Antioxidant and protective effects of extra virgin olive oil incorporated with diallyl sulfide against CCl4‐induced acute liver injury in mice

Abstract The present study delineates the effects of incorporation of 1% diallyl sulfide (DAS) into extra virgin olive oil (EVOO) on the physico‐chemical characteristics, in vitro antioxidant, and in vivo hepatoprotective properties in CCl4‐induced acute liver injury in mice. Results showed that the DAS‐rich EVOO exhibited good oxidative stability over one‐month storage and preserved its original quality‐related parameters including major components (oleic acid, linoleic acid, and palmitic acid), and minor components (tocopherols, chlorophylls and carotenoids, tyrosol, hydroxytyrosol, elenolic acid, oleuropein and its aglycone, pinoresinol, vanilic acid, cinnamic acid, ferulic acid, luteolin, apigenin, and sterols). Compared with EVOO or DAS, the DAS‐rich EVOO displayed the highest DPPH and ABTS‐radical scavenging activities and showed the strongest cellular antioxidant activity (CAA). In connection with its free radical scavenging activity and CAA, DAS‐rich EVOO significantly normalized the serum ALT and AST levels and prevented the increase in interleukin‐6 in CCl4‐intoxicated mice. The manifest anti‐inflammatory and hepatoprotective effects of DAS‐rich EVOO were further supported by liver histopathological examinations. Overall, the EVOO enrichment with DAS could open up opportunities for the development of novel functional food with improved antioxidant and hepatoprotective properties.

However, the profitability of EVOO-derived phenolics remains a challenging task because of their low daily intake (Rubió et al., 2012), and their quantitative and qualitative variations which in turn depend on genetic (cultivar), physiological (ripening stage at harvest), environmental (climate and soil), agronomic, and technologicalfactors (Vallsetal.,2015).Thus,toensureanoptimalintake of EVOO-derived phenolics without affecting the energy content, the enrichment of EVOO with their own phenolics could represent an attractive option. In this context, earlier attempts showed that the enrichment of virgin olive oil (VOO) through its malaxation with extracts from olive leaves (Sánchez de Medina et al., 2011), olive pomace (Zribi et al., 2013), or a mixture of them (leaves and pomace) (Delgado-Adámez et al., 2014) greatly enhanced its oxidative stability and antioxidant activities. Nonetheless, the main drawbacks of such enrichment strategy include increased bitterness and pungency taste, reducing consequently its sensorial properties and the consumer acceptability. Likewise, because of the pro-oxidant properties of some phenolic compounds (e.g., pinoresinol, tyrosol, ligstroside aglycon, (+)-1-acetoxypinoresinol, and elenolic acid) (Carrasco-Pancorboetal.,2005),theirincorporationintoVOOcould promote lipid peroxidation, which negatively influence its nutritional value and biological activities (Rubió et al., 2012). To overcome these problems, attempts have been made to produce enriched olive oils with vegetables, aromatic plants, and spices (Reboredo-Rodríguez etal.,2017).Althoughthepromisingresultsintermofoxidativestability and antioxidant activities of the enriched VOO, their preparation that involves infusion, milling, and/or malaxation was usually associated with the coextraction of undesirable components such as waxes and bitters that could modify their sensory characteristics (Moldão-Martinsetal.,2004).
Despite these advances, the use of pure bioactive compounds for the enrichment of EVOO/VOO has not received much interest.
The DAS is a lipid-soluble compound responsible in part for the strong taste and odor of garlic and Allium plant species. It is alleged to have antioxidant (Kalayarasan et al., 2009), antimicrobial (Velliagounder et al., 2012), anticancer (Sriram et al., 2008), hepatoprotective (Shaik et al., 2008), neuroprotective (Lin et al., 2012), and anti-inflammatory (Kalayarasan et al., 2009) activities. Owing to these biological activities, it was hypothesized that the incorporation of DAS into EVOO could greatly improve the antioxidant and hepatoprotective properties of the enriched EVOO. Therefore, the present study was designed to produce and char-acterizeanEVOOenrichedwithDASthroughdirectincorporation ofDASintoEVOO.TheDAS-richEVOOwasfutherevaluatedforits in vitro antioxidant activity and its protective effect against CCl 4induced hepatotoxicity in mice.

| Preparation of DAS-rich EVOO
The monovarietal Chemlali EVOO from the crop season 2017-2018 controlandDAS-richEVOOwereplacedinclosedamberglassbottles, stored at room temperature for one month, and then essayed for their physico-chemical characteristics and biological activities.
For the determination of total phenol content (TPC), a 200µl aliquotofsampleextractorthestandardcaffeicacid(CA)wasdiluted withpurifiedwatertoatotalvolumeof2.5ml,mixedwith0.25mlof 10-folddilutedFolin-Ciocalteureagentand,after3min,with0.5ml ofa35%(w/v)solutionofsodiumcarbonate,andfinallydilutedwith purified water up to final volume of 5 ml. After 2 h incubation at room temperature and in the dark, the absorbance of the reaction mixture was measured at 725 nm (Jasco Corp., Tokyo, Japan). The TPC was expressed as mg CA equivalents per kg of oil (mg CAE/ kg oil).

