Enhanced in vitro and in vivo antioxidant activities of olive pomace by solid- state fermentation using the yeast Kluyveromyces marxianus

Enhanced in vitro and in vivo antioxidant activities of olive pomace by solid- state fermentation using the yeast Kluyveromyces marxianus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced in vitro and in vivo antioxidant activities of olive pomace by solid- state fermentation using the yeast Kluyveromyces marxianus Abeer E. Mahmoud, Mamdouh M. Ali, Shadia A. Fathy, Amira T. Mohammed, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7255027/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: This study investigated the potential role of solid-state fermentation (SSF) to enhance the in vitro and in vivo antioxidant activities of olive pomace (OP, the environmentally polluting solid residue produced after olive oil extraction) using the GRAS (Generally Recognized As Safe) yeast Kluyveromyces marxianus . Fermented OP (FOP) was produced by SSF of OP by K. marxianus . Both unfermented OP (UFOP) and FOP were extracted using methanol. Results: FOP methanolic extract (FOPME) demonstrated significantly higher antioxidant activity than UFOP methanolic extract (UFOPME) in ABTS radical scavenging, metal chelating, H 2 O 2 scavenging, lipid peroxidation inhibition, reducing power, and total antioxidant capacity. On the other hand, UFOPME demonstrated slightly higher superoxide anion radical scavenging and RBCs-protecting activities than FOPME. Prophylactic and therapeutic antioxidant activities of UFOPME and FOPME were assessed in vivo against oxidative stress associated with diethylnitrosamine (DENA)-induced hepatocellular carcinoma (HCC) in rats. Relative to the normal group, the administration of DENA/CCl 4 in the HCC group significantly increased hepatic nitric oxide and malondialdehyde and significantly decreased total antioxidant concentration. Both UFOPME and FOPME protected against and relieved the oxidative stress associated with DENA/CCl 4 -induced HCC in rats. Conclusion: SSF of OP using the GRAS yeast K. marxianus significantly enhanced most of its in vitro antioxidant activities alongside demonstrating simultaneous protective/therapeutic effects against oxidative stress associated with DENA/CCl 4 -induced HCC in rats. Olive pomace solid-state fermentation Kluyveromyces marxianus phenolic compounds antioxidant activity oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Numerous pathologies associate to increased production of free radicals within biological systems which leads to oxidative stress. An antioxidant is a substance that can scavenge reactive species, inhibit their production, or positively regulate antioxidant defense systems [ 1 ]. Antioxidants can provide an electron to a free radical-containing compound without undergoing destabilization, thus discontinuing the free radical chain reaction. Naturally obtained free radical scavengers are effective, safe, and potent; thus, they can serve as lead compounds for the development of novel drugs. Owing to their chemical structure, phenolic compounds are very efficient and promising free radical scavengers [ 2 ]. Olive pomace (OP) represents a particularly challenging oil processing waste that is produced in huge amounts in Mediterranean countries. Sustainable olive oil production requires continuous pomace disposal, which is not an easy mission regarding the toxic physico-chemical characteristics of OP. Nevertheless, merely 2% of total phenolics found in olive fruits transfer to the extracted olive oil, while the other 98% retain in olive oil by-products. About 45% of the total phenolic content of olive fruit retain in OP [ 3 , 4 ]. Regarding its high polyphenolic content, OP can be considered a cheap and renewable source of pharmaceuticals rather than an environmentally polluting agro-industrial waste [ 5 ]. The approval of phenolic-rich extracts of OP as Generally Recognized As Safe (GRAS) by the US Food and Drug Administration (FDA) opened new perspectives for their usage in food, feed, and pharmaceutical sectors [ 4 ]. Several companies have started to commercialize olive phenolic extracts formulated in different ways that offer numerous health benefits without adverse health effects [ 6 ]. Previous work of Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [ 7 ] suggested that solid-state fermentation (SSF) of OP using the GRAS yeast, Kluyveromyces marxianus represents a novel technique for OP disposal that valorizes the pomace into a promising value-added product with potent anticancer activity studied in vitro . Also, Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [ 8 ] reported that the valorized OP had an enhanced phenolic profile with increased rutin, vanillin, cinnamic acid, quercetin, catechin, and syringic acid contents. The present work aimed to study the impact of SSF of OP using K. marxianus on pomace antioxidant activity. The antioxidant activities of unfermented (UFOPME) and fermented (FOPME) OP methanolic extracts were compared both in vitro and in vivo against oxidative stress associated with DENA/CCl 4 -induced HCC in rats. Moreover, the enhanced antioxidant activity of pomace after fermentation was explained according to the altered phenolic and phytochemical profiles reported by Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [ 7 ] and Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [ 8 ] to get a deeper and more comprehensive view of the effect of SSF of OP with the yeast K. marxianus . This study shows a significant academic advance by demonstrating both the protective and therapeutic effects of FOP against oxidative stress linked to diethylnitrosamine (DENA)-induced hepatocellular carcinoma (HCC). This application has been notably underexplored. It uses Kluyveromyces marxianus NRRL Y-8281, a rarely studied yeast strain recognized as GRAS, in SSF to greatly improve the antioxidant potential of OP. Moving beyond earlier compositional analyses, this research includes: (i) thorough in vitro tests such as radical scavenging, metal chelation, and lipid peroxidation inhibition; (ii) systematic in vivo validation using a rat model of HCC; and (iii) direct comparison of preventive versus therapeutic effectiveness in reducing oxidative stress. By evaluating both unfermented and fermented OP extracts, the study confirms significant biochemical improvement and highlights SSF as an important method for sustainable waste use. These findings offer meaningful originality by connecting eco-biotechnology with clinical potential, turning agricultural byproducts into valuable antioxidant agents. 1.1. Materials: 1.1.1. Olive oil pomace (OP): OP was obtained during its harvesting season by a local olive-pressing factory (three-phase decanter system), located in Al-Arish, North Sinai, Sinai Peninsula, Egypt. It was stored at-20℃ until used. 1.1.2. Experimental organisms: 1.1.2.1. Experimental microorganism: Kluyveromyces marxianus NRRL Y-8281 yeast strain used in the present study was purchased from the Agricultural Research Service (Peoria, Illinois, USA). 1.1.2.2. Experimental animals: The hepatocellular carcinoma (HCC) animal model study was conducted on 80 adult male albino western rats in addition to another 60 rats used for the determination of the LD 50 of extracts (average body weight of 130 ± 20g). Rats were housed in the Animal House Colony of the National Research Centre, Dokki, Giza, Egypt. The animal house was ventilated with a 12-hour light/dark cycle at an ambient temperature of 25°C-30°C throughout the experimental period, with free access to tap water and a standard rodent chow. Animals were allowed 7 days for acclimatization before the initiation of the experiment. 1.1.2.2.1. Ethics approval: All procedures of animal handling and sacrificing complied with the standard guidelines of the institutional ethics committee for animal care at the National Research Centre, Egypt (ethic No. 18/036). 1.1.3. Chemicals: 1, 10-Phenanthroline (Panreac, Espain). ABTS (Sigma-Aldrich, USA). Ascorbic acid (Sigma-Aldrich, USA). Carbon tetrachloride (CCl 4 ) (Sigma-Aldrich, USA). Diethylnitrosamine (DENA) (Sigma-Aldrich, Japan). Ferrozine (Sigma-Aldrich, USA). Linoleic acid (Fluka, Germany). Methanol and ethanol (Piochem, Egypt). Thiobarbituric acid (Merck, Germany). Trichloroacetic acid (SRL, India). All other chemicals and reagents used in this study were of analytical grade and purchased from Sigma-Aldrich Chemical Co. (Germany). 