**3. Discussion**

the hydrophobic environment. The same head and tail concept has been extended to evalu‐ ate octylprotocatechuate (**3**) and dodecyl protocatechuate (**7**), and both were found to show even slightly more potent inhibitory activity than that of the corresponding alkyl gallates. On the other hand, dodecyl 3,5-dihydroxybenzoate (**17**) which does not have an ability to chelate ironwas alsoevaluated for comparison. This resorcinol derivative was also found to inhibitthe enzymatic linoleic acid (**30**) peroxidation, but to a much lesser extent compared to dodecyl gallate (**16**) or dodecyl protocatechuate (**7**). It appears that the activity of dodecyl gallate (**16**), which has the ability to chelate iron, is conspicuously potent. Compared to an acardic acid (C15:0) (**15**), dodecylgallate (**16**) is nearly 200-fold more potent (Table 2). In the case of alky gallates, the hydrophilic ligands with a longer alkyl side chain of up to C14 in length tend to be the more potent inhibitors [14]. Since their hydrophilic head portions are the same, the data are consequently interpreted to mean that changes in the hydrophobic tail portions correlated to the activity.On the one hand, dodecyl 3,4-dihydroxy-5-methoxy‐ benzoate (**18**) still exhibited this inhibitory activity but to a much lesser extent compared to that of dodecyl gallate (**16**). This noticeable inferior activity can be explained by a steric hin‐ drance to its approach to the active site in the enzyme, but dodecyl gallate (**16**) still reduces the ferric form of the enzyme to an inactive ferrous form. It would thus seem that a relative‐ ly small change in chemical structure of these molecules affects their biological activity to a large extent. The activity of alkyl 3,4-dihydroxybenzoates was compared with those of alkyl 2,3-dihydroxybenzoates, and thus the differences should be due to imply the hydrophilic head portions. Noticeably, dodecyl protocatechuate (**7**) showed potent lipoxygenase inhibi‐ tory activity with an IC50 value of 0.06 µM, whereas 3,4-dihydroxyphenyltridecanoate (**32**) also exhibited the inhibitory activity but to a much lesser extent. In these catechol (**35**) struc‐ tures, the head and tail portions are connected as esters, either -COOR or -OCOR. Thus, the ester groups, either an electron withdrawing (-COOR) or an electron donating (-OCOR), were connected at the C-5 position to the catechol (**35**) moiety. The results obtained indicate that –COO Resters exhibited more potent inhibitory activity compared to the corresponding

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen


**Table 2.** Lipoxygenase-1 inhibitory related activity of selected compounds

Anacardic acid (C15:0) **(15)**

Relationships

192

Dodecyl 3,4,5-trihydroxybenzoate **(16)** Dodecyl 3,5-dihydroxybenzoate **(17)**

Dodecyl 3,4-dihydroxycinnamate **(19)** Dodecyl 4-hydroxybenzoate **(20)** Dodecyl 3-hydroxybenzoate **(21)**

Dodecyl 3,4-dihydroxy-5-methoxybenzoate **(18)**

**Compounds Tested IC50 (μM)**

The data obtained so far indicates that alkyl 3,4-dihydroxybenzoates can be expected as su‐ perior as lipoxygenase inhibitors similar to the alkyl 3,4,5-trihydroxybenzoates. It should be

14.3 0.07 60.3 10.5 3.3 200 200

Alkyl protocatechuate antioxidants may act in a variety of ways including direct quenching of reactive oxygen species, inhibition of enzymes involved in the production of the reactive oxygen species, and chelation of low valent metal ions such as Fe2+, Fe3+ or Cu2+. In connec‐ tion with this, both pyrogallol (**33**) and catechol (**35**) groups have iron binding properties *in vitro*. Alkyl gallates and protocatechuates may suppress the superoxide- driven Fenton reac‐ tion, which is currently believed to be the most important route to active oxygen species [25]. More specifically, alkyl gallates and protocatechuates may prevent cell damage in‐ duced by H2O2 since this can be converted to the more reactive oxygen species, hydroxy rad‐ icals, in the presence of these metal ions. Thus, metal chelation may play a large role in determining the antioxidant activity [26]. Interestingly, the inhibition of iron absorption *in vivo* has been positively correlated with the presence of galloyl groups but not catechol (**35**) groups [27].

Alkyl protocatechuates act as both lipoxygenase inhibitors and scavengers. Safety is a primary consideration for antioxidants in food products. After ingestion, the alkyl proto‐ catechuates are likely hydrolyzed, at least in part, to protocatechuic acid and the corre‐ sponding alcohols that are common in edible plants. For example, pecan nuts contain protocatechuic acid as a one of predominant phenolic acids [28]. If alkyl protocatechu‐ ates can reach the sites where antioxidants are needed, a more lipophilic alkyl side chain may partition into lipophilic membranes of cells and organelles, where it presumably ex‐ erts its antioxidant activity, similar to the phytyl chain in tocopherols and tocotrienols [29]. The site of antioxidant location is known to be important, however, it is not clear if alkyl protocatechuates can reach, without being metabolized, the sites where antioxidants are needed for protection from oxidative damages. It should be noted, however, that the role of alkyl protocatechuates in the human body is unknown when orally ingested. It is not clear if alkyl protocatechuates are absorbed into the system through the intestinal tract and delivered to the places where lipoxygenase inhibitors are needed without being metabolized.
