρ(r) bcp , low ρ(r) bcp values (<0.1).Inspection of Table 2 reveals clearly that the ρ(r) bcp values of the O 16 •••H 12 hydrogen bonds in 1-7 are greater, while the corresponding values of O 15 •••H 14 are lower than those in shikonin, respectively.It suggests that the strength of O 16 •••H 12 hydrogen bonds in 1-7 are stronger, while the corresponding strength of O 15 •••H 14 hydrogen bonds are weaker than those in shikonin, respectively.Furthermore, the E HB values of O 16 •••H 12 hydrogen bonds in 1-7 are greater, while the corresponding values of O 15 •••H 14 are lower than those in 1, respectively.