1.1.4. Instruments: Cooling centrifuge (SiGMA 3–18 KS, Germany). Homogenizer (Kinematica Polytron, PT 10–35 GT, China). Incubator shaker (Thermo Fisher Scientific, Model MAXQ 481R HP, USA). Laminar Flow: (heal force, hf safe 1200, China). Rotary Evaporator (Heidolph, Germany). Shaking water bath (DAIHAN Scientific, MAXturdy-18, Korea). Spectrophotometer (JASCO V-730, Japan). Sterilizer (Tomy, SX-700, Japan). 1.2. Methods: 1.2.1. Culture maintenance and inoculum preparation: The yeast strain was grown and maintained according to Wickerham LJ [ 9 ]. The inoculum was prepared according to Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [ 7 ]. 1.2.2. Solid-state fermentation (SSF): The protocol of Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [ 7 ] was adopted for SSF. Incubation was done in a static incubator for 48 hours at 45 ℃. 1.2.3. Preparation of phenolic-rich extracts: Both unfermented olive pomace methanolic extract (UFOP) and fermented olive pomace methanolic extract (FOP) were prepared according to Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [ 7 ]. Both extracts were reconstituted to obtain different extract concentrations (2, 4, 6, 8, and 10 mg/ml) using methanol, distilled water, or phosphate-buffered saline (PBS) according to the instructions of each method applied in this study. 1.2.4. Assessment of in vitro antioxidant activities of extracts: 1.2.4.1. ABTS radical scavenging activity: ABTS (2, 2′-azinobis (3-ethylbenzthiazolin-6-sulfonic acid) radical scavenging activity was assessed according to Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C [ 10 ]. Both extracts were reconstituted in distilled water. The percentage inhibition was calculated using the formula: Scavenging activity (%)=[1-( \(\:\frac{\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) ) X 100%] (Eq. 1) 1.2.4.2. Reducing power: The reducing power of both extracts was assessed using the reducing power method described by Oyaizu M [ 11 ]. Extracts were reconstituted in methanol. Ascorbic acid was used as a standard, and results were expressed as mg ascorbic acid equivalents per liter (mg AscE /L). 1.2.4.3. Metal chelating activity: The chelation of ferrous ions was estimated using the method of Dinis TC, Madeira VM, Almeida LMJAob and biophysics [ 12 ]. Extracts were reconstituted in methanol. The metal chelating activity was calculated according to Eq. (1). 1.2.4.4. Assessment of antioxidant activities against reactive oxygen species: 1.2.4.4.1. Hydrogen peroxide (H 2 O 2 ) scavenging activity: The method of Mukhopadhyay D, Dasgupta P, Roy DS, Palchoudhuri S, Chatterjee I, Ali S and Dastidar SG [ 13 ] was adopted for the assessment of the hydrogen peroxide scavenging activity of extracts. Extracts were reconstituted in methanol. Hydrogen peroxide scavenging activity was calculated according to Eq. (1). 1.2.4.4.2. Superoxide anion (O 2 - ) scavenging activity: For assessing superoxide anion radical scavenging activity, the method reported by Jiao Z, Liu J and Wang S [ 14 ] was followed. Extracts were reconstituted in methanol. The scavenging activity was calculated as a percentage inhibition of control according to Eq. (1). 1.2.4.5. Assessment of protective activities: 1.2.4.5.1. Lipid peroxidation inhibition: Lipid peroxidation inhibition activity was assessed according to the method reported by Matkowski A and Piotrowska M [ 15 ]. Extracts were reconstituted in PBS. The percentage of peroxidation inhibition was calculated according to Eq. (1). 1.2.4.5.2. RBCs protecting activity: The anti-hemolytic activity was assessed by following the spectrophotometric method of Yang Z-G, Sun H-X and Fang W-H [ 16 ]. Extracts were reconstituted in PBS. Protecting activity was calculated according to Eq. (1). 1.2.5. Assessment of in vivo antioxidant activities of extracts against HCC-associated oxidative stress: 1.2.5.1. Determination of median lethal dose (LD 50 ) of extracts: This study was designed to assess the acute oral toxicity produced when UFOPME and FOPME were administered by oral gavage to rats using the method described by Wilbrandt W [ 17 ]. 1.2.5.2. Experimental design: After acclimatization for 1 week on based diet, the experimental animals were weighed and randomly divided into 8 groups, with 10 animals in each group for a study period of 16 weeks. The eight groups were designed as follows: Group (1): Normal group: animals were fed on a standard diet and given the vehicle (saline) throughout the experiment. Group (2): HCC group: in which HCC was induced in rats by intraperitoneal injection of a single dose of diethylnitrosamine (DENA) in saline at a dose of 200 mg/kg b.w., 2 weeks later rats received carbon tetrachloride in corn oil (1:1) (as a promoter for carcinogenesis) subcutaneously at a dose of 3 ml/kg/week for 10 weeks [ 18 ]. Group (3): UF control group: in which rats were daily orally treated with a safe dose of 283.3 mg UFOPME/kg b.w. (1/10 of the LD 50 ) for 30 days, then the animals were given the vehicle till the end of the experiment. Group (4): F control group: in which rats were daily orally treated with a safe dose of 283.3 mg FOPME /kg b.w. (1/10 of the LD 50 ) for 30 days, then the animals were given the vehicle till the end of the experiment. Group (5): UF pre-treated group (Pre-UF): in which rats were orally pre-treated daily with the safe dose of UFOPME for 30 days before HCC induction (as in group 2). Group (6): F pre-treated group (Pre-F): in which rats were orally pre-treated daily with the safe dose of FOPME for 30 days before HCC induction (as in group 2). Group (7): UF post-treated group (Post-UF): in which HCC-bearing rats were orally treated daily with the safe dose of UFOPME for 30 days. Group (8): F post-treated group (Post-F): in which HCC-bearing rats were orally treated daily with the safe dose of FOPME for 30 days. At the end of the experiment (16 weeks), the animals were anesthetized by injection of ketamine/xylazine mixture at a dose of 100 mg/kg-10 mg/kg BW, sacrificed by decapitation, and dissected, then their livers were excised and washed with saline. Biopsies of livers were immediately homogenized in ice-cold PBS (pH 7.4) using a tissue homogenizer. The homogenates were centrifuged at 1700 rpm for 30 minutes at 4 ℃ and the resulting supernatants were stored at-80 ℃ for assessment of hepatic oxidative stress markers. 1.2.5.2.1. Hepatic oxidative stress markers: 1.2.5.2.1.1. Hepatic malondialdehyde (MDA) level: Malondialdehyde (MDA) level in liver homogenate, as lipid peroxidation end product, was determined by the endpoint colorimetric method of Satoh K [ 19 ] and Ohkawa H, Ohishi N and Yagi K [ 20 ] using a commercial assay kit (Biodiagnostic, Egypt) where MDA reacts with thiobarbituric acid in an acidic medium at a temperature of 95°C for 30 minutes to form a pink product that can be measured at 534 nm. The assay was done according to the manufacturer’s guidelines. Calculation of results : MDA concentration (nmol/g tissue used) = \(\:\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{s}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}}\times\:\frac{10}{\text{g}\:\text{t}\text{i}\text{s}\text{s}\text{u}\text{e}\:\text{u}\text{s}\text{e}\text{d}}\) (Eq. 2). 1.2.5.2.1.2. Hepatic nitric oxide (NO) level: Nitric oxide (NO) level in liver homogenate was determined by the endpoint colorimetric method of Montogomery H and Dymock JF [ 21 ] using a commercial assay kit (Biodiagnostic, Egypt). The kit is based on the measurement of endogenous nitrite concentration (the final products of NO in vivo ) as an indicator of NO production. It depends on the addition of Griess reagent, which converts nitrite into a deep purple azo compound with a bright reddish-purple color that can be measured at 540 nm. The assay was done according to the manufacturer’s guidelines. Calculation of results : Nitrite concentration (μmol/L) = \(\:\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{s}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}}\times\:\text{S}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\) (Eq. 3). 1.2.5.2.1.3. Hepatic total antioxidant capacity: The total antioxidant capacity level in liver homogenate was determined by the endpoint colorimetric method of Koracevic D, Koracevic G, Djordjevic V, Andrejevic S and Cosic V [ 22 ] using a commercial assay kit (Biodiagnostic, Egypt) where the determination of the antioxidative capacity is performed by the reaction of antioxidants in the sample with a defined amount of exogenously provided H 2 O 2 . The antioxidants in the sample eliminate a certain amount of the provided H 2 O 2 . The residual H 2 O 2 is determined colorimetrically by an enzymatic reaction that involves the conversion of 3, 5, dichloro-2-hydroxybenzensulphonate to a colored product that can be measured at 505 nm. The assay was done according to the manufacturer’s guidelines. Calculation of results : Total Antioxidant concentration (mM/L) = A blank -A sample X 3.33 (Eq. 4). 