DFT study on the antioxidant activity of rosmarinic acid

Journal of Molecular Structure: THEOCHEM 719 (2005) 177–183 www.elsevier.com/locate/theochem

DFT study on the antioxidant activity of rosmarinic acid Huai Caoa,b,*, Wei-Xian Chenga,b, Cong Lia,b, Xu-Lin Pana,b, Xiao-Guang Xiea,b, Tao-Hong Lia,b a

Modern Biological Research Center, Yunnan University, No. 2 North Cuihu Road, Kunming 650091, China b Department of Chemistry, Yunnan University, Kunming 650091, China Received 19 November 2004; revised 7 January 2005; accepted 23 January 2005

Abstract A theoretical calculation based on the density functional theory (DFT) has been performed to understand the antioxidant activity of rosmarinic acid in view of a molecular structure. According to the geometry of the ground state molecule and its free radical, the HOMO and LUMO, the O–H bond dissociation energy (BDE), and the single electron density distribution of the radicals, we interpreted the capacity of scavenging the radicals for rosmarinic acid by its structure. Compared with flavonoids, the higher antioxidant activity of rosmarinic acid may be from the abstraction of hydrogen atoms of the ortho-position hydroxyls on the rings A and B. This abstraction can occur continuously to form a semiquinone structure, or even to form a quinone structure. The activity of the ring A is similar with the ring B. However, the ring B is a stronger electron donor than the ring A, and, the radical formed from the H-abstraction of the ring A is more stable than that of the ring B. We also found that there is a good relationship between the BDE, which is used to show the stability of the parent molecule and the unpaired electron delocalization correlated to the stability of the free radical. These theoretical researches will be helpful to the development for the antioxidant compounds. q 2005 Elsevier B.V. All rights reserved. Keywords: Rosmarinic acid; DFT; Antioxidant activity; Free radical structure

1. Introduction Free radicals can result in the food souredness, the oil rottedness, and the most of industrial product aging. BHT, BHA, and TBH are extensively used as antioxidants at present in order to reduce the harm caused by free radicals. However, these antioxidants will be unfavorable to be used in the fields of foods and health products because they would induce some tumors and other toxicity in animal body [1]. Many experiments indicated that free radicals are necessary to support the life though they are also dangerous to exist in the biological cells and tissues. Under the normal physiological conditions, the free radicals in the body will undergo a process of the producing and scavenging continuously so as to sustain the physiological equilibrium. When the free radicals generated in the body are short and the concentrates are low, the body metabolism may be in disorder and some diseases can be caused [2]. Nevertheless, body can give rise * Corresponding author. Tel.: C86 871 5033496; fax: C86 871 5036373. E-mail address: [email protected] (H. Cao).

0166-1280/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2005.01.029

to surplus free radicals under the majority circumstances. More and more experiments have shown the common illness and injury mechanism of virus pneumonia: the loading-host immune cells (such as phagocyte, neutrophilic granulocyte) can generate the surplus free radical molecules to injure the lung tissue during the interaction between the virus and the immune cells which permeate into the lung. This can lead to the losing of lung function and even the death of loading-host on account of the lung dropsy and bleeding [3]. A model experiment tested that there was no virus subsisting in the lung tissue of the infected mouse [4]. Virus itself is only the troublemaker, but those immune cells generating surplus free radical molecules are the main reason to injure the lung cells. Therefore, it may be a potential therapeutic strategy suited for the middle and last stages of the virus infection to synthesize and sieve the more high-effect, economical, and low-toxic natural antioxidants. Phenolic antioxidant is a potent compound applied in the fields of commerce and biology to inhibit the material oxidation. Based on the studies of the antioxidant mechanism in the experiment and theory, it was universally considered that the scavenging free radicals would be from

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OH O O

5

A

4'

8

O

11

10

5'

1' 13

6'

4

2

HO

12

OH

B 9

1

2'

7

6

HO

3'

OH

3 Fig. 1. Rosmarinic acid.

the H-abstraction reactions [5,6]. ArOH C RO,2 / ArO,

C ROOH

RO,2 C ArO, / nonradical products

(1) (2)

