A range of fatty acids differing in
chain length (C8 to C22), degree and location of unsaturation (saturated vs
unsaturated and terminal vs nonterminal olefins) and the presence of hydroxyl
moiety were conjugated with coniferyl alcohol. Antioxidant activity of phenolic
compounds depends on the polarity of their microenvironment (McClements and Decker 2000; Foti et al. 2004; Figueroa-Espinoza and Villeneuve 2005). Thus, the antioxidant activity of
the synthesized capsiconinoid analogues were studied in different experimental
models, such as 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging
activity in polar methanolic medium, inhibition of lipid (fish oil) oxidation
by thiobarbituric acid reactive substances (TBARS) assay, and autoxidation of
linoleic acid in Tween 20 micellar medium. The aim of the work is to evaluate
the influence of the chain length and type of fatty acids of the prepared coniferyl
esters of fatty acids on their antioxidant activities.
Phenolipids are diversified group of
compounds present abundantly in nature and are known for many potent biological
activities (Stasiuk and Kozubek 2010). They have been used for decades as
industrial antioxidants in cosmetic and food industries (Shahidi and Zhong
2010). Capsicum annum L. is found to
be source of two well known natural phenolipids, capsinoids and capsaicinoids.
Other than these two phenolipids, capsiconinoids have also been isolated from
the fruits of pepper, Capsicum baccatum L.
(Kobata et al. 2008). First synthesis of capsiconiate and dihydrocapsiconiate was
carried out by lipase-catalyzed transesterification of coniferyl alcohol with
methyl esters of 8-methylnon-6-enoic and 8-methylnonanoic acids (Kobata et al.
2008). But, the synthesis of natural capsiconinoid is complicated and costly
due to the specific nature of the alkyl side chain. However, linear analogues
of capsiconinoids such as coniferyl stearate, coniferyl palmitate and coniferyl
oleate have been isolated, synthesized and evaluated for antioxidant activities
(Lee et al. 2004; Appendino et al. 2004).
The present work was taken up to
synthesize a series of linear analogues of capsiconinoids differing in alkyl
chain length and functional moieties. The objective is to study the effect of
hydrophobicity as well as the presence of functional moiety in the hydrophobic
chain on antioxidant activity of the linear capsiconinoid analogues. Coniferyl
alcohol and fatty acids were esterified using immobilized lipase Candida antarctica (Novozyme 435) in t-butanol at 55 ?C for 4 h. The
structures of the compounds were confirmed by NMR (1H and 13C)
and high resolution mass spectrometry. Altogether 10 compounds were
synthesized by varying the alkyl chain length as well as functionality in the
alkyl chain. Out of them three compounds (e-g)
were reported earlier whereas seven compounds are reported in this work (a to d & h to j) (Fig. 2).
The antioxidant activity of the synthesized compounds was
determined by using three in vitro assays: DPPH radical scavenging assay (polar medium), thiobarbituric acid reactive
substances (TBARS) assay (nonpolar medium) and autoxidation of linoleic acid (micellar
DPPH free radical scavenging activity
The DPPH radical scavenging capacity of all the synthesized compounds (Table 1) indicated
good radical scavenging activity similar with BHT, but significantly
better than ?-tocopherol (p<0.001). Among the synthesized capsiconinoid analogues coniferyl alcohol conjugated to saturated fatty acids, shorter chain homologues (compounds a, b and d) have shown almost similar activity. However, with the increase in alkyl chain length beyond coniferyl laurate (compounds e, f and h) a decreasing trend in activity was observed, but still showing significant superiority over ?-tocopherol. Even in unsaturated homologues, similar trend was observed (compounds c, g and i). This decrease in activity with the increase in hydrophobicity might be correlated with the decrease in solubility of the analogues in polar methanolic medium. However, coniferyl ricinoleate (j) was found to exhibit the maximum radical scavenging activity compared to all the coniferyl esters and reference compounds. The reason might be the presence of hydroxyl group at the hydrophobic chain influencing the solubility characteristics in polar methanol medium. Lee et al. (2004) also found that the antioxidant activities of coniferyl palmitate and oleate are as high as BHT or BHA as per DPPH assay. 3.2.2. Lipid oxidation as measured by TBARS assay Fish oil, being rich in polyunsaturated fatty acids was taken as medium in TBARS assay in evaluating the antioxidant activity of the prepared capsiconinoid analogues. BHT and ?-tocopherol were used as reference antioxidants. The results demonstrated a gradual increase in activity with the increase in saturated fatty acid chain length till coniferyl stearate (compound f). Beyond coniferyl stearate a drastic fall in activity was observed (compound h) (Table 2). Similar was the trend among the unsaturated homologues of capsiconiate. Thus, longer chain homologues like coniferyl stearate (f), coniferyl oleate (g) and coniferyl ricinoleate (j) provided maximum protection to fish oil among the tested capsiconinoids. Activities of all the capsiconinoid analogues were similar or better than the reference antioxidant BHT, but lower than ?-tocopherol. 3.2.3. Autoxidation of linoleic acid in Tween 20 micellar media Antioxidants are widely employed in cosmetic and food products to inhibit lipid peroxidation. Most of such products exist in a formulated complex emulsion system. This particular assay model mimics such complex emulsion system, wherein the rate of autoxidation of linoleic acid was carried out in Tween 20 micellar medium in the presence of capsiconinoid analogues and the reference antioxidants (1 mM). The formation of conjugated diene from the oxidation of linoleic acid in Tween 20 micellar medium was very rapid and was measured at 234 nm. In the control sample, the formation of conjugated diene was found to reach maximum in 36 h upon 120 h study (Fig. 3). The autoxidation of linoleic acid was found to decrease profoundly due to the addition of capsiconinoid analogues and reference compounds. This was evident from the negligible formation of conjugated diene in 36 h. Coniferyl esters attached to undecanoic acid (b) exhibited the maximum inhibition of autoxidation of linoleic acid, significantly higher (p < 0.001) than all the studied compounds except coniferyl undecenoate (c), and laurate (d). Coniferyl undecenoate (c) and laurate (d) exhibited very similar activity. Among higher chain analogues, coniferyl ricinoleate (j) exhibited the maximum inhibition, significantly higher (p < 0.001) than other higher chain analogues, but significantly lower than b. BHT was the most active compound among all the studied antioxidants. Porter (1980) and Porter et al. (1989) proposed "polar paradox" hypothesis to explain the effectiveness of an antioxidant in bulk oil and emulsion. The paradox is that the hydrophilic antioxidants are more effective in bulk oil, whereas hydrophobic antioxidants are more effective in oil-in-water emulsion. Thus in hydrophobic medium like oil, increased hydrophobicity of an antioxidant decreases its activity. On the other hand, the antioxidant activity increases with the increased lipophilicity in an emulsion system. But, there are limitations to this theory as proposed by Laguerre et al. (2009). They proposed a nonlinear dependency of lipophilicity on antioxidant activity, which has culminated into a new "cut-off effect" hypothesis. According to this new hypothesis, the activity of an antioxidant in an emulsion system increases with the increased hydrophobicity, reaching a maximum for the dodecyl chain, beyond which activity decreases drastically. This nonlinear effect was supported by various reports using a range of phenolics (Laguerre et al. 2009; Lucas et al. 2010; Shahidi and Zhong 2011; González et al. 2015). It would be interesting to analyze the results of capsiconinoid analogues on the basis of their hydrophobicity.