1.2.6. Statistical analysis: Data are represented as mean ± standard deviation. Statistical analysis was carried out using the SPSS 16.0 program according to IBM’s statistics guide [ 23 ]. Statistical results were reported according to APA-7 guidelines. Two-way ANOVA was conducted to examine the effect of fermentation and extract concentration on antioxidant activities. Residual analysis was performed to test for the assumptions of the two-way ANOVA. Outliers were assessed by inspection of boxplots; normality was assessed using Shapiro-Wilk’s normality test for each cell of the design; and homogeneity of variances was assessed by Levene’s test. Differences were considered significant at a significance level of p < 0.05 [ 24 ]. All pairwise comparisons were run for each simple main effect with reported 95% confidence intervals using statistical significance, receiving a Bonferroni adjustment, and being accepted at a significance level of p < 0.025 level [ 25 ]. The EC 50 of extracts was calculated using regression analysis [ 26 ]. One-way ANOVA was conducted to detect statistically significant differences between different groups of the HCC animal model in all biochemical parameters. The test assumptions were checked out. For each group, outliers were assessed by inspection of boxplots; normality was assessed using Shapiro-Wilk’s normality test and homogeneity of variances was assessed by Levene’s test. For data sets with homogeneity of variances, one-way ANOVA/Tukey HSD post hoc analysis was used. Differences were considered significant at a significance level of p < 0.05 [ 24 ]. For data sets that violate homogeneity of variance, one-way Welch ANOVA/Games-Howell post hoc analysis was used. Differences were considered significant at a significance level of p < 0.05 [ 27 ]. . Results and Discussion Notably, UFOPME and FOPME were previously subjected to GC/MS analysis by Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [ 7 ] and HPLC analysis by Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [ 8 ]. Previously published GC/MS analysis revealed that fermentation of OP using K. marxianus led to the biosynthesis of carvacrol, thymol, eugenol, and caryophyllene oxide since they were detected only in the methanolic extract of FOP. In addition, the fatty acid esters (oleic acid methyl ester, oleic acid ethyl ester, and methyl palmitate) have been identified as major volatile compounds in both extracts [ 7 ]. On the other hand, published HPLC results confirmed the alteration of the phenolic profile of OP after fermentation by reinforcing its content of rutin, vanillin, cinnamic acid, quercetin, catechin, and syringic acid by 69.22%, 39.35%, and 31.44%, 22.78%, 7.06%, and 5.81%, respectively. However, fermentation decreased OP gallic, caffeic, and p -coumaric acid contents by 59.24%, 55.25%, and 53.96%, respectively [ 8 ]. Figure (1) demonstrates the effect of extract concentration of both UFOPME and FOPME on ABTS •+ scavenging activity. EC 50 was found to equal 4.58 and 1.17 mg/ml for UFOPME and FOPME, respectively. Both UFOPME and FOPME demonstrated concentration-dependent ABTS •+ scavenging activity (Fig. 1). Two-way ANOVA (no outliers, p > 0.05 and p = 0.206 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that the two independent factors, fermentation and extract concentration, significantly affect ABTS •+ scavenging activity with high effect size, where, for the first factor, fermentation, F (1, 20) = 592.246, p < 0.001, partial η 2 = 0.967, and for the second factor, extract concentration, F (4, 20) = 171.118, p < 0.001, partial η 2 = 0.972. Also, the interaction between the two independent factors significantly affects ABTS •+ scavenging activity ( F (4, 20) = 87.708, p < 0.001, partial η 2 = 0.946). The mean difference in ABTS •+ scavenging activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations except for concentration 10 mg/ml. Fermentation had a strong effect on OP’s scavenging activity, as revealed by the decreased EC 50 value after fermentation (EC 50 is 4.58 and 1.17 mg/ml for UFOPME and FOPME, respectively). An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in ABTS radical scavenging activity for UFOPME at different concentrations (F(4, 20) = 246.134, p < 0.001, partial η2 = 0.98), as for FOPME (F(4, 20) = 12.692, p < 0.001, partial η2 = 0.717). Pairwise comparisons demonstrated that for UFOPME, all concentrations were significantly different from each other, while for FOPME, there were no significant differences between concentrations 4, 6, 8, and 10 mg/ml. The results of UFOPME coincide with those of Tapia-Quirós P, Montenegro-Landívar MF, Vecino X, Alvarino T, Cortina JL, Saurina J, Granados M and Reig M [ 28 ] and Albahari P, Jug M, Radić K, Jurmanović S, Brnčić M, Brnčić SR and Vitali Čepo D [ 29 ]. Phenolic compounds can scavenge ABTS •+ through hydrogen atom donation and/or electron transfer mechanisms. It is worth noting increasing the number of hydroxyl groups in the aromatic ring does not necessarily lead to an increase of ABTS •+ scavenging activity of the compound. Structure-activity relationship of individual phenolic compounds reported before by Nenadis N, Wang L-F, Tsimidou M and Zhang H-Y [ 30 ] can explain the increased ABTS •+ scavenging activity of FOPME despite the decrease of caffeic and p -coumaric acid content after fermentation, as reported by Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [ 8 ]. This is because quercetin possesses radical scavenging activity nearly 67.3 times more than caffeic acid and 34.1 times more than p -coumaric acid, the increase of quercetin after fermentation probably compensated for the decrease of both compounds [ 30 ]. The enhanced ABTS •+ scavenging activity of FOPME can also be attributed to the microbial biosynthesis of the monoterpenoids carvacrol, thymol, and eugenol, since all these compounds can scavenge ABTS •+ radicals [ 31 , 32 ]. Reducing agents play a key role in oxidative stress reliving and macromolecule injury repair [ 33 ]. Reducing power assays can show that the tested compounds are electron donors, and thus they can reduce the oxidized intermediates [ 34 ]. The reducing power of UFOPME ranged from 5.88 ± 0.82 mg AscE /L to 49.21 ± 1.22 mg AscE /L; while the reducing power of FOPME ranged from 10.42 ± 0.24 mg AscE /L to 90.86 ± 0.12 mg AscE /L with a concentration-dependent increasing pattern of reducing power for both extracts and FOPME expressing higher activity than UFOPME at all studied concentrations (Fig. 2). Fermentation of OP with K. marxianus strongly enhanced its reducing power (Fig. 2) with a high effect size (partial η 2 = 0.992). Results align with Pasten A, Uribe E, Stucken K, Rodríguez A and Vega-Gálvez A [ 35 ] who reported a reducing power of OP methanolic extract equivalent to 101.75 ± 5.47 trolox equivalents/gds evaluated using ferric reducing antioxidant power assay. While Andrés AI, Petrón MJ, Adámez JD, López M and Timón ML [ 36 ] reported a reducing power of 0.46 mg AscE /L at a concentration of 1 mg/ml for the aqueous extract of OP. Two-way ANOVA test (no outliers, p > 0.05 and p = 0.092 for Shapiro-Wilk’s test and Levene’s test, respectively) demonstrated that both independent factors, fermentation ( F (1, 20) = 2415.823, p < 0.001, partial η 2 = 0.992) and extract concentration ( F (4, 20) = 1663.672, p < 0.001, partial η 2 = 0.997) significantly affect the reducing power with high effect sizes. Moreover, the interaction between the two independent factors had a significant effect on reducing power ( F (4, 20) = 171.665, p < 0.001, partial η 2 = 0.972). The mean difference in reducing power between UFOPME and FOPME was found to be statistically significant at all studied concentrations. