Do different chain lengths and configurations of gallate affect its antioxidant activity?

Abstract

10 gallic acid (GA) esters antioxidants with different ester group structures were prepared and characterized. The antioxidant effects of these antioxidants were evaluated under accelerated storage conditions and compared with commercial antioxidants. The results showed that the antioxidant effects of octyl gallate with 3 different configurations were superior to those of butylhydroquinone (TBHQ) and GA, but there was no significant difference in the antioxidant effects of these 3 configurations. In addition, FT-IR, ¹H NMR, and ¹³C NMR showed that omega-3 unsaturated fatty acids were the first to be oxidized. Docosahexaenoic acid is more distributed in the sn-2 position, while eicosapentaenoic acid is more distributed in the sn-1,3 region, and fatty acids located at sn-2 were the least resistant to thermal oxidation. This study provides basic data for selecting suitable antioxidants to improve the oxidative stability of fish oil during storage, and also provides design rules and techniques on how to develop new and efficient antioxidants.

Introduction

Fish oil is a general term for all oily substances in deep-sea and freshwater fish, such as tuna oil, whale oil, and sardine oil (Guil-Guerrero & Belarbi, 2001). Unlike conventional animal fats and oils, fish oils are rich in unsaturated fatty acids (UFA). According to the number of carbon–carbon double bonds, UFA can be categorized into monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), of which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the two most important PUFA in the ω-3 family (Morais et al., 2015). As the main functional components of fish oil, the PUFA of the ω-3 series are beneficial for the promotion of mental development in infants and children (Huanhuan et al., 2022), the prevention of Alzheimer’s disease, and the regulation of blood lipids and the improvement of hypertension (Benjamin et al., 2015). However, PUFA in fish oils are highly susceptible to oxidative degradation by light, oxygen, moisture, metal ions, and other factors. The resulting oxidation products at all levels not only reduce the nutritional value of the product and affect the colour and taste of the product, but also affect the health of the consumer (Agregán et al., 2017). The oxidation of fish oils relates to their nutritional value and safety, so preventing the oxidation of fish oils is a problem that needs to be solved by current research.

There are many ways to reduce the oxidation of fish oil, which can be roughly divided into two antioxidant mechanisms: physical reduction of oxygen content and addition of auxiliary antioxidant components. Commonly used antioxidant methods for fish oils include protection from light and heat, low temperature, vacuum and inert gas storage, etc. The addition of antioxidants is relatively affordable and effective, which can effectively chelate metal ions, scavenge oxygen radicals, etc., to achieve the purpose of extending the shelf life (Qiaoxian et al., 2017). The commonly used antioxidants dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), and TBHQ have a certain degree of toxicity and carcinogenicity, and long-term consumption of them can damage human health. It has been shown that BHT and BHA are carcinogenic in some aspects, while TBHQ has been banned in Japan and some European countries (Roby et al., 2013). Therefore, it is of great importance to synthesize low-toxic, efficient, and economical antioxidants to inhibit the oxidation of fish oil.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA), a polyphenolic organic compound, is found in large quantities in common plants, vegetables, nuts, and fruits (Meftahi & Aboutaleb, 2023). GA and its alkyl esters have been widely used as food antioxidants. In terms of antioxidant, GA at certain concentrations protects and inhibits body proteins, lipids, and DNA (Sneha et al., 2015) from oxidative stress damage. Its antioxidant effect may be the role of phenolic hydroxyl and free radicals hydrogen ions combined to achieve the effect and enhance the activity of antioxidant enzymes and reduce the content of oxidation products (Guan et al., 2023). GA contains three phenolic hydroxyl and one carboxyl group, a total of four strong polar groups, and these strong polar groups make GA has good antioxidant effect, but also make the solubility of GA in the oils and fats low and affect its practical application (Dimitrios, 2006). However, the Polar Paradox Theory states that strongly polar antioxidants are more effective in less polar media (e.g., oils) than less polar antioxidants (Mickaël et al., 2015; Ying & Fereidoon, 2012). In order to enhance the solubility of some strongly polar antioxidants in oil and to reduce their polarity, the antioxidants are often solubilized, e.g., by esterification of the antioxidants with non-polar long-chain alcohols or acids (Atikorn et al., 2012; Castro-Gonzà et al., 2020). The antioxidant capacity of lipophenolic compounds is related to their hydrophobicity, which is determined by the length of the alkyl chain of the lipophenol, and shows different degradation effects in different systems such as bulk oils and emulsions (João et al., 2016; Natthaporn et al., 2017). In other words, the optimal length of the phenolic ester chain to obtain the best antioxidant effect varies for different food matrices (Atikorn et al., 2010; Leann et al., 2015). This implies that there is a need to determine the appropriate phenolic ester chain lengths for optimal antioxidant effects in various applications. However, to the best of our knowledge, few studies have been reported on the differences in the in situ antioxidant properties of lipophenolic antioxidants with different alkyl chain lengths or configurations in the heat treatment of fish oils, whereas the relevant studies are limited to bulk oils, liposomes, and emulsion systems.