The total reaction is H atom transfer and it contains a part of electron and proton transfer [7,8]. , ArOH C RO,2 / ½ArOHdC /RO,dK 2 / ½ArO

/ROOH/ ½ArO, C ROOH

(3)

It is analogous to an oxidation-reduction couple [9]. ArOH/ ArO, C eK C HC

(4)

It is clear from the above reaction mechanism that the antioxidant activity depends on the two factors: the H-abstraction level of the ground state molecule and the stability of the formed free radical. Rosmarinic acid (RA, Fig. 1) is a natural phenolic compound extracted from Rosemarinus officinalis L. It contains two phenolic rings of which both have the two ortho-position hydroxyl groups. There are a carbonyl, an unsaturated double bond and a carboxylic acid between the two phenolic rings. Its structure is different from the flavonoids, which have been studied extensively. It has many biological activities such as inhibiting the HIV-1, antitumor, antihepatitis and protecting the liver, inhibiting the blood clots and antiinflammation etc. [10]. Some experiments have reported the strong capacity of RA scavenging the free radicals, which showed that the antioxidant activity is over three times than Trolox that RA can inhibit the activity of Xanhine Oxidase, and it is used to scavenge the surplus free radicals in the body. In addition, RA can reduce Mo(VI) to Mo(V), preventing the product of free radicals caused by the metal [11]. Here, we calculated the geometry and electron structure of RA by using DFT method. Our aim is to provide a theoretical explanation to the relationship between the antioxidant activity and the molecular structure. This may illustrate the antioxidant mechanism of phenolic compounds, and it

will be of benefit to search and develop this kind of compounds.

2. Methods The precision of DFT which involves the electron relation effect is in common better than Hartree–Fock method in which the electron spin is not considered, especially calculating the molecules with hydrogen bonds [12]. Firstly, the original structures of RA molecule and the corresponding free radicals were optimized by AM1 [13], then a full optimization to these structures were performed at 6-31G* basis set by B3LYP method. At last, we calculated the single electron distribution of the free radical by using B3LYP/6-311G*. The parameters describing the molecule properties contain: the energies and the disposition of the frontier orbitals HOMO and LUMO, the geometry, the distribution of the single electron density of the free radicals and the bond disassociation energy (BDE) of phenol hydroxyl groups. All calculations were carried out with the program GAUSSIAN-98W [14] on PIV1.6G PC.

3. Results and discussion Fig. 2 shows the geometries of RA molecule and the free radicals formed after H-abstraction in different phenol hydroxyls. Observing the main torsion angles, we found that the ring A and 9-carbonyl in the molecule are almost located on an identical plane (q7,8,9,O9Z175.78), which will be favorable to form a large conjugating system. But the ring B and 9-carbonyl are not on the same plane so that there is a torsion angle (q9,10,11,13Z63.58). The torsion angle between 11-carboxyl and the ring B is bigger (q1,13,11,12Z124.98). The sp3 hybridization of 11-C would result in the whole molecule deviating the plane that the ring A is little relative with the ring B. There is a weaker hydrogen bond between the two ortho-position hydroxyls in the rings A and B.

H. Cao et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 177–183

179

H 0.969 O

2.124

H 0.973

1.379

O O O

1.392

1.457

H

1.412

2.112

1.365 1.469

O

1.406

1.364 1.405

1.524

1.350

1.393

1.357

1.388

1.358

1.206

1.218 0.973

OH

1.391

1.399

1.546

O 1.428

1.514

1.397

1.406

1.376 1.383

O

1.413

0.969

H

I

H 0.969 O 2.124 H 0.973 1.379

O O

. O

1.216 1.433

1.973 1.260

1.464 1.469

H 0.991

1.389

1.361

1.405

1.391

1.399

1.546

O 1.429

1.475

O

1.406

1.364

1.525

1.348

1.386

OH 1.357

1.206

1.513

1.397

1.433

1.329

O

1.392

1.388

II

H 0.969 O 2.125 H 0.973 1.378

O O 1.217

O 0.989

1.447 1.475

1.360 1.474

1.389

1.363 1.391

1.399

1.545

O 1.430

O

1.406

1.405

1.525

1.356

1.413

1.334

H 1.984

1.379

OH 1.357

1.206

1.513

1.397

1.438

1.251

.