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in reducing power for UFOPME at different concentrations ( F (4, 20) = 400.033, p < 0.001, partial η 2 = 0.988), as for FOPME ( F (4, 20) = 1435.303, p < 0.001, partial η 2 = 0.997). Pairwise comparisons demonstrated that for both extracts, all concentrations were significantly different from each other. The reducing power of phenolic compounds depends mainly on the degree of hydroxylation and conjugation. Generally, flavonoids with a catechol moiety and an enolic group in the C-ring show stronger reducing activity. Thus, the enhanced reducing power after fermentation can be attributed to the increase in quercetin and rutin concentrations rather than catechin. This is because favonols have higher reducing power than favanols due to the presence of the double bond between the 2-and 3-positions of the C-ring and also the double-bonded oxygen atom at the 4-position of the C-ring [ 37 , 38 ]. On the other hand, the reducing power of phenolic acids depends on the number and position of hydroxyl groups. Regarding phenolic acids and aldehydes, Mathew S, Abraham TE and Zakaria ZA [ 39 ] reported that they follow the order of gallic acid > caffeic acid > p -coumaric acid ≈ vanillin in their reducing power. The lower reducing power of vanillin than caffeic acid is attributed to the methoxyl group, which greatly reduces the reducing power. Also, quercetin is a more potent reducing compound than caffeic and gallic acids [ 37 ]. Thus, the decreased concentrations of phenolic acids (gallic, caffeic, and p -coumaric acids) after fermentation did not adversely affect the reducing power of FOPME, as it was compensated for by the increase in quercetin. On the other hand, the enhanced reducing power of FOPME can also be attributed to the exclusive presence of carvacrol, thymol, and eugenol since the three volatile compounds were reported to exhibit reducing activity in the potassium ferricyanide-reducing power assay [ 32 , 34 , 40 ]. Transition metals play a major role in the generation of ROS in living organisms. Chelating agents, such as phenolic compounds, may inactivate metal ions and potentially inhibit metal-dependent oxidative processes [ 41 ]. Fermentation of OP with K. marxianus positively affected its chelating activity (Fig. 3). This was approved by decreasing EC 50 value from 4.76 mg/ml for UFOPME to 1.765 mg/ml after fermentation. Two-way ANOVA (no outliers, p > 0.05 and p = 0.079 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that fermentation ( F (1, 20) = 1537.728, p < 0.001, partial η 2 = 0.987) as well as extract concentration ( F (4, 20) = 881.879, p < 0.001, partial η 2 = 0.994) significantly affected metal chelating activity with high effect sizes. Also, the interaction between the two factors had a significant effect on metal chelating activity ( F (4, 20) = 204.086, p < 0.001, partial η 2 = 0.976). The mean difference in metal chelating activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in metal chelating activity for UFOPME at different concentrations ( F (4, 20) = 966.406, p < 0.001, partial η 2 = 0.995), as for FOPME ( F (4, 20) = 119.560, p < 0.001, partial η 2 = 0.96). Pairwise comparisons demonstrated that all concentrations were significantly different from each other for both extracts. The value of EC 50 of UFOPME is lower than that reported by Moudache M, Silva F, Nerín C and Zaidi F [ 42 ] who reported a value of 8.41 ± 0.98 mg/ml for the methanolic extract of OP. On the other hand, Andrés AI, Petrón MJ, Adámez JD, López M and Timón ML [ 36 ] reported a very low EC 50 value (0.09 ± 0.03 mg/ml) for the aqueous extract of OP. The metal-chelating activity of phenolic compounds depends on the number of catechol groups, galloyl groups, or pyrone oxygens present in the compound [ 43 ]. Stability constants for polyphenol-iron interactions provide insight into their antioxidant behavior. The stability constant for different polyphenol-iron interactions was reported to follow the order of gallic acid < rutin ≈ quercetin < catechin [ 44 ]. Based on these outlined reports, the enhanced metal chelating activity after fermentation can be attributed to the increased concentration of catechin (one metal-binding site), rutin (has two metal-binding sites), and quercetin (has three metal-binding sites) [ 44 , 45 ] rather than the increase in syringic content since syringic acid does not complex with metal ions as its structure lacks catechol and galloyl groups [ 41 ]. The decreased concentrations of p -coumaric (has no metal binding site), caffeic (one metal binding site), and gallic (two overlapped metal binding sites) acids after fermentation did not negatively affect the iron chelating activity of FOPME. This is probably because phenolic acids seem to play a weaker role in iron binding than other polyphenolic compounds since they either do not bear catechol or galloyl moieties; or bear small numbers of metal chelating groups [ 41 ]. Hydrogen peroxide (H 2 O 2 ) scavenging activity results demonstrated that fermentation greatly enhanced H 2 O 2 scavenging activity of OP since EC 50 was reduced from 4.89 mg/ml for UFOPME to 2.6 mg/ml for FOPME (Fig. 4). Two-way ANOVA (no outliers, p > 0.05 and p = 0.642 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that fermentation ( F (1, 20) = 4988.684, p < 0.001, partial η 2 = 0.996) as well as extract concentration ( F (4, 20) = 8244.717, p < 0.001, partial η 2 = 0.999) significantly affected H 2 O 2 scavenging activity with high effect size. Also, the interaction between the two factors had a significant effect on H 2 O 2 scavenging activity ( F (4, 20) = 425.839, p < 0.001, partial η 2 = 0.988). The mean difference of H 2 O 2 scavenging activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations except for concentration 10 mg/ml. An analysis of simple main effects for extract concentration was performed. There was a statistically significant difference in H 2 O 2 scavenging activity for UFOPME at different concentrations ( F (4, 20) = 5080.027, p < 0.001, partial η 2 = 0.999), as for FOPME ( F (4, 20) = 3590.529, p < 0.001, partial η 2 = 0.999). Pairwise comparisons demonstrated that for UFOPME, all concentrations were significantly different from each other, while for FOPME, there were no significant differences between concentrations 6, 8, and 10 mg/ml. The finding that OP extract can scavenge H 2 O 2 in a concentration-dependent manner align with that reported by Morsi MKE-S, Galal SM, Alabdulla OJCJoFS and Technology [ 46 ]. Previously published structure-activity relationship (SAR) studies confirmed that the H 2 O 2 scavenging activity of phenolic acids depends mainly on the available number of hydroxyl groups, and the presence of these groups in ortho position to each other enhances their activity more than para positions [ 47 ]. The enhanced H 2 O 2 scavenging activity after fermentation can be attributed to the increased concentration of quercetin. These findings align with previously reported SAR results showing quercetin is a stronger H 2 O 2 scavenger than gallic acid. Both compounds also show a concentration-dependent response [ 13 , 48 ]. Despite being a weak oxidant, superoxide anion can combine with molecules such as nitric oxide to generate powerful oxidative species that are harmful to human health. Some antioxidants can inhibit the chain propagation reaction caused by superoxide radicals through hydrogen donation [ 49 ]. Results of the superoxide anion scavenging assay demonstrated that fermentation of OP with K. marxianus demonstrated an adverse, moderate effect on superoxide anion scavenging activity at all studied concentrations (Fig. 5). EC 50 was found to equal 7.368 mg/ml for UFOPME and 8 mg/ml for FOPME. Two-way ANOVA (no outliers, p > 0.05 and p = 0.096 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that fermentation ( F (1, 20) = 63.498, p < 0.001, partial η 2 = 0.760), as well as extract concentration (F(4, 20) = 1522.577, p < 0.001, partial η2 = 0.997), significantly affected superoxide anion radical scavenging activity with high effect sizes. Also, the interaction between the two factors had a significant effect on superoxide anion radical scavenging activity ( F (4, 20) = 7.598, p = 0.001, partial η 2 = 0.603). The mean difference in superoxide anion radical scavenging activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations except for concentration 2 mg/ml. