Relationship structure-antioxidant activity of hindered phenolic compounds

The relationship between the structure and the antioxidant activity of 21 hindered phenolic compounds was investigated by Rancimat and DPPH· tests. 3-Tert-butyl-5-methylbenzene-1,2-diol is the strongest antioxidant in the Rancimat test but not in the DPPH· test because its two hydroxyl groups have very strong steric synergy. 2,6-Ditert-butyl-4-hydroxy-methylphenol exhibits a strong antioxidant activity as 2,6-ditertbutyl-4-methoxyphenol does in lard. 2,6-Ditert-butyl-4hydroxy-methylphenol also exhibits stronger activity than 2-tert-butyl-4methoxyphenol. The methylene of 2,6-ditert-butyl-4-hydroxy-methylphenol can provide a hydrogen atom to active free radicals like a phenolic hydroxyl group does because it is greatly activated by both the aromatic ring and hydroxyl group. Five factors affect the antioxidant activities of the phenolic compounds: how stable the phenolic compound free radicals are after providing hydrogen atoms; how many hy drogen atoms each of the phenolic compounds can provide; how fast the phenolic compounds provide hydrogen atoms; how easily the phenolic compound free radicals can combine with more active free radicals, and whether or not a new antioxidant can form after the phenolic compound provides hydrogen atoms.


INTRODUCTION
Autoxidation is the main process of fat, oil and lipid-based food deterioration, which results in a loss in nutrition, and the production of an offflavor and other harmful substances (Pokorny et al., 2001).Many hindered phenolic antioxidants such as 2,6-ditert-butyl-4-methylphenol (BHT), 4-hydroxy-3-tert-butylanisole (BHA), tert-butylated hydroquinone (TBHQ) are widely used as antioxidants in the food industry, especially for bulky oils and fatty foods.These synthetic antioxidants are highly active and cheap.They are colorless, odorless, tasteless and non-toxic (Zhang et al., 2004).The addition of antioxidants is the most effective, convenient and economical way to retard lipid autoxidation (Li et al., 2006).Therefore, the antioxidant activity of natural and synthetic phenolic compounds attracts the attention many researchers.
Determination of the oxidative stability of oils and fats can be used to measure the activity of antioxidants in oil.Various chemical tests and accelerated methods have been reported for the determination of the oxidative stability of oils and fats.The active oxygen method (AOM) has traditionally been use d for such determinations (Weng and Wu, 2000).This method is tedious and involves the use of large quantities of toxic chemicals and laborious titration (Läubli and Bruttel, 1986;Läubli et al., 1988).
The Metrohm Rancimat is a rapid automated method (Gordon and Mursi, 1994) which is fairly consistent with the AOM method (Läubli and Bruttel, 1986;Läubli et al., 1988) and can avoid the disadvantages mentioned above.These methods allow for the determination of the induction period of oils and fats.
The ability of phenolic compounds to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Bonde et al., 1997;Mensor et al., 2001;Philip 2004), superoxide anion radical, and the hydroxyl radical (Lebeau et al., 2000) is commonly used to study antioxidant activity.A new parameter can be used to characterize the activity of phenolic antioxidants in eliminating DPPH•.The parameter is 1/ (EC 50 t 1/2 ), in which EC 50 stands for the concentration of antioxidants and t 1/2 is the time taken to eliminate half the amount of DPPH•.This parameter can be used to compare activities of antioxidants better than EC 50 .
The antioxidant structure-activity relationship of phenolic compounds was previously investigated by some researchers.Zhang 1998Zhang , 1999aZhang , 1999bZhang , 2000;;Zhang et al., 1999Zhang et al., , 2000) ) used several models to calculated the parameters, such as the difference in heat of formation between an antioxidant and its free radical (ΔHOF) and the highest occupied molecular orbit (HOMO) energy level to express the free radical scavenging activity of phenolic compounds, which are mainly flavonoids.Their results were highly consistent with the experiment results.They studied the phenolic hydroxyl, alkoxyl and carbonyl group and conjugated systems affecting the antioxidant activity of phenolic compounds.Burton andIngold 1981, 1986;Burton et al., 1983, Burton et al., 1985), Lucarini (1996) and Wright et al., (2001) studied how various groups (mainly alkyl, hydroxyl and alkoxyl groups) were effective on the H-O bond dissociation energy of phenolic compounds.Burton (1980) investigated how the stereoelectronic factors of substituents affect the antioxidant activity of phenols.Weng (1993) discussed how the conjugated systems, ortho-and para-groups affect the antioxidant activity of phenolic compounds.Duan et al. (1998) found that some phenolic compounds can form new compounds which have strong antioxidant activity after they have acted as antioxidants.
In this paper, our study focused on the relationship between antioxidant structure and activity of these phenolic compounds by using the Rancimat test in lard and the DPPH assay.

Antioxidant activity evaluated by Rancimat test
The antioxidant activity of hindered phenolic antioxidants at different concentrations in lard was determined by Rancimat (Metrohm AG, Herisau, Switzerland) based on the method by Guo et al. (2005).The air flow rate was controlled at 20 L/h, the temperature was controlled at 100 °C and lard was used as a substrate.0.02% Antioxidant (w/w) was added to each lard sample (3 ± 0.02g) separately.Each sample was treated in duplicate.

