STUDY AND DESIGN OF EUGENOL DERIVATIVES AS POTENT ANTIOXIDANT USING QUANTUM MECHANICAL METHOD
Neera Sharma*, Dinesh Kumar
Department of Chemistry, Hindu College, University of Delhi, Delhi, India
The main component of clove oil (Eugeina caryophyllata), Eugenol (4-allyl-2-methoxy phenol) is a phenolic compound. Among many natural and synthetic antioxidants compounds, mono and polyphenols have been the focus of both experimental and computational research on the mechanism of free radical scavenging. A series of eugenol derivatives have been studied for their antioxidant properties. Electronic parameters such as energy of highest occupied molecular orbital (HOMO), energy of lowest occupied molecular orbital (LUMO), the energy gap (ELUMO – EHOMO), dipole moment and atomic charges were calculated using PM3 semi-empirical method. A correlation between these parameters related to structure and antioxidant activity of eugenol derivatives has been established. These results have been used to design new more effective eugenol derivatives as potential antioxidants.
Key words: Antioxidants, eugenol, polyphenols, computational study, semi-empirical method.
The physico-chemical properties and structural features of phenolic molecules which determine their capacity to act as antioxidant have been extensively studied both experimentally1-3 and computationally4,5. They have attracted much attention in the last decade due to their properties and beneficial health effects6-9. A large number of polyphenolic compounds have been identified in various plant species7 as secondary metabolites. They protect them against oxidative damage, harsh climatic conditions, ultraviolet radiation and aggression by pathogens. These molecules play an important role in neutralizing the destructive reactivity of undesired reactive oxygen/nitrogen species produced as by product during metabolic processes in the body. Eugenol is a natural pharmacologically active aromatic substance present in essential oils of several plants10-12 and known for its aroma and medicinal values13-17. It posses large number of biological activity18,19 such as anti-inflammatory, analgesic, anesthesic, antipyretic, antiplatet, anticonvulsant, anti bacterial, anti fungal, antiviral, antioxidant etc. Several studies showed antioxidant capacity of eugenol and its derivative, inhibit the lipid peroxidation20 induced by reactive oxygen species (ROS). ROS are highly reactive oxygen containing molecules, which include free radicals also. The process of cellular damage and its antioxidant inhibition occurs through the chemicals reactions which follow the free radical mechanism as shown below21 (Fig. 1):
Fig. 1: Free radical mechanism of cellular damage and its antioxidant inhibition.
The reaction is initiated by some radical species (In˙) which may reacts with the (lipid) substrate RH (mainly by H-atom abstraction) and results in the formation of an alkyl radical R˙. In the chain propagation step this alkyl radical R˙, reacts with oxygen to form a peroxyl radical (ROO˙). ROO˙ radical generated, attacks another molecule of the substrate in cyclic manner to form a hydroperoxide ROOH (the oxidised substrate) and another radical R˙. The chain reaction proceeds for many cycles before two radical species incidentally quench each other in a termination step. The role of antioxidant is of impairing this radical chain reaction by transferring an H atom and is divided into two main groups depending on their mechanism of interference19. 1) When they interfere with initiation process (they retard initial formation of radical species), are called Preventive antioxidant. 2) When they slow or block autoxidation by competing with the propagation reactions, i.e. they react with peroxyl radicals more rapidly than the oxidizable substrate to form species that do not propagate the oxidation chain, are called Chain-breaking antioxidant. Chain-breaking antioxidants are by far most important antioxidants, because preventive antioxidants are completely ineffective after the process has started. Phenols are the prototypical Chain-breaking antioxidants22. It is known that reactive species are kept at physiological levels by phenolic antioxidant by quenching free radical species through hydrogen atom transfer from Ar–OH group. During this process, the phenolic group (ArOH) is converted into phenoxyl radical (ArO˙) which is more stable due to electron delocalisation (Fig. 2) within adjacent benzene rings23 or dimerization.
Fig. 2: Stabilization of phenoxyl radical (ArO˙) via electron delocalisation after H-transfer from ArOH.