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in superoxide anion radical scavenging activity for UFOPME at different concentrations ( F (4, 20) = 864.784, p < 0.001, partial η 2 = 0.994), as for FOPME ( F (4, 20) = 665.391, p < 0.001, partial η 2 = 0.993). Pairwise comparisons demonstrated that for both extracts, all concentrations were significantly different from each other. The results of the superoxide anion scavenging assay align with those reported by Squillaci G, Marchetti A, Petillo O, Bosetti M, La Cara F, Peluso G and Morana A [ 49 ] who declared that the superoxide anion scavenging activity of OP can range between 23.33 (± 0.01) and 75.72 (± 1.78)% according to the extracting solvent and extraction temperature. Previously reported SAR studies can explain the slightly increased EC 50 (decreased activity) after fermentation. It was reported that phenolic compounds with more than one hydroxyl group in their aromatic ring have stronger inhibitory potency against superoxide anion than monohydroxy substituents due to increased hydrogen donating ability, thus, gallic and caffeic acids are stronger superoxide anion scavengers than p -coumaric acid. It was also stated that the phenolic compounds follow the order of gallic acid > caffeic acid > vanillin, then p -coumaric acid in superoxide anion scavenging activity [ 39 ]. Vanillin was also reported to have weak superoxide anion scavenging activity by Bezerra DP, Soares AKN and de Sousa DP [ 50 ]. On the other hand, the flavonoid quercetin has no superoxide anion scavenging activity, unlike the phenolic acid, and gallic acid as Abdullah MZ, Mohd Ali J, Abolmaesoomi M, Abdul-Rahman PS and Hashim OH [ 51 ] reported. Also, regarding the flavonoid, rutin, Kong KW, Mat-Junit S, Aminudin N, Ismail A and Abdul-Aziz A [ 52 ] reported that its superoxide scavenging activity is surpassed by gallic acid. Meanwhile, catechin was reported to possess a prooxidant activity in superoxide anion-generating systems that leads to induced radical formation rather than scavenging [ 53 ]. In the light of these SAR studies, the slightly increased EC 50 after fermentation can be explained. This adverse effect can be attributed to the decreased concentration of phenolic acids (gallic, caffeic, and p -coumaric acids) since quercetin has no activity toward superoxide anions, and rutin, as well as vanillin, are less active than phenolic acids. Moreover, the adverse effect can be attributed not only to decreased phenolic acids concentration but also to increased catechin concentration after fermentation which induces superoxide anion radical generation rather than scavenging. Primary products of lipid peroxidation, derived from oxidative stress, can contribute to cancer by forming harmful adducts with macromolecules including proteins, lipids, and DNA [ 54 ]. Phenolic compounds can inhibit lipid peroxidation by several mechanisms, including radical scavenging (both radicals initiating and produced by the lipid peroxidation process), metal chelation, and interaction with biological lipid membranes [ 55 ]. Fermentation of OP using K. marxianus strongly increased its protective activity against lipid peroxidation (Fig. 6). EC 50 was found to be 4.76 mg/ml for UFOPME and 1.76 mg/ml for FOPME. Two-way ANOVA (no outliers, p > 0.05 and p = 0.230 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that both fermentation ( F (1, 20) = 703.819, p < 0.001, partial η 2 = 0.972) and extract concentration ( F (4, 20) = 19803.747, p < 0.001, partial η 2 = 1.000) can significantly affect lipid peroxidation inhibition activity. Also, the interaction between the two factors had a significant effect on lipid peroxidation inhibition activity ( F (4, 20) = 40.391962, p < 0.001, partial η 2 = 0.89). The mean difference in lipid peroxidation inhibition activity between UFOPME and FOPME was found statistically significant at all studied concentrations. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in lipid peroxidation inhibition activity for UFOPME at different concentrations ( F (4, 20) = 10579.694, p < 0.001, partial η 2 = 1.000), as for FOPME ( F (4, 20) = 9264.445, p < 0.001, partial η 2 = 0.999). Pairwise comparisons demonstrated that for FOPME, all concentrations were significantly different from each other, while for UFOPME, there was no significant difference between concentrations 6 and 10 mg/ml. The lipid peroxidation inhibiting activity of phenolic compounds depends on the combination of their polarity and portioning in mediums, the degree of hydroxylation (or radical scavenging groups), and the number of metal chelating sites. Therefore, structure alone cannot predict the lipid peroxidation inhibition behavior [ 56 ]. Quercetin is more efficient than catechin in lipid peroxidation inhibition since its apolarity (lipophilicity) is higher and it has a higher number of metal chelating sites and hydroxyl groups. On the other hand, despite the fact that p -coumaric acid is apolar more than caffeic acid, its protecting activity is lower. This is attributed to the increased number of hydroxyl groups in caffeic acid. For the same reason (increased hydroxyl groups), catechin is superior to caffeic acid regarding its protecting activity, although the former is a mono-chelator and weaker apolar. Therefore, the combination of the three ruling factors makes the order of compounds in lipid-protecting activity as quercetin > catechin > caffeic acid > p -coumaric acid [ 56 ]. This explanation align with the finding of increased lipid-protecting activity in FOPME. Regarding other phytochemical classes, the enhanced lipid protective effect of FOPME can also be attributed to the exclusive presence of the monoterpenoids carvacrol and thymol [ 40 ], the phenylpropanoid, eugenol [ 32 ], and the terpenoid caryophyllene oxide [ 57 ]. RBCs are a good model for studying antioxidant effects since they are susceptible to endogenous and exogenous oxidative damage because of their specific role as oxygen carriers. Oxidative stress negatively affects RBCs, as they are anucleated cells with poor repair and biosynthetic mechanisms [ 58 ]. Oxidation of the RBC membrane’s lipids and proteins by oxidants and free radicals may lead to loss of membrane stability and subsequent cell hemolysis [ 59 ]. Phenolic compounds can protect RBCs against radical-induced hemolysis [ 58 , 59 ]. Both extracts demonstrated good antihemolytic activity, with EC 50 values of 3.27 mg/ml and 3.61 mg/ml for UFOPME and FOPME, respectively (Fig. 7). The difference in EC 50 values was not statistically significant. Two-way ANOVA (no outliers, p > 0.05 and p = 0.057 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that the two independent factors, fermentation and extract concentration, significantly affect RBCs protective activity, where, for the first factor, fermentation, F (1, 20) = 28.543, p < 0.001, partial η 2 = 0.588, and for the second factor, extract concentration, F (4, 20) = 47.988, p < 0.001, partial η 2 = 0.906. Also, the interaction between the two independent factors significantly affects RBCs protective activity ( F (4, 20) = 5.062, p = 0.006, partial η 2 = 0.503). The mean difference of RBCs protecting activity between UFOPME and FOPME was found to be statistically significant at concentrations 6, 8, and 10 mg/ml only. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in RBCs protecting activity for UFOPME at different concentrations ( F (4, 20) = 30.866, p < 0.001, partial η 2 = 0.861), as for FOPME ( F (4, 20) = 22.184, p < 0.001, partial η 2 = 0.816). Pairwise comparisons demonstrated that for UFOPME, there were no significant differences between concentrations of 4 and 8 mg/ml; 4 and 10 mg/ml, 6 and 8 mg/ml, and 8 and 10 mg/ml. While for FOPME, there were no significant differences between concentrations 2 and 4 mg/ml; 2 and 8 mg/ml; 2 and 10 mg/ml; and 4 and 8 mg/ml. Also, two-way ANOVA results demonstrated that the concentration of OP extracts is the ruling factor in their antihemolytic activities. However, the decreased protective activity of both extracts at high concentrations was not significantly different from low concentrations, as revealed by multiple pairwise comparisons. The EC 50 of UFOPME is higher than that reported by Madureira J, Dias MI, Pinela J, Calhelha RC, Barros L, Santos-Buelga C, Margaça FMA, Ferreira ICFR and Cabo Verde S [ 60 ] who reported an EC 50 for OP extract of 32.2 ± 0.6 µg/ml. the difference may be due to the different solvents used for the extraction of phenolic compounds. Both quercetin and rutin were reported to have dose-dependent antihemolytic activity, with higher protective activity associated with rutin [ 59 ]. Moreover, vanillin was reported to have antihemolytic activity [ 61 ]. Also, rutin, catechin, quercetin, caffeic acid, and gallic acid have the same activity in descending order, which contradicts the finding that