| DPPHradicalscavengingactivity
The DPPH radical activity of DAS (1% in methanol), olive oil, and DAS-richoliveoilwasdeterminedaccordingtotheslightlymodified method of Kim et al. (2005). Briefly, 100 µl of sample extract was added to 1.9 ml of 0.1 mM DPPH methanol solution. The mixture was vortexed vigorously and allowed to stand for 1h in the dark, thereaf-tertheabsorbanceofthereactionmixturewasmeasuredat515nm and compared with the absorbance of blank control (methanol).

| Ferricreducingantioxidantpower (FRAP)assay
In the aforementioned assays (DPPH, ABTS, and FRAP), trolox was used as a positive control and the results were expressed as µmol trolox equivalents per kg of oil (µmol TE/Kg oil).

| Statistical analysis
Data are presented as mean ± standard error of the mean (SEM).
Mean values for physico-chemical parameters and antioxidant assays were compared using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. For serum biochemical markers, the comparison between groups was performed using unpaired

| In vitro antioxidant activity
The antioxidant activity of control EVOO, DAS-rich EVOO, and 1% DAS (in methanol) was evaluated through 4 complementary assays.

| D ISCUSS I ON
In recent years, several landmark studies have shown that enrichment of EVOO/VOO can improve their oxidative stability, nutritional quality, and organoleptic properties (Benkhoud et al., 2021;Rubió et al., 2012). In this study, it has been demonstrated that the enrichmentofEVOOwithDASpreservesitsqualityintermofmajor (e.g., fatty acids) and minor compounds (e.g., phenolics, pigments, sterols, and vitamin E), probably by inhibiting lipid peroxidation and the formation of free radicals. This result indicates that the incor-

poration of DAS into EVOO effectively maintains its antioxidant
properties as appreciable amounts of antioxidants (e.g., tocopherols, phenolics,chlorophylls,andcarotenes)weredetected.Supportsto this assumption are given by Kim et al. (1997) who reported that the diallyl disulfide (DADS)-and diallyl trisulfide (DATS)-rich garlic homogenates effectively inhibit lipid oxidation in an oil-water system.
Six years later, Yin and Cheng (2003) showed that exogenous ap-plicationofDASsignificantlydelayedlipidoxidationingroundbeef owing to its reducing or chelating ability.
of AST suggesting that DAS-rich EVOO had protective effects on CCl 4 -intoxicated mice through liver cell membrane stabilization.
Microscopic examinations underpin these observations and clearly showed the recovery of liver appearance (normal brown liver) and histologic parameters including the well-shaped hepatocytes with well-defined cell wall, prominent nuclei, granulated cytoplasm, etc. (Figure 3p). These ameliorative effects were not recorded for EVOO and DAS treatments (Figure 3n). Given its superior antioxidant activity in vitro, it can be suggested that the hepatoprotective effects of DAS-rich EVOO against CCl 4 -induced liver damage are mediated through its ability to neutralize the free radicals (CCl 3 • and CCl 3 OO•), prevent lipid peroxidation, maintain the functional integrity of liver biomembranes, and reduce enzymes leakage. The decrease in relevant pro-inflammatory cytokine IL-6 is suggested too.DatafromFigure2.showingthatDAS-richEVOOwasmoreef-fectiveinreducingIL-6thanthatofEVOOandDASprovidesupport for such assumption. The mechanism behind the inhibitory effect of DAS-rich EVOO on IL-6 production is understood, but may be likely associated with its capacity to interfere with the expression of nuclearfactorkappaB(NF-κB)whichisresponsiblefortheexpres-sionofIL-6(Zhouetal.,2009).Additionalindepthexperimentsare needed to decipher the exact mechanism involved in such inhibitory effectofDAS-richEVOO.
The anti-inflammatory activity of some EVOO components has been extensively investigated (Cárdeno et al., 2013;Cicerale et al., 2012). For example, the secoiridoids oleuropein and it agly- In the present study, it is possible that DAS and one or more bioactive components of the EVOO will synergistically contribute to theprotectiveeffectofDAS-richEVOOagainstCCl 4 -induced hepatotoxicity, which is in tune with our previous conclusions regarding the in vitro antioxidant activity. Synergy involving DAS has been previously observed with pomegranate fruit extract (PFE) (George etal.,2011).Intheirexperimentalmodel,authorsshowedthatDAS and PFE impart better cancer chemopreventive effect than either of these agents alone because of the enhanced antioxidant and anti-inflammatoryofthesystemDAS/PFE.

Particularly,strikingisthelowprotectiveeffectofEVOOandDAS
in CCl 4 -intoxicated mice, despite their well-known anti-inflammatory and hepatoprotective properties (Kalayarasan et al., 2009). These unexpected results might be due to the low concentrations used and/or metabolization of the bioactive components.

| CON CLUS ION
ThekeyresultsofthisstudyrevealedthattheincorporationofDAS preserves the original composition and enhanced the oxidative sta-

bilityoftheDAS-richEVOO.Italsoimprovesitsantioxidantactivity
in vitro and underpins its anti-inflammatory and hepatoprotective effect on CCl 4 -intoxicated mice, which was shown by the decrease in serumALT,AST,andcreatininelevels,aswellasimprovedliverhistology. Further studies are needed to find out the exact mechanism ofheptaoprotectiveactionofDAS-richEVOO,anditsexploitation for the prevention and treatment of liver injury.

ACK N OWLED G M ENTS
This work was financed by the Tunisian Ministry of Higher Education andScientificResearch(MHESR)(GrantNumberLR15INRAP02).

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest. Writing-review & editing (lead).

E TH I C S A PPROVA L
This study was performed in strict accordance with protocols ap-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.