A study of eugenol structure activity revealed that in addition to the phenolic ring, the side chain has an important role in anti-oxidant activity24. Keeping in view, a series of eugenol derivatives were selected to perform structure-antioxidant activity relationship. The theoretical molecular properties and trolox equivalent antioxidant capacity (TEAC) of some of eugenol derivatives used as antioxidant are reported in the literature25. In the present work quantum chemical studies using PM3 semi-empirical method have been done on some eugenol derivatives to calculate their molecular properties and correlated with reported experimental values.
Theoretical calculations were carried out by using Parametric Method 3 (PM3) semi-empirical method to obtain the optimized geometries, electronic parameters such as energy of highest occupied molecular orbital (HOMO), energy of lowest occupied molecular orbital (LUMO), the energy gap (ELUMO – EHOMO), dipole moment, atomic charges of all the eugenol derivatives studied here. The quantum chemical package HyperChem Release 5.1 Pro26 was used for the semi-empirical molecular-orbital calculations.
RESULTS AND DISCUSSION
The chemical structure and optimized geometry of eugenol derivatives are presented in Table1.
Table 1: Structure and Optimized Geometry of Eugenol and Its Derivatives.
Electronic parameters for these molecules like energy values of highest occupied molecular orbital (HOMO), lowest occupied molecular orbital (LUMO), frontier molecular orbital energy gap (ELUMO – EHOMO), dipole are shown in Table 2. Literature TEAC values25 of some eugenol derivates are also given in Table 2. It has been reported earlier that highest atomic charge is present on Ar–OH oxygen atom and are correlated with metal binding  properties of eugenol derivatives. In view of this, atomic charges on Ar–OH oxygen atom and oxygen atom of other –OH groups are also presented in Table 2.
Table 2: Electronic Parameters for the Optimized Structure of Eugenol and Its Derivatives.
The scavenging activity of phenolic compound is determined by the bond dissociation energy27 of O–H bond, which in turn is mainly governed by the stability of phenoxyl free radical generated after H-transfer (Fig. 2). Usually, the factors that stabilize the free radical will increase the antioxidant activity. The inductive effect of substituents and delocalization of unpaired electron determines the stability of phenoxyl radical28.
The frontier orbitals (HOMO and LUMO) are the main orbital which are involved in the chemical reactions. The HOMO energy characterizes the ability of electron transfer and represents the free radical scavenging efficiency of phenolic compound in the process of inhibition of auto-oxidaion29. The –OH group is easily attacked by either electrophilic or nucleophilic agents such as alkyl radical, peroxy radical, superoxide and metal ions. It has been reported in the literature16 that antioxidant activity of phenolic compounds increases with increase in EHOMO and decrease in the energy gap ∆E, (ELUMO – EHOMO). On comparing these values14, it has been found that Isoeugenol (3), has higher EHOMO and lower ∆E value as compared to eugenol (1) due to the displacement of double bond position in the allyl chain, para to –OH group, which causes extended conjugation, while molecule (2) has less capacity. Introduction of hydroxymethyl (–CH2OH) group at C2 in compound (4) enhances antioxidant capacity due to intramolecular hydrogen bonding30 (Fig. 3). Two structures (A and B) can be drawn for six member ring formed after hydrogen bonding for compound (4). On the basis of computational data obtained, structure A is found to be predominant on structure B and can produced more stable phenoxyl radical A˙ than radical B˙ after H-transfer (Fig. 3).
Fig. 3: Possible structures (A and B) of compound (4) and their respective free radicals (A˙ and B˙) stabilized through intramolecular six member H-bonds.
Computationally, dimeric compound dieugenol (5) has higher antioxidant activity than that of monomer i.e. eugenol (1). It shows more favourable data for better antioxidant activity may due to the presence of sterically crowded two phenolic moieties, not due to extended conjugation in biphenyl system because of non-planer structure obtained (Fig. 4) from PM3 optimized geometry (Tortional angle = -51°). In support, ESR spin trapping experiment13 reveals that the trapping potency of dieugenol is almost 5 times higher than eugenol, this indicates that bulky substituents at the neighbouring position of hydroxyl group in eugenol derivatives enhance the antioxidant ability.