Inspired by this above, in this paper, GA was first esterified with alcohols of different chain lengths to obtain a series of GA ester derivatives (Figure 1). As shown in Figure 1, the GA ester derivatives with different chain lengths were abbreviated as BG, PG, H1G, H2G, OG, and NG. The antioxidant activity of the GA derivatives was evaluated by using the 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) and iron reduction methods, and the octyl gallate with the highest antioxidant activity was selected. Secondly, four octyl gallate derivatives with different configurations, denoted as 1-OG, 2-OG, 3-OG, and 4-OG, were synthesized by the reaction of four cheap octanols with GA, and three octyl gallates with better antioxidant activities were screened by simple DPPH and Fe reduction tests. In addition, the oxidative stability of fish oil before and after the addition of these compounds during accelerated storage at 140 °C in the dark for up to 12 days was investigated. The oxidation of fish oil under accelerated conditions was monitored by determining the peroxide value (POV), thiobarbituric acid reactant (TBARS) content, and p-anisidine value (pAV) of the fish oil, with the aim of investigating the differences in antioxidant activity among the different configurational octyl gallate esters.

Materials and methods

Materials

Fish oil was purchased from Zhejiang Xinglong Hemp Industry Co., synthetic raw materials were purchased from Wuhan Sinopharm Group Chemical Reagent Co. The chemical reagents used in the experiments, both analytical and optical grade, were commercially available. The synthesized new compounds were purified using a silica gel column (Merck, Kieselgel 60, 100–400 mesh) with ethyl acetate/petroleum ether as eluent. NMR spectra of the new compounds were obtained on a Bruker spectrophotometer (DPX 300, USA) with CDCl₃ as solvent. IR (KBr) spectra of new compounds and fish oil samples were analyzed on a BIORAD Tensor 27 spectrometer (Brucker Optics, Germany) with 0.5 cm⁻¹ resolution.

Synthesis of GA and its derivatives and testing of free radical scavenging ability.

The synthesis of several GA derivatives and the associated free radical scavenging capacity tests, including DPPH and FRAP reduction, were used as prerequisites for the antioxidant capacity tests (Supplementary material).

Oxidation stability test

Sample preparation

TBHQ (0.0499 g), GA (0.0510 g), OG (0.0847 g), 2-OG (0.0847 g), 4-OG (0.0847 g) were weighed in 10 ml centrifugal tubes and were dissolved by sonication with the addition of 5 ml of DMF (N,N-dimethylformamide) in that order. 5 ml of DMF without any antioxidant were used for control. The resulting solution and fresh fish oil (300 g) was added in 500 ml sequentially numbered flasks respectively, and the solution was dissolved by ultrasonication for 30 min and then the lid was closed, and then it was stored at 140 °C in the oven protected from light for 12 days. The samples were taken at intervals of 2 days, four were taken in each group, and each time 10 g was taken. The samples were labelled as 0, 2, 4, 6, 8, 10, and 12 days, sequentially.

Statistics and analysis

All analytical tests (except IR analysis) were performed in three replicates and data were reported as mean values and their standard deviations (mean ± SD). ANOVA test was used to compare the means of the parameters and Tukey’s multiple comparison test was used to test for significant differences between the means (p < .05). All statistical analyses were performed by SPSS version 26 software (Chicago, IL, USA).