O

1.444

1.367

III O

0.990

H 1.975

1.334

O H

O

O

1.394

1.405

2.125 1.372

0.972

1.459 1.390

H

1.524

1.350

1.371 1.466

O 1.422

.

O

1.467

1.359

1.205

1.217

0.969

OH 1.389

1.259 1.399

1.439

1.429

1.550 1.510

1.373

1.411

1.362

O

1.411

1.385

IV

Fig. 2. Geometries of ground state molecule and its radicals (I ground state molecule, II 1-radical, III 2-radical, IV 4 0 -radical).

˚ , respectively. The The bond lengths are 2.112 and 2.124 A hydrogen bond between the ortho-position hydroxyls would stabilize the ground state molecule and will impede the H-abstraction reaction to occur. On the other hand, the free radicals formed in the H-abstraction reaction contain

stronger intramolecular hydrogen bond, which makes the reaction occur easily. II, III and IV can form the intramolecular hydrogen bonds of which the lengths are ˚ , respectively, the intramolecular 1.973, 1.984 and 1.975 A hydrogen bonds can stabilize the free radicals well, the total

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H. Cao et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 177–183

Fig. 3. The HOMO (a) and LUMO (b) electron densities of ground state molecule.

Table 1 Frontier orbital energies of ground state molecule and its radicals, the bond dissociation energies of hydroxyls (BDEZERCEHKE0, ER denotes energy of free radical, EH denotes energy of hydrogen, E0 denotes energy of ground state molecule [19]) Compounds Rosmarinic acid 2-radical 1-radical 4 0 -radical

a b a b a b

Quinone-structure

3HOMO/au

3LUMO/au

Etotal/Hartree

BDE/kJ molK1

K0.205 K0.211 K0.211 K0.210 K0.210 K0.208 K0.218 K0.215

K0.066 K0.077 K0.148 K0.082 K0.146 K0.065 K0.128 K0.144

K1297.328 K1296.708

314.9

K1296.704

325.7

K1296.707

316.5

K1296.082

646.0

effects are to make the H-abstraction occur easily. As an important factor, being similar with the most of orthoposition phenol hydroxyl compounds, the higher antioxidant activity of RA is the intramolecular hydrogen bond effect after abstracted H. The free radical formed after 3 0 -position H-abstraction in the ring B is similar with that of 4 0 -position, so it is not listed here. The free radicals usually exist in a semiquinone structure. Comparing III with the ground state molecule I (Fig. 2), in the 2-free radical, the bond length of benzyl˚ is turned into 1.251 A ˚ , which is closer to the oxygen 1.376 A bond length of CaO. The bonds of C1–C6 and C3–C4 showed a decreasing change from 1.392, 1.413 to 1.370 ˚ , respectively, but the bonds of C1–C2, C2–C3, and 1.367 A

C4–C5 and C5–C6 showed an increasing from 1.412, 1.383, ˚, 1.406, and 1.393 to 1.475, 1.444, 1.438 and 1.412 A respectively. This change with a single-double bond tending illustrates the semiquinone structure characteristics. About the bond lengths of 4 0 - and 1-position free radicals, the same changes took place too. The semiquinone resonance structures are favorable to stabilize the free radicals. The frontier orbital energies, 3HOMO and 3LUMO are also the important parameters of molecular electron structure. The lower is the 3HOMO, the weaker is the molecule donating electron ability. On the contrary, the higher 3HOMO implies that the molecule is a good electron donor. 3LUMO presents the ability of a molecule receiving electron [15]. The HOMO disposition of a phenolic compound can indicate its H 0.969

O

2.124

H

1.378

O O O

1.468

1.362 1.467

1.220 1.556

1.525 1.336

1.479

1.389

O 1.431

1.364 1.404

1.474

1.391

1.399

1.545 1.514

1.475

1.218

O

0.973

O

1.406

1.356

1.206

1.215 1.346

OH

1.347

Fig. 4. A quinone structure of rosmarinic acid.

1.397

O

(c) (b) (a) HO

HO

O

O

O

Fig. 5. The forming of semiquinone resonance and quinone structures of RA.