Fig. 4: Optimized geometry of compound (5) with tortional angle -51° between two phenyl rings.
Molecule (6) does not have –OH hydrogen atom and posses less antioxidant capacity also shown from TEAC value. Validating the effect of substituent at para position and role of extended delocalisation in phenoxyl radical, three molecules (7, 8, 9) are designed with phenyl ring at para position. Presence of p-phenyl group found to increase the EHOMO and decrease in the energy gap ∆E, and shows better antioxidant capacity than other derivatives. Further, substituting p-phenyl ring with electron releasing (NH2) and electron withdrawing (COOH) groups provide the extra stability to phenoxyl radical through extended delocalization and thus increase the antioxidant activity. This observation is supported from the high values of EHOMO and low energy gap ∆E values as compared to other molecules. Abstraction of proton from –COOH group in molecule (9) is also possible which is evident from highest charge value on oxygen atom.
Recent development of computational facilities greatly facilitated theoretical research on the antioxidant properties of phenolic compounds. Through PM3 semi-empirical quantum mechanical calculations, a correlation between parameters related to structure of some eugenol derivatives and their antioxidant activity could be established. Relative stability of phenoxyl radical and antioxidant activity depends on the substituent on the neighbouring carbon atom, number of –OH groups, groups present at other positions which causes resonance stabilization through extended conjugation. Quantitative structure-activity relationship (QSAR) can be used for design of new more effective phenolic antioxidant and identification of most useful natural antioxidants.
The part of the computational work was carried out in the laboratory of Prof. R.C. Rastogi, Department of Chemistry, University of Delhi. We thank Prof. Rastogi for the help.
- 1) Heijnen CGM, Haenen GRMM, Vekemans JAJM and Bast A: Peroxynitrite scavenging of flavonoids: structure activity relationship. Environmental Toxicologyand Pharmacology 2001; 10:199-206.
- 2) Lucarini M, Mugnaini V, Pedulli GF and Guerra M: Hydrogen-Bonding Effects on the Properties of Phenoxyl Radicals. An EPR, kinetic, and computational study. Journal of American Chemical Society 2003; 125:8318-8329.
- 3) Cao G, Sofic E and Prior RL: Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radical Biological Medical 1997; 22:749-760.
- 4) van Acker SABE, Koymans LHM and Bast A: Molecular pharmacology of vitamin E. Free Radical Biological Medical 1993; 15:311-328.
- 5) Wright JS, Johnson ER and DiLabio GA: Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. Journal of American Chemical Society 2001; 123:1173-1183.
- 6) Husain RS, Cillard J and Cillard P: Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 1987; 26:2489-2491.
- 7) Manach C, Scalbert A, Christine M, Christian R and Jimenez L: Polyphenols: food sources and bioavailability. The American Journal of Clinical Nutrition 2004; 79:727-47.
- 8) Carrasco A H, Espinoza C L, Cardile V, Gallardo C, Cardona W, Lombardo L, Catalán M K, Cuellar F M and Russod A: Eugenol and its synthetic analogues inhibit cell growth of human cancer cells (Part I).Journal of the Brazilian Chemical Society 2008; 19:543-548.
- 9) Rojo L, Vazquez B, Roman JS and Deb S: Eugenol functionalized poly(acrylic acid) derivatives in the formation of glass-ionomer cements. Dental Materials 2008; 24:1709–1716.
- 10) Toda S, Ohnishi M, Kimura M and Toda T: Inhibitory effects of eugenol and related compounds on lipid peroxidation induced by reactive oxygen. Planta Medica 1994; 60:282.
- 11) Kumaravelu P, Dakshinamoorthy DP, Subramaniam S, Devraj H and Devraj NS: Effect of eugenol on drug-metabolizing enzymes of carbon tetrachloride-intoxicated rat liver. Biochemical Pharmacology 1995; 49:1703-1707.
- 12) Escobar RG: Eugenol: propiedades farmacológicas y toxicológicas. Revista Cubana de Estomatología 2002; 39:139-156.
- 13) Ogata M, Hoshi M, Urano S and Endo T: Antioxidant activity of eugenol and related monomeric and dimeric compounds. Chemical and Pharmaceutical Bulletin 2000; 48:1467-1469.