UFOPME has higher antihemolytic activity than FOPME, while the latter has higher contents of rutin, catechin, and quercetin [ 62 ]. The median lethal dose (LD 50 ) required to kill 50% of both UFOPME-administered rats and FOPME-administered rats was found to equal 2833.3 mg/kg b.w. (Table 1 and Table 2 for UFOPME and FOPME, respectively). Consequently, animals were administered a safe dose of 283.3 mg/kg b.w. (which represents 1/10 of LD 50 ) throughout the experiment. Table (1): Calculation of median lethal dose (LD 50 ) for unfermented olive pomace methanolic extract (UFOPME) Dose (mg/ kg b.w.) No. of animals No. of dead animals Z d Z × d 250 6 0 0 250 0 500 6 0 0 250 0 1000 6 0 0 500 0 2000 6 2 1 1000 1000 4000 6 4 3 2000 6000 Table (2): Calculation of median lethal dose (LD 50 ) for fermented olive pomace methanolic extract (FOPME) Dose (mg/ kg b.w.) No. of animals No. of dead animals Z d Z × d 250 6 0 0 250 0 500 6 0 0 250 0 1000 6 0 0 500 0 2000 6 2 1 1000 1000 4000 6 4 3 2000 6000 Levels of nitric oxide (NO) and malondialdehyde (MDA) in rats’ liver tissue were assessed as markers for oxidative stress that occurs in association with DENA/CCl 4 – induced HCC, and results are reported in Figure (8). For MDA, one-way ANOVA test (no outliers, p > 0.05 and p = 0.545 for Shapiro-Wilk’s test and Levene’s test, respectively) demonstrated that MDA levels were statistically significantly different between different groups, F (7, 25) = 51.019, p < 0.001. Tukey HSD post hoc analysis revealed that the increase in tissue MDA level from the normal group to the HCC group was statistically significant ( p < 0.001), Also, the decreases from the HCC group to the pre-UF group, pre-F group, post-UF group, and post-F group were statistically significant ( p < 0.001). On the other hand, the decrease from the normal group to the UF control group, F control group, and post-F group, as well as the increase from the normal group to the pre-UF group, pre-F group, and post-UF group, did not demonstrate statistical significance ( p > 0.05). For NO, one-way ANOVA test (no outliers, p > 0.05 and p = 0.618 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that NO level was statistically significantly different between different groups, F (7, 25) = 25.022, p < 0.001. Tukey HSD post hoc analysis revealed that the increase in tissue NO level from the normal group to the HCC group was statistically significant ( p < 0.001), Also, the decreases from the HCC group to the pre-UF group, pre-F group, post-UF group, and post-F group were statistically significant ( p ≤ 0.001). On the other hand, the decrease from the normal group to the UF control group, F control group, post-UF group, and post-F group, as well as the increase from the normal group to the pre-F group and pre-UF group, did not demonstrate statistical significance ( p > 0.05). Results demonstrate a significant increase ( p < 0.001) of NO and MDA (the end product of lipid peroxidation) levels in liver tissue after DENA/CCl 4 administration compared to the normal group, indicating the occurrence of oxidative stress in the liver as a result of increased both reactive species production and lipid peroxidation. Similar results were reported by Hassan HA, Ghareb NE and Azhari GF [ 63 ], Shawki AK, El-Desouky MA, Fouad SM, Ahmed AFM, Aboulhoda BE and Ahmed WA [ 64 ] and Abdel-Hamid NM, Hassan MK, Ahmed AAM, Abd Allah SG and Anber NH [ 65 ] for DENA/CCl 4 -induced HCC in rats. On the other hand, the administration of UFOPME or FOPME alone did not have any marked impact on NO or MDA levels in liver tissue, compared to the normal group, ( p > 0.05) reflecting no deleterious effects for both extracts on the oxidative homeostasis in liver tissue. Oral gavage of UFOPME and FOPME either pre- or post-DENA/CCl 4 administration restored NO and MDA levels to normal, as revealed by insignificant differences with the normal group ( p > 0.05) suggesting potential protective and therapeutic effects, respectively, against DENA/CCl 4 -induced oxidative stress in liver tissue. This can be partially attributed to the enhanced antioxidant defensive system as measured by total antioxidant capacity (Fig. 9). Total antioxidant capacity was evaluated in liver tissue and considered as a collective representation of the antioxidant defense system (Fig. 9). One-way Welch ANOVA test (no outliers, p > 0.05 and p = 0.018 for Shapiro-Wilk’s test and Levene’s test, respectively) results demonstrated that total antioxidant capacity was statistically significantly different between different model’s groups, Welch's F (7, 11.118) = 11.588, p < 0.001). Games-Howell post hoc analysis revealed that the decrease in tissue total antioxidant capacity from the normal group to the HCC group was statistically significant ( p = 0.027). Also, the increase from the HCC group to the pre-UF group, pre-F group, post-UF group, and post-F group was statistically significant ( p = 0.049, 0.024, 0.029, and 0.023, respectively). On the other hand, the difference between the normal group and UF control, F control, pre-UF, pre-F, post-UF, and post-F was not statistically significant ( p > 0.05). Results demonstrate a significant decrease in total antioxidant capacity in liver tissue after DENA/CCl 4 administration ( p = 0.027) compared to the normal group, indicating an impaired liver antioxidant defense system. Decreased total antioxidant capacity of liver tissue after DENA/CCl 4 induction of HCC was also reported by Hassan HA, Ghareb NE and Azhari GF [ 63 ]. On the other hand, administration of UFOPME or FOPME alone resulted in enhanced total antioxidant capacity in liver tissue. This enhancement was not statistically different from the normal group ( p > 0.05) indicating that the enhanced total antioxidant capacity remains in the normal range. Oral gavage of UFOPME and FOPME either pre- or post-DENA/CCl 4 administration restored liver total antioxidant capacity to normal, as revealed by the insignificant difference with the normal group ( p > 0.05) suggesting potential protective and therapeutic effects, respectively, against DENA/CCl 4 -induced oxidative stress in liver tissue. The insignificant differences between the four treated groups and the normal group make it impossible to choose between UFOPME and FOPME or between pre- and post-treatment. So, it can be concluded that at the studied concentration, both OP extracts are good exogenous antioxidant agents that exert comparable protective and therapeutic activities, which can relieve DENA/CCl 4 -induced oxidative stress in liver tissue, restoring the oxidative state to normal. The decreased oxidative stress markers after administration of different OP extracts may be attributed to the presence of quercetin, as it was experimentally proven to lower NO and MDA levels in liver homogenate when administered before or after HCC induction by thioacetamide in rats [ 66 ]. The same observation was augmented by Seufi AM, Ibrahim SS, Elmaghraby TK and Hafez EE [ 67 ] and Vasquez-Garzon VR, Macias-Perez JR, Jimenez-Garcia MN, Villegas V, Fattel-Fazenta S and Villa-Trevino S [ 68 ], who reported the ability of quercetin to lower MDA in liver homogenate in HCC in rats induced by DENA and DENA/2-acetylaminofluorene, respectively. Also, rutin was proven to reduce MDA levels in the liver homogenate of DENA-induced HCC in rats [ 69 ]. Independently of phenolic compounds, the carvacrol present in FOPME was reported to lower MDA levels and improve the overall liver antioxidant state in DENA-induced HCC in rats [ 70 ], and the same effect was reported for thymol in acetaminophen-induced toxicity in HepG-2 cell lines [ 71 ]. Also, thymol was reported to lower MDA and enhance liver antioxidant status in doxorubicin-induced hepatotoxicity in rats [ 72 ]. Conclusion SSF of olive oil pomace by the Generally Regarded As Safe Kluyveromyces marxianus NRRL Y-8281 yeast strain is a precious eco-friendly and applicable technique that not only allows sustainable olive oil production but also valorizes the environmental pollutant olive pomace into pomace enriched with potent in vitro and in vivo antioxidant compounds that can find their applications in different industrial sectors, including food, chemical, pharmaceutical, and medical sectors. Moreover, the present study can contribute to attaining the 2030 global sustainability goals of the United Nations by considering environment preservation, competing climate change, and industrial and agricultural sustainability. Declarations CRediT authorship contribution statement: A.E. , M.M., S.A. , AT. and D.M. contributed equally to the manuscript. Declaration of Competing Interest : The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Consent for publication: Not applicable. Availability of data and materials: Not applicable. Acknowledgements: The authors thank the National Research Centre funding program (13010603), for supporting this research. Also, the authors want to thank The Science, Technology & Innovation Funding Authority (STDF). References Gulcin İ: Antioxidants: a comprehensive review. Arch Toxicol 2025, 99: 1893-1997. Haider K, Haider MR, Neha K, Yar MS: Free radical scavengers: An overview on heterocyclic advances and medicinal prospects. Eur J Med Chem 2020, 204: 1-16. 