Results and discussion

Evaluation of the test compounds on fish oil stability during accelerated storage

Usually, POV is a measure of the degree of oxidative rancidity of fats and oils, which monitors the production of primary oxidation products (Shan et al., 2019), TBARS and pAV belong to the detection indexes of secondary oxidation products, and the higher the content of TBARS and PAV in the sample, the more serious the oxidative degradation of fat (Mei et al., 2014). As can be seen in Figure 2A, after 12 days of accelerated storage at 140 °C, the POV value of the control, TBHQ, GA, 2-OG, OG, and 4-OG groups were 108.25 ± 0.58, 62.54 ± 0.42, 57.74 ± 0.65, 42.23 ± 0.41, 41.53 ± 0.22, and 52.26 ± 0.40 meq kg⁻¹, respectively, which means the lipid peroxides content of TBHQ, GA, 2-OG, OG, and 4-OG groups was significantly lower than that of the control group (p < .05). This indicated that the addition of the five antioxidants could effectively inhibit the generation of primary oxides and inhibit the oxidative spoilage of fish oil. Moreover, throughout the accelerated storage period, the POV values were positively correlated with the storage time from 0 to 12 days (p < .05), and the antioxidant activities of 2-OG, OG, and 4-OG were superior to those of TBHQ and GA. The POV values of 4-OG were slightly higher relative to those of OG and 2-OG, but there was little difference between the POV values of OG and 2-OG. As can be seen in Figure 2B, after 12 days of accelerated storage at 140 °C, the TBARS contents of the control, TBHQ, GA, 2-OG, OG, and 4-OG groups were 18.53 ± 0.32, 18.63 ± 0.31, 19.35 ± 0.53, 19.56 ± 0.82, 22.35 ± 0.42, and 48.58 ± 0.32 mg MAD kg⁻¹, it can be seen that the content of lipid secondary oxidation products in the TBHQ, GA, 2-OG, OG, and 4-OG groups was significantly lower than that in the control group (p < .05), which suggests that the incorporation of the five antioxidants significantly inhibited the generation of lipid secondary oxides. In particular, the generation rate of lipid secondary oxides was much smaller in days 0–4 than in days 4–10, because the primary oxidation products generated in the system on days 0–4 underwent a drastic secondary oxygenation on days 4–10, while a general decrease occurred from days 10 to 12, which is consistent with the findings (Chen et al., 2020). It may be due to the instability of hydroperoxides and the volatility of small molecule secondary oxidation products such as aldehydes, which decompose at a rate that exceeds the rate of production at high processing temperatures or for long periods of time, resulting in a tendency for the lipid secondary oxide content to decrease (Leann & Eric, 2016; Wang et al., 2011). As can be seen in Figure 2C, the pAV values also increased with time during accelerated storage. After 12 days of accelerated storage at 140 °C, the pAV values of the control, TBHQ, GA, 2-OG, OG, and 4-OG groups were 18.53 ± 0.32, 18.63 ± 0.31, 19.35 ± 0.53, 19.56 ± 0.82, 22.35 ± 0.42, and 48.58 ± 0.32. The addition of all five antioxidants can effectively inhibit the generation of secondary oxidation products. It is worth noting that the pAV values of samples containing 2-OG, OG, and 4-OG changed slowly during the accelerated storage process and their pAV values were much lower on the 12th day, which indicated that the 2-OG, OG, and 4-OG as antioxidants were the most effective in inhibiting the generation of secondary oxidation products, and all of them had a stronger antioxidant effect than that of TBHQ and GA. The final pAV values of 4-OG were slightly higher than those of OG and 2-OG, while OG and 2-OG were not significantly different from each other.