- 14) Atsumi T, Fujisawa S and Tonosaki K: A comparative study of the antioxidant/prooxidant activities of eugenol and isoeugenol with various concentrations and oxidation conditions. Toxicology in Vitro 2005; 19:1025-1033.
- 15) Nagababu E, Rifkind JM,Sesikeran B and Lakshmaiah N: Assessment of antioxidant activities of eugenol by in vitro and in vivo Methods in Molecular Biology 2010; 610:165-180.
- 16) Mohamed ME: Structure – Antioxidant activity relationship study of eugenol derivatives using semi-empirical method. New York Science Journal 2013; 6:102-106.
- 17) d Avila Farias M, Oliveira PS, Dutra FSP, Fernandes TJ, de Pereira CMP, de Oliveirad SQ, Stefanello FM, Lencinac CL and Barschak AG: Eugenol derivatives as potential anti-oxidants: is phenolic hydroxyl necessary to obtain an effect? Journal of Pharmacy and Pharmacology, 2014; 66:733-746.
- 18) Chen F, Shi Z, Neoh KG and Kang ET: Antioxidant and antibacterial activities of eugenol and carvacrol-grafted chitosan nanoparticles. Biotechnology and Bioengineering 2009; 104:30-39.
- 19) Baskaran Y, Periyasamy V and Venkatraman AC: Investigation of antioxidant, anti-inflammatory and DNA-protective properties of eugenol in thioacetamide-induced liver injury in rats. Toxicology, 2010; 268:204-212.
- 20) Okada N, Satoh K, Atsumi T, Tajima M, Ishihara M, Sugita Y, Yokoe I, Sakagami H and Fujisawa S: Radical modulating activity and cytotoxic activity of synthesized eugenol-related compounds. Anticancer Research 2000; 20:2955-2960.
- 21) Amorati R, Foti MC and Valgimigli L: Antioxidant activity of essential oils. Journal of Agricultural and Food Chemistry 2013; 61:10835-10847.
- 22) Burton GW, Doba T, Gabe E, Hughes L, Lee FL, Prasad L and Ingold KU: Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols. Journal of American Chemical Society 1985; 107:7053-7065.
- 23) Kitagawa S, Fujisawa H and Sakurai H: Scavenging effects of dihydric and polyhydric phenols on superoxide anion radicals, studied by electron spin resonance spectrometry. Chemical and Pharmaceutical Bulletin 1992; 40:304-307.
- 24) Ito M, Murakami K and Yoshino M: Antioxidant action of eugenol compounds: role of metal ion in the inhibition of lipid peroxidation. Food and Chemical Toxicology 2005; 43:461-466.
- 25) Arenas DRM, Acevedo AM, Méndez LYV and Kouznetsov VV: Scavenger activity evaluation of the clove bud essential oil (Eugenia caryophyllus) and eugenol derivatives employing ABTS+• Sci Pharm, 2011; 79:779-791.
- 26) HyperChem Release 5.1, Hypercube, Inc, USA, 1997.
- 27) Bordwell FG, Zhang XM, Satish AV and Cheng JP: Assessment of the importance of changes in ground-state energies on the bond dissociation enthalpies of the O-H bonds in phenols and the S-H bonds in thiophenols. Journal of American Chemical Society 1994; 116:6605-6610.
- 28) Tomiyama S, Sakal S, Nishiyama T and Yamada F: Factors influencing the antioxidant activities of phenols by an ab Initio Bulletin of the Chemical Society of Japan 1993; 66:299-304.
- 29) Nagaoka SI, Kuranaka A, Tsuboi H, Nagashima U and Mukai K: Mechanism of antioxidant reaction of vitamin E: charge transfer and tunneling effect in proton-transfer reaction. Journal of Physical Chemistry 1992; 96:2754-2761.
- 30) Wright JS, Carpenter DJ, McKay DJ and Ingold KU: Theoretical calculation of substituent effects on the O-H bond strength of phenolic antioxidants related to vitamin E. Journal of American Chemical Society 1997; 119:4245-4252.