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Karakaya S, Yilmaz SV, Özdemir Ö, Koca M, Pınar NM, Demirci B, Yıldırım K, Sytar O, Turkez H, Baser KHC: A caryophyllene oxide and other potential anticholinesterase and anticancer agent in Salvia verticillata subsp. amasiaca (Freyn & Bornm.) Bornm. (Lamiaceae). J Essent Oil Res 2020, 32: 512-525. Paiva-Martins F, Gonçalves P, Borges JE, Przybylska D, Ibba F, Fernandes J, Santos-Silva A: Effects of the olive oil phenol metabolite 3,4-DHPEA-EDAH2 on human erythrocyte oxidative damage. Food Funct 2015, 6: 2350-2356. Asgary S, Naderi G, Askari N: Protective effect of flavonoids against red blood cell hemolysis by free radicals. Exp Clin Cardiol 2005, 10: 88-90. Madureira J, Dias MI, Pinela J, Calhelha RC, Barros L, Santos-Buelga C, Margaça FMA, Ferreira ICFR, Cabo Verde S: The use of gamma radiation for extractability improvement of bioactive compounds in olive oil wastes. Sci Total Environ 2020, 727: 138706. Tai A, Sawano T, Yazama F, Ito H: Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays. Biochim Biophys Acta Gen Subj 2011, 1810: 170-177. Tabart J, Kevers C, Pincemail J, Defraigne J-O, Dommes J: Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chem 2009, 113: 1226-1233. Hassan HA, Ghareb NE, Azhari GF: Antioxidant activity and free radical-scavenging of cape gooseberry ( Physalis peruviana L.) in hepatocellular carcinoma rats model. Hepatoma Res 2017, 3: 27-33. Shawki AK, El-Desouky MA, Fouad SM, Ahmed AFM, Aboulhoda BE, Ahmed WA: Camel (Camelus Dromedarius) Milk Antibodies Ameliorated Diethylnitrosamine-Induced Hepatocellular Carcinoma in Wistar Rats. Egypt J Chem 2021, 64: 4611-4623. Abdel-Hamid NM, Hassan MK, Ahmed AAM, Abd Allah SG, Anber NH: Liver Proliferating Cell Nuclear Antigen, BAX/Bcl-2 Ratio, Collagen, and Polysaccharide Accumulation as Diagnostic Tools in Experimental Hepatocellular Carcinoma. J Contemp Med Sci 2022, 8: 59-64. Salama YA, El-karef A, El Gayyar AM, Abdel-Rahman N: Beyond its antioxidant properties: Quercetin targets multiple signalling pathways in hepatocellular carcinoma in rats. Life Sci 2019, 236: 1-12. Seufi AM, Ibrahim SS, Elmaghraby TK, Hafez EE: Preventive effect of the flavonoid, quercetin, on hepatic cancer in rats via oxidant/antioxidant activity: molecular and histological evidences. J Exp Clin Cancer Res 2009, 28: 1-8. Vasquez-Garzon VR, Macias-Perez JR, Jimenez-Garcia MN, Villegas V, Fattel-Fazenta S, Villa-Trevino S: The Chemopreventive Capacity of Quercetin to Induce Programmed Cell Death in Hepatocarcinogenesis. Toxicol Pathol 2013, 41: 857-865. Pandey P, Rahman M, Bhatt PC, Beg S, Paul B, Hafeez A, Al-Abbasi FA, Nadeem MS, Baothman O, Anwar F, Kumar V: Implication of nano-antioxidant therapy for treatment of hepatocellular carcinoma using PLGA nanoparticles of rutin. Nanomedicine 2018, 13: 849-870. Subramaniyan J, Kumar M, Asokkumar S, Subramanian R, Dhas K, Sattu K, Divya G, Devaki T: Potential preventive effect of CAR against diethylnitrosamine- induced hepatocellular carcinoma in rats. Mol Cell Biochem 2011, 360: 51-60. Palabiyik SS, Karakus E, Halici Z, Cadirci E, Bayir Y, Ayaz G, Cinar I: The protective effects of carvacrol and thymol against paracetamol-induced toxicity on human hepatocellular carcinoma cell lines (HepG2). Hum Exp Toxicol 2016, 35: 1252-1263. Ahmed OM, Galaly SR, Mostafa M-AMA, Eed EM, Ali TM, Fahmy AM, Zaky MY: Thyme Oil and Thymol Counter Doxorubicin-Induced Hepatotoxicity via Modulation of Inflammation, Apoptosis, and Oxidative Stress. Oxid Med Cell Longev 2022, 2022: 6702773. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7255027","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502126565,"identity":"33a231ea-e3f7-48f7-b649-9bc51f34bf7e","order_by":0,"name":"Abeer E. Mahmoud","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACCSjNz8DARqIWyQaStRgcIFYL/+wGxoc//thFG99IfvbgQwWDPL/YAQKW3DnAbMzblpy77UaaueGMMwyGM2cnELDmRgKbNGMDM1BLgpk0bxtDgsFtAlrkbySw//zxpz5384z0b8RpMQDawsDDdjh3g0QOkbYYAv0CVHk8d8aZN2WSM85IEPaL3O0Gxo8//lTn9renb5P4UGEjzy9NQAswmD9AaAGwSgk8KjG1HiBF9SgYBaNgFIwkAAB6ykLX/h/L3QAAAABJRU5ErkJggg==","orcid":"","institution":"National Research Centre","correspondingAuthor":true,"prefix":"","firstName":"Abeer","middleName":"E.","lastName":"Mahmoud","suffix":""},{"id":502126566,"identity":"d02dde4e-1148-46b7-b40e-c3fd637fb79a","order_by":1,"name":"Mamdouh M. Ali","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Mamdouh","middleName":"M.","lastName":"Ali","suffix":""},{"id":502126567,"identity":"2b1a632e-ae0b-42bf-ac9f-ef0c8f4468e9","order_by":2,"name":"Shadia A. Fathy","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Shadia","middleName":"A.","lastName":"Fathy","suffix":""},{"id":502126568,"identity":"e2ac705e-713a-4bd0-9a52-fb35d47be920","order_by":3,"name":"Amira T. Mohammed","email":"","orcid":"","institution":"National Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Amira","middleName":"T.","lastName":"Mohammed","suffix":""},{"id":502126569,"identity":"64ef2792-b669-46c9-93b5-6df2e8828fd5","order_by":4,"name":"Doaa M. Ibrahim","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Doaa","middleName":"M.","lastName":"Ibrahim","suffix":""}],"badges":[],"createdAt":"2025-07-30 16:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7255027/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7255027/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89383013,"identity":"d7860575-3f10-43b9-9b1f-257e066b02db","added_by":"auto","created_at":"2025-08-19 12:15:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27061,"visible":true,"origin":"","legend":"\u003cp\u003eABTS radical scavenging activity of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003e- Means bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/ebe6e79da143e3816922f132.jpg"},{"id":89383787,"identity":"22ed0553-703f-40f1-aeb1-a2ca992f9186","added_by":"auto","created_at":"2025-08-19 12:23:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25885,"visible":true,"origin":"","legend":"\u003cp\u003eReducing power of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003e- Means bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/f2bbea2a02c60df4d87f4267.jpg"},{"id":89383025,"identity":"d0032a03-c6a6-4b25-a38f-7bf254f43441","added_by":"auto","created_at":"2025-08-19 12:15:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24427,"visible":true,"origin":"","legend":"\u003cp\u003eMetal chelating activity of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003e- Means bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/9c0b772abfa216bac062af44.jpg"},{"id":89383023,"identity":"db4a08f8-bf33-4ddf-ba04-461ec32f359d","added_by":"auto","created_at":"2025-08-19 12:15:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27154,"visible":true,"origin":"","legend":"\u003cp\u003eHydrogen peroxide scavenging activity of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003e- Means bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/944181bbc8bc32ab51dca623.jpg"},{"id":89383019,"identity":"d29b0cf5-367d-4b44-8ea0-f11b1e92c292","added_by":"auto","created_at":"2025-08-19 12:15:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":23944,"visible":true,"origin":"","legend":"\u003cp\u003eSuperoxide anion radical scavenging activity of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003e- Means bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/90593056a30cdb4c8ef230e0.jpg"},{"id":89383022,"identity":"eef0dee6-6a8e-4ead-aa30-320523f70795","added_by":"auto","created_at":"2025-08-19 12:15:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24637,"visible":true,"origin":"","legend":"\u003cp\u003eLipid peroxidation inhibition activity of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003e- Means bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/0289d7a251dc4d9b40b59ce2.jpg"},{"id":89383789,"identity":"7403b0f9-90b5-4447-a932-51534463027b","added_by":"auto","created_at":"2025-08-19 12:23:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":22752,"visible":true,"origin":"","legend":"\u003cp\u003eRBCs protecting activity of different concentrations of unfermented (UFOPME) and fermented (FOPME) olive pomace methanolic extracts\u003c/p\u003e\n\u003cp\u003e- Data are represented as mean ± SD of three different batches.