Based on the results of testing the POV, TBARS, and pAV values during accelerated storage, the antioxidant activity of gallate ester-based derivatives was stronger than that of GA and TBHQ. However, the antioxidant effects of branched ester-based gallate ester-based antioxidants were not much different from those of straight-chain ester-based ones on fish oil. In general, there are many factors affecting the antioxidant effect of antioxidants, and it is difficult to exert antioxidant effect when the antioxidants cannot be concentrated in the oxidation sites, This involves the dispersion of the antioxidants in the oils, and the degree of dispersion is related to the difference in solubility of the antioxidant molecules and the oils, binding energy of the antioxidant molecule with the fat molecules, and the mobility of antioxidants in the oils. Generally, the closer the values of the solubility parameters of the antioxidant and the oil, and the smaller the difference, the better the compatibility of the antioxidant molecules with the oil and their dispersion in the oil (Luo et al., 2019). According to Jie’s previous study (Jie et al., 2021), GA derivatives biologically reduce the polarity of the antioxidant due to the introduction of alkyl chains, which results in less intermolecular electrostatic interactions, lowering the value of the polar component and allowing better solubility of the antioxidant molecules in oils and fats. Thus, this explains the higher antioxidant activity of gallate derivatives relative to GA in antioxidant activity tests. The magnitude of the binding energy quantitatively characterizes the magnitude of intermolecular interaction forces. The larger the binding energy, the stronger the intermolecular interactions and the more difficult it is to disperse them (Luo et al., 2017). Jie simulated the binding energy between the antioxidant and the oil as well as between the antioxidant’s own molecules and found that the value of the binding energy between the antioxidant and the oil becomes larger with the growth of the antioxidant’s alkyl chain length, which is due to the fact that an increase in the relative molecular weight results in a larger van der Waals interaction between the two (Jie et al., 2021). The binding energy between antioxidant molecules and oil is less than or approximates the binding energy between antioxidant molecules due to hydrogen bonding between the molecules of GA, which will be unfavourable for the dispersion of antioxidant molecules in the oil, and may even result in the agglomeration of antioxidant molecules, therefore, in this aspect, gallate derivatives have higher antioxidant activity compared to GA.

The mobility of antioxidant molecules in oil also has a very important effect on the antioxidant effect. The location of free radicals in oil is random, and antioxidants need to move to the vicinity of the free radicals in order to be effective, so theoretically the better the mobility of antioxidants, the better the antioxidant effect. Jie simulated the mobility of antioxidant molecules in oil. The growth of antioxidant molecules in the relative molecular mass, the binding energy of molecules and the oil rises, the movement is difficult, and the mobility of its consequent decline, the antioxidants with lower relative molecular mass show stronger mobility. This explains the fact that the antioxidant activity of nonyl gallate is not as good as octyl gallate. In conclusion, the medium chain length modified OG better balanced the contradiction of dispersibility as well as mobility, which might be the reason why it showed the best antioxidant effect. The different configurations of octyl gallate have the same relative molecular mass, which makes the binding energy and mobility of octyl gallate to oil molecules little difference, so they show similar effects in the antioxidant activity tests.

However, the antioxidant activity of 4-OG was slightly lower than that of OG and 2-OG in the antioxidant test, which may be attributed to the fact that 4-OG has two longer alkane chains, which increase the spatial resistance of antioxidant migration in oils and fats, resulting in a slight decrease in its antioxidant activity compared with that of OG and 2-OG. On the other hand, Laguerre’s study showed that in systems containing both aqueous and oil phases, the oil–water interface is considered as the site where oxidation occurs. The chain length of phenolic ester antioxidants determines their hydrophobicity (Laguerre et al., 2017). Therefore, phenolic ester antioxidants with a certain hydrophobicity threshold (determined by their alkyl chain length) are enriched at the site of oxidation (oil–water interface), and thus octyl gallate modified with medium chain length has a better antioxidant effect.

Infrared spectra of virgin fish oil and accelerated oxidized fish oil

Figure 3 shows the IR spectra of fish oil containing various antioxidants in the range between 400 and 4,000 cm⁻¹ for accelerated oxidation at 140 °C. The treated samples have the same bands as the fish oil without antioxidants due to the fact that the concentration of various antioxidants is not enough to be detected in the IR spectra (Kurtulbaş et al., 2018). Most of the spectral information was observed in the region of 600 to 1,800 cm⁻¹ and 2,800 to 3,150 cm⁻¹. Figure 3A shows a representative IR spectrum of the unheated initial sample with visible peaks at 3,015, 2,930, 2,850, 1,748, 1,460, 1,379, 1,244, 1,150, 1,099, and 739 cm⁻¹. These characteristic peaks were assigned based on previous IR studies of oil (Kanchan et al., 2020; Kurtulbaş et al., 2018). A slight increase in peak intensity was observed at 2,930, 2,850, 1,748, 1,460, 1,379, 1,150, and 1,099 cm⁻¹, while a decrease in peak intensity was observed at 3,015, 1,244, and 739 cm⁻¹.