Antioxidant activity evaluated by DPPH• test
The antioxidant activities of the hindered phenolic compounds were measured in terms of hydrogen-donating or radical-scavenging ability (Lebeau et al., 2000;Li et al., 2006) using the DPPH• method.All spectrophotometric measurements were performed with a UV-2012 PC spectrophotometer (UNICO Corp, Shanghai, China).In t erms of hydrogen-donating or radical-scavenging ability, the EC 50 value is defined as the concentration of substrate that causes a 50% loss in DPPH• activity.The EC 50 of phenolic compounds were calculated by linear regression of plots, where the abscissa represented the concentration of tested samples and the ordinate represented the average percentage of scavenging capacity from triplicates.A new parameter, 1/(EC 50 t 1/2 ), is used to characterize the activity of phenolic antioxidants for eliminating DPPH• by Qiu et al. (2005).

Antioxidant activity by Rancimat test
The antioxidant activities of the hindered phenolic compounds were tested on the Rancimat at 100 °C.To explain the effect of the compound structures on the antioxidant activities, the antioxidant protection factors (Pf) have been calculated according to equation 1.

Pf = IP (induction periods of lard added antioxidant) / IP (induction period of lard) Eq 1
The compounds used to study antioxidant activities in the experiment are shown in Figure 1.The Pf values of the compounds are shown in Table 1.A higher value of Pf means a stronger antioxidant activity of a compound.If Pf < 1, the compound had pro-oxidant activity; if Pf = 1, the compound had no antio xidant activity at all; if 2 > Pf > 1, the compound had some (weak) antioxidant activity; if 3 > Pf > 2, the compound had an obvious antioxidant activity and if Pf > 3, the compound had a strong antioxidant activity (Wang et al., 2000).
When lard is used as the substrate and Rancimat as assaying instrument, the temperature is controlled at 100 °C, and the air flow rate is controlled at 20 L•min −1 , the antioxidant activities of the compounds shown in figure 1 are reduced in the following order (Table 1) The results in Table 1 show that all 21 compounds in Figure 1 have very different levels of antioxidant activity.It is interesting that compound 21 (3-tert-butyl-5-methylbenzene-1,2-diol) presents extremely powerful antioxidant activity (Pf = 16.55).Compound 12 (TBHQ) also demons trates superstrong antioxidant activity (Pf = 10.85), but much weaker than compound 21.Compounds 6, 4 and 13 show very strong antioxidant activities (Pf = 6.54, 6.17 and 5.97 respectively).Compound s 18, 14, 19, 7, 15 and 3 demonstrate strong antioxidant activities (Pf = 5.09, 4.52, 4.39, 3.86, 3.26 and 3

Antioxidant activity evaluated by the DPPH• test
The determination of the radical scavenging activities of hindered ph enolic antioxidants was carried out at 517 nm and the results are shown in Table 1.It is interesting to note that the two strongest antioxidants, compounds 21 and 12 exhibited much weaker antioxidant activity when they were evaluated by Rancimat in oil.when they were evaluated by the EC 50 of DPPH•-scavenging, they were ranked as numbers 14 and 8. Compounds 20 and 21 are much weaker antioxidants than their mother compounds 18 and 19, because the free radicals of compounds 20 and 21 cannot combine bulky free radical DPPH• while their mother compounds can.This is explained in our previous research work (Huang et al., 2013).
It has been proven theoretically that the new parameter, 1/(EC 50 t 1/2 ), is related to the reaction velocity constant k, which is equal to [ln (1/2)]/ (EC 5 t 1/2 ), which can correctly characterize the ability of the antioxidants to eliminate DPPH• in quantity (Qiu et al., 2005).This means that the bigger the reciprocal [1/(EC 50 t 1/2 )] of the arithmetic product EC 50 and t 1/2, the larger the k value is, and the stronger the phenolic compound is (Table 1).
According to the 1/(EC 50 t 1/2 ) in the DPPH test, the antioxidant activities of the 21 compounds listed in Table 1 reduce in the following order, and these result are somewhat similar to the Rancimat test.In conclusion, there are five factors which affect the antioxidant activities of the phenolic compounds: 1.After the phenolic compound provides hydrogen atoms, it depends how stable the phenolic compound free radicals are.This is especially true in the case of the autoxidation of bulky relative saturated oils and fats, such as lard and palm oil because the free radicals produce very slowly and the concentration of free radicals is relatively low.2. It also depends on how many hydrogen atoms each of the phenolic compounds can provide.Of course, it is true both in the cases of Rancimat and DPPH• test.3. Another factor is how fast the phenolic compounds provide hydrogen atoms.In the DPPH• test and autoxidation of highly unsaturated oils such as concentrated eicosapentaeoic acid and docosahexaeonic acid (Cao and Weng 1995), the speed of phenolic compounds providing hydrogen atoms to relatively more active free radicals is particularly important.4. How easily the phenolic compound free radicals can combine with more active free radicals.5.And whether a new antioxidant can form or not after the phenolic compound provides hydrogen atoms.
Factors ( 4) and ( 5) are both important for the evaluation of the phenolic compounds by the Racimat test and DPPH• test.
It has also been concluded that the DPPH•scavenging method is more sensitive to stereohindrance than the Rancimat method because firstly free radicals are produced much more slowly in the Rancimat test and their concentration is much lower in comparison to the DPPH• scavenging test, and secondly, DPPH• is much more bulky than ROO•.

FIGURE 2 .
FIGURE 2. Illustration of the fact that compound 4 demonstrates very strong antioxidant activity and much stronger than compound 5.

FIGURE 4 .
FIGURE 4. Illustration of the fat that compound 2 exhibits stronger antioxidant activity than compound 7 in the DPPH test (according to the EC50 value).

TABLE 1 .
The protection factors (Pf) of the lard containing different compounds at 0.02% concentration by the Rancimat test and the EC 50 , half elimination time (t 1/2 ), the new parameter 1/(EC 50 t 1/2 ) in the DPPH• assay.