(d) O .O O

OH

OH

OH

H

O

O

O

O

OH

OH

OH

H

O

O

O

O

OH

OH

OH

H

O

O

O

O

OH

OH

OH

O

O

O

O

(e)

OH

OH

OH

H. Cao et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 177–183

181

active site of scavenging free radicals qualitatively because the H-abstraction reaction undergoes the electron transfer. The HOMO electron density (Fig. 3(a)) in the ground molecule of RA is mainly distributed over C1 0 , C3 0 , C4 0 , C5 0 , C6 0 , O3 0 and O4 0 in the ring B. O3 0 and O4 0 of the two hydroxyls donate electron easily so that it is possible to lose electron during the H-abstraction reaction and form the electropositive free radical, then the proton transfer occurs. Moreover, the ring B would play a role through providing electrons to coordinate with some metal irons, and inhibiting those free radicals aroused by the metal iron reactions [16]. The LUMO electron density (Fig. 3(b)) is distributed over the ring A, the double bond (C7aC8) and the CaO, forming a conjugating system, it is advantageous for dispersing the electrons and bringing some effects on free radicals like OK 2 . Though the factors, which affect on the H-abstraction reaction between the phenolic antioxidants and the free radicals, are complicated, hydrogen atom transfer (HAT) and single electron transfer (SET) are the two main mechanisms known at present [17]. Hence, the BDE and 3HOMO of RA, as well as the single electron distribution of its free radicals are the important indexes to character the antioxidant efficiency. The BDEs of 1-OH, 2-OH in the ring A and 4 0 -OH in the ring B are shown in Table 1. These values have an order of 2-radicalz4 0 -radical!1-radical, and demonstrate that the H-abstraction reactions of the three hydroxyls occur easily because the lower BDEs compared with 498.0 kJ molK1 of the HO–H bond in water [18], especially 2-OH in the ring A and 4 0 -OH in the ring B. There were the experiments in which the ortho-position phenolic hydroxyls of flavonoid compounds could undergo an abstraction of the two hydrogen atoms and form a quinone structure [20]. However, there has been no theoretical backing. We calculated the BDE of the quinone structure formed by losing the two hydrogen atoms in the ring A. It is 646.0 kJ molK1 and only 6.0 kJ molK1 different from the BDE sum of losing 2-H and 1-H, respectively. The 323.0 kJ molK1 of average value to the each hydroxyl remains to be lower. Fig. 4 shows the optimization geometry of the quinone structure. It is shown that, compared with the ground state molecule, the lengths of C1–O1 and C2–O2 ˚, reduced from 1.357 and 1.376 to 1.220 and 1.218 A respectively, being shorter than the normal CaO bond ˚ . The two bonds C3–C4 and C5–C6 changed from 1.227 A ˚ , respectively, which 1.413 and 1.393 to 1.347 and 1.362 A are close to the CaC bond. Moreover, another four bonds, C2–C3, C4–C5, C1–C2 and C1–C6, their lengths increased from 1.383, 1.406, 1.412 and 1.392 to 1.474, 1.475, 1.556 ˚ , respectively. The changes of the bond lengths and 1.468 A show clearly that the original conjugating benzyl ring structure have transformed into a quinone structure after the H-abstraction of the ortho-position phenolic hydroxyls took place. In addition, analyzing on the frontier orbital energies 3HOMO and 3LUMO (Table 1), the 3HOMO of the ground state molecule is K0.205, and 3LUMO is K0.066 au.

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H. Cao et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 177–183