The characteristic absorption peaks at 739 and 1,244 cm⁻¹ involve wobbling and bending vibrations of -(CH₂)n- and -HC=CH-(cis), and the intensity of the peaks decreases upon accelerated oxidation, with the molecular chain of the oil molecule breaking and the molecular weight becoming smaller, the unsaturation of the oils was decreasing, with the strongest decreasing trend in the control group and little difference in the decreasing trend in the groups with added antioxidants. Peaks at 1,099 and 1,150 cm⁻¹ are the ester groups (-C-O) stretching vibration, and the absorption peaks become narrower during oxidation, the change in peak intensity here corresponds to the formation of secondary oxidation products and hydrolysis of the carbonyl ester functional group to ketone. 1746 cm⁻¹ is the triglyceride carbonyl (-C=O) stretching vibration peak, where the absorption peak is the strongest, which is caused by the increase of functional groups due to the generation of aldehydes, ketones, and acids during the oxidation process. It was evident that the control group showed the most significant increase in the intensity of absorption peaks, followed by GA > OG > 2-OG > TBHQ >  4-OG. 2,850 cm⁻¹ is the symmetric stretching vibration of methyl (-CH₃), and 2,930 cm⁻¹ is the asymmetric stretching vibration of methylene (-CH₂), and the intensity of the absorption peaks of both of them is enhanced during the oxidation process, which is a slight decrease in the peak intensity. The intensity of the absorption peaks increased during oxidation, which may be due to the disappearance of the cis-alkene C-H double bond in UFA (Kanchan et al., 2020), the most significant increase in intensity was in the control group. The C-H stretching vibration on the unsaturated C-atom of the grease olefin at 3,015 cm⁻¹, and the absorption peaks declined during oxidation, and the intensity of the absorption peaks here correlates with the unsaturated fatty acid content in fish oil, which suggests that the unsaturated fatty acid content decreased. Each small change in the absorption peaks of the functional group characteristic of the infrared spectra of fish oils reflects the internal changes of the oils, i.e., the UFA of fish oils are gradually decomposed during the accelerated storage process, but the oxidation products (aldehydes, ketones and acids, etc.) are gradually formed. Combined with the changes in the characteristic peaks throughout the IR spectrum, the oxidation tendency of fish oils without antioxidants was stronger during accelerated storage, while the oxidation tendency of fish oils with antioxidants such as OG, 2-OG, 4-OG, TBHQ, GA was relatively smooth, the inclusion of antioxidants can effectively inhibit the oxidation of UFA.

NMR of initial fish oil and accelerated oxidation of fish oil with added antioxidants

