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Distinct association between bioactive compounds and antioxidant ac- tivity might be related to the presence of various active compounds in the plant. The synergistic effects of different compounds, the experimental conditions and the mechanism of the antioxidant reactions method used may affect this association. Moreover, most methods have their own limi- tations in the determination of antioxidant activity (Jayaprakash and Patil, 2007).

Phenolic compounds are the most prevalent antioxidant phytochem- icals in the plant kingdom. There are about 5000 known plant phenolics, and many of them exhibit significant antioxidant activity, both singlet oxygen-quenching activity and radical scavenging activity (Guo et al., 1999; Robards et al., 1999). However, there is a wide degree of variation between different phenolic compounds in their effectiveness as antioxi- dants, which is determined by several structural features (Ou et al., 2002). Also, there are a number of different mechanisms by which phenolics may act as antioxidants: via free radical scavenging, hydrogen donation, singlet oxygen quenching, metal ion chelation or as a substrate for attack by superoxide (Hamilton et al., 1997; Rice-Evans et al., 1997a; Robak and Gryglewski, 1998). The antioxidant activity of phenolic compounds is due mainly to their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers, heavy metal chelators and radical quenchers (Kaur and Kapoor, 2002).

The numbers and position of the hydrogen-donating hydroxyl groups on the aromatic ring of the phenolic molecules control the free radical and antioxidant activity of the phenolics. This is also affected by other

factors such as the glycosylation of aglycones and other H-donating groups (-NH, -SH), etc. For phenolic acids (hydroxyl benzoic acid, hydroxyphe- nyl acetic and hydroxycinnamic acids) and their ester derivatives, it is known that antioxidant activity depends on the number of hydroxyl groups in the molecule that are affected by steric hindrance from their carboxylate group (Rice-Evans et al., 1997a). The antioxidant activity of phenolic acids increases with additional hydroxyl groups. The closeness of the carboxylate group and hydroxyl groups on the phenolic ring in hydroxybenzoic acids affects their donor proton ability negatively. As a result, higher antioxidant activities are usually observed on hydroxycin- namic acids such as coumaric, caffeic and ferulic acid as compared to their hydroxybenzoic acid counterparts (Fig. 8.4). Further, ortho substi- tution with electron donating alkyl or methoxy groups increases the sta- bility of the aryloxyl radical, and hence its antioxidant potential (Rice-Evans

et al., 1996a). The alkoxyl radical scavenging ability in a lipid peroxida-

tion system increased in the following order: salicylic < vanillic <chlorogenic < caffeic < gallic acids (Milic et al., 1998).

The antioxidant activities of flavonoids (flavonols, isoflavones, etc.) that have a diphenylpropane skeleton depend on the structure, degree of hydoxylation and substitution pattern of hydroxyl groups. Antioxidant potency is related to structure in terms of electron delocalization of the aromatic nucleus. Hydroxylation of the B-ring is the major requirement for consideration of activity (Herrmann, 1976; Miller, 1996b). Hydroxyl radical scavenging activity increases with the number of hydroxyl groups substituted on the B-ring, especially at C-3′ (Ratty and Das, 1988).

A single hydroxyl substituent generates little or no antioxidant ac- tivity. Flavonones such as naringenin and hesperitin with only one hy- droxyl group on the B-ring have negligible antioxidant activity. But all flavonoids with 3′,4′-dihydroxy substitution possess antioxidant activity (Dziedzic and Hudson, 1983). Quercetin and cyanidin with 3′,4′-dihydroxy substitution in the B-ring and conjugation between the A- and B-rings have antioxidant potential four times that of trolox. The essential requirement

R1 R4 R3 R2 R1 CH. CHCOOH Cinnamic acids

Cinnamic acid esters R2

R3

COOH

Benzoic acids Gallic acid R1=R2=R3=OH

Protocatechuic acid R1=H, R2=R3=OH

Vanillic acid R1=H, R2=OH, R3=OCH3

Syringic acid R2=OH, R1=R3=OCH3

Ferulic acid R1=R2=H, R3=OH, R4=OCH3

p-Coumaric acid R1=R2=R4=H, R3=OH

o-Coumaric acid R2=R3=R4=H, R1=OH

Caffeic acid R1=R2=H, R3=R4=OH

Sinapic acid R1=H, R3=OH, R2=R4=OCH3

for effective radical scavenging is the 3′,4′-orthodihydroxy configuration in B-ring and the 4-carbonyl group in C-ring. The presence of 3-OH groups or 3- and 5-OH groups giving a catechol-like structure in C-ring is also beneficial for the antioxidant activity of flavonoids. The presence of the C-2–C-3 double bond configured with a 4-keto arrangement is known to be responsible for electron delocalization from B-ring, and it increases the radical scavenging activity. In the absence of the O-dihydroxy struc- ture in B-ring, a catechol structure in A-ring can compensate for flavonoid antioxidant activity. The presence of glycosylations on the molecule may decrease its antioxidant activity.

The relationship between the chemical structure of flavonoids and their radical scavenging activities was analysed by Bors et al. (1990). Quercetin has a catechol structure in B-ring as well as a 2,3-double bond in conjuction with a 4-carbonyl group in C-ring, allowing for delocaliza- tion of the phenoxyl radical electron to the flavonoid nucleus. The com- bined presence of a 3-hydroxy group with a 2,3-double bond additionally increases the resonance stabilization for electron delocalization; hence, it has a higher antioxidant value. Quercetin and luteolin have an iden- tical number of hydroxyl groups with 3′,4′- and 5,7-dihydroxyl groups in B- and A-rings, respectively. Flavonols (quercetin, myricetin, kaempferol and isorhamnetin) have a hydroxyl group in position 3. The 3,4-position of dihydroxylation of the phenolic ring in caffeic acid showed increased antioxidant activity as compared to p-coumaric acid (Kim and Lee, 2004). Caffeic acid is expected to have higher antioxidant activity because of additional conjugation in the propenoic side chain, which might facilitate electron delocalization by resonance between the aromatic ring and the propenoic group.

A linear correlation between the content of total phenolic compounds and their antioxidant capacity has been demonstrated (Cai et al., 2004; Katsube et al., 2004; Djeridane et al., 2006; Katalinic et al., 2006).