For the semiquinone 1- and 2-radical, the 3HOMO is K0.210 and K0.211 au, and the 3LUMO is K0.146 and K0.148 au. When being formed the quinone structure, its 3HOMO and 3LUMO are K0.215, K0.144 au, respectively. This indicates that the semiquinone would provide an electron again to continue the H-abstraction, combining with the BDE and the geometry, it can be seen that the quinone structure shows a little stability. According to the discussion above, it would be inferred that the antioxidant activity of RA is resulted from the H-abstraction reaction. This reaction may occur firstly at 2-position of the ring A (or 4 0 -position of the ring B) and come into being a semiquinone free radical (Fig. 5(a) and (b)) which will have the three resonance structures (Fig. 5(b–d)) as the electron resonance effect can stabilize a free radical. After that, the hydrogen atom of the orthoposition hydroxyl is abstracted again and the quinone structure is formed (Fig. 5(d and e)). The semiquinone free radical and the quinone structure are the proper explanations for the higher antioxidant activity of RA. The spin density and the single electron density are the important parameters to characterize the stability of the free radicals because the energy of a radical can be efficiently decreased if the unpaired electron is highly delocalized through a conjugating system [21]. In the studies on the antioxidant activity of flavonoids, a basic conclusion is that the activity is determined by the ring B, the presence of ortho-catechol group being the most important for a high antioxidant capacity [22]. Nevertheless, there are some controversies about the role of the 2,3-double bond in conjugation with a 4-oxo function in the ring C of flavonoids, which seem to set the unpaired electron delocalization against the O–H bond breaking (BDE) in stabilizing the free radical [23]. As that mentioned before, RA is an interesting molecule, it contains the two rings with the ortho-catechol structure, but the para-positions of 2- and 4 0 -OH, namely 5- and 1 0 -position, have the different groups. So, it will be necessary to consider the unpaired electron delocalization for the different free radicals. Both the single electron density and the electron spin density of different free radicals of RA showed a similar variation: O2!O4 0 !O1 (Fig. 6). This order conforms to BDEs of the hydroxyls in the relevant positions, having indicated a good relationship between H-abstraction and unpaired electron delocalization. After H-abstraction, the single electron of free radical mainly distributes over the O atom and the ortho- and para-position carbon atoms. The C7aC8 double bond, the 9-position CaO and the single electron on O2 can form a large p-like system which is beneficial to decrease some single electron distribution on the O2. So, the 2-radical has a better unpaired electron delocalization and the BDE of its parent molecule is the lowest. Moreover, the atom O of the ortho-OH also contributes to the single electron dispersing. Thus, not only the hydrogen bond effect but also the induce effect, the ortho-OH can stabilize the free radical well.

H O O

H

OH

O

O O

0.226 0.313

H

.O

O

0.263

0.187

0.301

I

H O O

H

OH

O

O

.O

0.364

0.204

O H 0.301

0.257

O

II O O

H

H

.

O 0.354

0.262

OH

O 0.312

O

0.183

O H O

III

H O

0.023

O

H

OH

O

O 0.091

O

0.134 0.177

H 0.052

.O

0.240

O

0.127

0.026

0.101

I′

H O O

H

OH

O

O

.O

0.311

0.107

0.062

0.030

O

0.023

H O

0.165

0.177

0.108

II′

0.100

O O H

OH

0.155

O

H

.

O 0.297 0.066

0.169

O

0.101

O

0.037

H O

III′

Fig. 6. The spin density and the single electron density (I, II, III spin density, I 0 , II 0 , III 0 single electron density).

H. Cao et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 177–183

To sum up, bearing analogy with the phenolic compounds which have the ortho-OH [24], the main active groups of RA are the two phenolic hydroxyls in the rings A and B. Its antioxidant activity is mainly expressed by the H-abstraction reaction, and the semiquinone and the quinone structure formed as following. Compared with the flavonoids, in which the main active position is in the ring B [22], the hydroxyl H-abstraction capacity is almost identical for the two rings of RA. Besides, the intramolecule hydrogen bond and the semiquinone resonance effects in the free radical, there is a distinction between the rings A and B. It seems from the HOMO disposition that the ring B is a stronger electron donor, however, 11-carbonxylic has little contribution to the stabilization of the radical formed after H-abstraction in the ring B because they are not on the same plane, so the activity of ring B depends on the electron effect. And the ring A, through the free radical forming, manifests a conjugating effect with the double bond and the carbonyl to disperse the single electron on the atom O, which can stabilize the radical.

Acknowledgements We are grateful to Professor A.M. Tian for having provided us with their computational facilities. This work is supported by the Fund of Yunnan Province-Chinese Academy of Science Cooperation (2000YK-01) and the Science and Technology Plant of Education Committee of Chongqing (B3-6-61).

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