In this study, the oxidation process of fish oil containing various antioxidants under accelerated storage conditions was monitored by ¹H NMR and ¹³C NMR spectroscopy on days 0 and 12. As shown in Figure 4, the ¹H NMR spectra of each antioxidant-containing sample at day 12 were similar to those of the initial fish oil. This is due to the fact that the content of the various antioxidants was much lower than the fish oil content, and the characteristic peaks of the antioxidants were covered by the characteristic peaks of the fish oil (Tan et al., 2017). The triple peak at δ 0.83–0.95 ppm (A) was categorized as the terminal methyl group from saturated fatty acids, and the triplet of MUFA at δ 0.95–1.00 ppm (B) corresponds to the terminal ω-3 PUFA methyl characterization. The double peak at δ 1.14–1.43 ppm (C) belongs to the methylene groups of SA, MUFA, and ω-6 PUFA; the multiple peak at δ 2.75–2.90 ppm (J) corresponds to the bis-allylic hydrogens, which are highly reactive and susceptible to free radicals for the extraction of hydrogen, and the loss of the bis-allyl hydrogens indicates that the structure of the cis-double bond between the methylene groups is disrupted, and the formation of the lipid radicals. The multiple peaks at δ 5.20–5.30 ppm (L) belong to the olefinic hydrogen of the triacylglycerol glycerol backbone. As can be seen in Figure 4, there is a significant difference in the intensity of the characteristic peaks between 0.88–0.93 ppm (-CH₃) and 1.27–1.36 ppm (-CH₂-) after accelerated oxidation at high temperatures, and the integrals show a decrease in the peak area in these two regions, indicating a decrease in the content of PUFA. In the ¹H NMR of the fish oil stored for 12 days without any antioxidants, the decreasing trend of the characteristic peaks with chemical shifts at 0.88–0.93 ppm and 1.27–1.36 ppm was more drastic than that with the addition of the antioxidants of OG, 2-OG, 4-OG, TBHQ, and GA, respectively, which further proved that the addition of the antioxidants could effectively inhibit the oxidation of the fish oil. In addition, chemical shifts in the range of 1.52–1.70 ppm (-OCO-CH₂-CH₂-), 1.94–2.14 ppm (-CH₂-CH=CH-), 2.23–2.36 ppm (-OCO-CH₂-), 2.70–2.86 ppm (=HC-CH₂-CH=), 4.10–4.32 ppm (-CH₂OCOR), 5.26–5.40 ppm (-CH=CH-). These peaks are somewhat lower than those of the original fish oil, while the peaks at 4.04–4.10 ppm (-CHOH) are increased, which indicates the oxidative rupture of the unsaturated double bonds of the fish oil during the accelerated storage process and the formation of aldehydes, ketones, alcohols, acids, and other small molecules. Combined with the whole ¹H NMR spectrum, the overall oxidation degree of the fish oil without antioxidant was strong, and the decomposition trend of UFA was the most intense, while the oxidation degree of the fish oil with the addition of antioxidants such as OG, 2-OG, 4-OG, TBHQ, and GA was relatively low. The results of ¹H NMR of fish oil showed that after 12 days of high temperature treatment, the unsaturated fatty acid content of the fish oil without any antioxidant had a strong tendency to decrease, especially ω-3 PUFA, and the fish oil was oxidized intensely, whereas the addition of the antioxidants OG, 2-OG, 4-OG, TBHQ, GA improved the oxidative stability of the fish oil to a certain extent.

In Figure 4, the ¹³C NMR spectra can be observed in five spectral regions: the saturated methyl region (13–15 ppm), the methylene region (20–40 ppm), the glycerol region (60–75 ppm), the olefin region (121–141 ppm), and the carbonyl region (172–180 ppm). Within the methyl spectral range (13–15 ppm), the chemical shifts of the methyl peaks of n-6 PUFA (mainly C22:5n-6), saturated fatty acids (mainly C16:0 and C14:0), and n-3 PUFA (mainly C22:6n-3) were 14.08, 14.12, and 14.27 ppm, respectively, showing a distinctive characteristic peak divergence. In the methylene spectral region (20–40 ppm), the chemical shifts of the methylene peaks of saturated fatty acids and n-6 PUFA (mainly C22:6n-3 and C22:5n-6) were 22.58 and 22.68 ppm, respectively. The chemical shift of the methylene peaks of all the fatty acids (except C22:6n-3 and C22:5n-6) was 24.84 ppm. The chemical shifts of the methyl peaks of all the fatty acids (except C22:6n-3 and C22:5n-6) were 24.84 ppm, and the chemical shift of the methyl peaks of the saturated fatty acids was 24.84 ppm. The peaks at 29.05, 29.32, and 29.67 ppm correspond to the methylene carbons of all fatty acids (except C20:5n-3 and C22:6n-3).34.03 and 34.19 ppm are the methylene peaks of all fatty acids. In the glycerol spectral region (50–75 ppm), 62.10 and 68.88 ppm belonged to sn-1,3 and sn-2 carbon atoms on the triglyceride glycerol backbone, respectively. In the olefin spectral region (121–141 ppm), 127.90 and 128.08 ppm belonged to olefin carbon atoms on PUFA. 129.71–130.22 ppm belonged to olefin carbon atoms on UFA. The peak at 172.84 ppm in the carbonyl spectral region (172–178) corresponds to all carbonyl carbon atoms associated with the triglyceride sn-2 position except for C22:6n-3 and C22:5n-6. The peak at 173.26 is associated with all carbonyl carbon atoms linked to the triglyceride sn-1,3 position, excluding C22:6n-3 and C22:5n-6. Peak 173.30 belongs to the carbonyl carbon atom of the free fatty acid.

The distribution of the major ω-3 PUFAs in the sn-2 and sn-1,3 positions of the triglyceride (TAG) can usually be determined by ¹³CNMR, and this region can be used for the identification of the quality of fish oil (Tengku-Rozaina & Birch, 2014). A–J in the figure represent the characteristic peaks of SFA (sn-1,3), MuFA (sn-1,3), docosapentaenoic acid (DPA) (sn-1,3), EPA (sn-1,3), SFA (sn-2), MuFA (sn-2), eicosatetraenoic acid (ETA) (sn-2), DPA (sn-2), EPA (sn-2), EPA (sn-2), DHA (sn-1,3), DHA (sn-2), respectively, which are consistent with the previous studies (Tengku-Rozaina & Birch, 2014). Fish oil is rich in DHA and EPA, and according to the intensity of the characteristic peaks, it can be seen that DHA occupies a higher position in sn-2, while EPA occupies a higher position in sn-1, 3. After 12 days of accelerated storage, the DHA and EPA of the fish oil were oxidized and decomposed to a certain extent. Specifically, the decreasing trend of DHA eigenpeaks located at sn-2 was stronger, and the decreasing intensity of DHA eigenpeaks located at sn-1,3 was relatively weaker, which suggests that fatty acids located at sn-2 are less thermally stable, possibly because the sn-2 position is more easily oxidized and absorbed in vivo, and the intensity of the EPA eigenpeaks located at sn-2 and EPA eigenpeaks located at sn-1,3 also decreased to a certain extent, but the decreasing of the two eigenpeaks trends did not differ much. In addition, saturated fatty acids (SFA) at sn-1 and MuFA at sn-1,2,3 also underwent oxidative decomposition, and the peak intensities decreased to some extent. Combining the decreasing trends of all the characteristic peaks, the oxidation of ω-3 UFA was the most intense in fish oils without added antioxidants after accelerated storage, and the oxidation trends were GA > TBHQ > OG, 2-OG, 4-OG, respectively. The oxidative NMR of the fish oils showed that there was no significant difference in the antioxidative activities of the antioxidants in the different configurations, which is in agreement with the previous conclusions.

Conclusion

Ten octyl gallate antioxidants with different configurations were synthesized and characterized, and their antioxidant capacities were evaluated by free radical scavenging assay and fish oil thermal stability test. The results showed that the antioxidant activities of the three octyl gallate antioxidants with different configurations were stronger than those of TBHQ and GA, and there was no significant difference in the antioxidant effects of the gallate antioxidants with branched ester groups and those with straight-chain ester groups on fish oils. In addition, Fourier transform infrared spectrometer (FT-IR), ¹HNMR, and ¹³C NMR showed that omega-3 PUFAs were more susceptible to oxidation than other PUFAs, DHA is more distributed in the sn-2 position, while EPA is more distributed in the sn-1,3 region, and fatty acids located at the sn-2 position and the unsaturated position closest to the carbonyl group were the most thermally unstable, and the incorporation of antioxidants was able to significantly improve the oxidative stability of fish oil. The results of this study remind future researchers that when synthesizing similar ester derivatives with different branched chains as antioxidants, priority should be given to the reasonable availability of raw materials, short synthesis routes, convenient synthesis methods, etc., without paying too much attention to the configurations of antioxidants. This study provides basic data for selecting suitable antioxidants to improve the oxidative stability of fish oils during storage, as well as design rules and techniques on how to develop new and efficient antioxidants.

Data availability

Data will be made available on request.

Author contributions

Qing Li (Writing—original draft, Methodology), Shengqin Zhu (Data curation, Formal analysis, Conceptualization), Yanqing Li (Visualization, Methodology), Juan Liu (Visualization), Min Fu (Methodology), Zhiyong Xue (Writing—review & editing), and Lijuan Yu (Project administration, Supervision, Funding acquisition)

Funding

This work was supported by grants from Agriculture Science and Technology Program of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202305), PhD project initiated by Wuhan Polytechnic University (No. 2024RZ048), and Hubei Key Laboratory of Animal Nutrition and Feed Science (Wuhan Polytechnic University, No. 202316).

Conflicts of interest

The authors state that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References