\u003c/p\u003e\n\u003cp\u003eMeans bearing different letters are significantly different from each other as indicated by two-way ANOVA/Bonferroni-adjusted pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 and 0.025, respectively).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/d5283ce07a5bc093975a0d3c.jpg"},{"id":89383015,"identity":"95db42f0-6752-4f77-9703-0aa61aea95d7","added_by":"auto","created_at":"2025-08-19 12:15:23","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":39723,"visible":true,"origin":"","legend":"\u003cp\u003eHepatic levels of oxidative stress markers of different groups\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp;Data are presented as mean±standard deviation.\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp;Differences between groups were analyzed using one-way ANOVA/Tukey HSD post hoc tests.\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp;Means bearing \u003csup\u003ea\u003c/sup\u003e and \u003csup\u003eb\u003c/sup\u003e superscripts are significantly different from normal group and HCC group, respectively, at significance level \u003cem\u003ep\u0026lt;\u003c/em\u003e0.05.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/c55189c30e87e4b3d0b3d2c1.jpg"},{"id":91775176,"identity":"11c17b57-cc98-4457-93c6-dc0c52150f01","added_by":"auto","created_at":"2025-09-20 18:16:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4536744,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7255027/v1/438aa23e-2515-44ca-a212-14f73795264a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced in vitro and in vivo antioxidant activities of olive pomace by solid- state fermentation using the yeast Kluyveromyces marxianus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNumerous pathologies associate to increased production of free radicals within biological systems which leads to oxidative stress. An antioxidant is a substance that can scavenge reactive species, inhibit their production, or positively regulate antioxidant defense systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Antioxidants can provide an electron to a free radical-containing compound without undergoing destabilization, thus discontinuing the free radical chain reaction. Naturally obtained free radical scavengers are effective, safe, and potent; thus, they can serve as lead compounds for the development of novel drugs. Owing to their chemical structure, phenolic compounds are very efficient and promising free radical scavengers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOlive pomace (OP) represents a particularly challenging oil processing waste that is produced in huge amounts in Mediterranean countries. Sustainable olive oil production requires continuous pomace disposal, which is not an easy mission regarding the toxic physico-chemical characteristics of OP. Nevertheless, merely 2% of total phenolics found in olive fruits transfer to the extracted olive oil, while the other 98% retain in olive oil by-products. About 45% of the total phenolic content of olive fruit retain in OP [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Regarding its high polyphenolic content, OP can be considered a cheap and renewable source of pharmaceuticals rather than an environmentally polluting agro-industrial waste [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The approval of phenolic-rich extracts of OP as Generally Recognized As Safe (GRAS) by the US Food and Drug Administration (FDA) opened new perspectives for their usage in food, feed, and pharmaceutical sectors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Several companies have started to commercialize olive phenolic extracts formulated in different ways that offer numerous health benefits without adverse health effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious work of Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] suggested that solid-state fermentation (SSF) of OP using the GRAS yeast, \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e represents a novel technique for OP disposal that valorizes the pomace into a promising value-added product with potent anticancer activity studied \u003cem\u003ein vitro\u003c/em\u003e. Also, Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] reported that the valorized OP had an enhanced phenolic profile with increased rutin, vanillin, cinnamic acid, quercetin, catechin, and syringic acid contents. The present work aimed to study the impact of SSF of OP using \u003cem\u003eK. marxianus\u003c/em\u003e on pomace antioxidant activity. The antioxidant activities of unfermented (UFOPME) and fermented (FOPME) OP methanolic extracts were compared both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e against oxidative stress associated with DENA/CCl\u003csub\u003e4\u003c/sub\u003e-induced HCC in rats. Moreover, the enhanced antioxidant activity of pomace after fermentation was explained according to the altered phenolic and phytochemical profiles reported by Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] to get a deeper and more comprehensive view of the effect of SSF of OP with the yeast \u003cem\u003eK. marxianus\u003c/em\u003e. This study shows a significant academic advance by demonstrating both the protective and therapeutic effects of FOP against oxidative stress linked to diethylnitrosamine (DENA)-induced hepatocellular carcinoma (HCC). This application has been notably underexplored. It uses \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e NRRL Y-8281, a rarely studied yeast strain recognized as GRAS, in SSF to greatly improve the antioxidant potential of OP. Moving beyond earlier compositional analyses, this research includes: (i) thorough \u003cem\u003ein vitro\u003c/em\u003e tests such as radical scavenging, metal chelation, and lipid peroxidation inhibition; (ii) systematic \u003cem\u003ein vivo\u003c/em\u003e validation using a rat model of HCC; and (iii) direct comparison of preventive versus therapeutic effectiveness in reducing oxidative stress. By evaluating both unfermented and fermented OP extracts, the study confirms significant biochemical improvement and highlights SSF as an important method for sustainable waste use. These findings offer meaningful originality by connecting eco-biotechnology with clinical potential, turning agricultural byproducts into valuable antioxidant agents.\u003c/p\u003e\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1. Materials:\u003c/h2\u003e\u003cdiv id=\"Sec3\" class=\"Section3\"\u003e\u003ch2\u003e1.1.1. Olive oil pomace (OP):\u003c/h2\u003e\u003cp\u003eOP was obtained during its harvesting season by a local olive-pressing factory (three-phase decanter system), located in Al-Arish, North Sinai, Sinai Peninsula, Egypt. It was stored at-20℃ until used.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e1.1.2. Experimental organisms:\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section4\"\u003e\u003ch2\u003e1.1.2.1. Experimental microorganism:\u003c/h2\u003e\u003cp\u003e\u003cem\u003eKluyveromyces marxianus\u003c/em\u003e NRRL Y-8281 yeast strain used in the present study was purchased from the Agricultural Research Service (Peoria, Illinois, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section4\"\u003e\u003ch2\u003e1.1.2.2. Experimental animals:\u003c/h2\u003e\u003cp\u003eThe hepatocellular carcinoma (HCC) animal model study was conducted on 80 adult male albino western rats in addition to another 60 rats used for the determination of the LD\u003csub\u003e50\u003c/sub\u003e of extracts (average body weight of 130\u0026thinsp;\u0026plusmn;\u0026thinsp;20g). Rats were housed in the Animal House Colony of the National Research Centre, Dokki, Giza, Egypt. The animal house was ventilated with a 12-hour light/dark cycle at an ambient temperature of 25\u0026deg;C-30\u0026deg;C throughout the experimental period, with free access to tap water and a standard rodent chow. Animals were allowed 7 days for acclimatization before the initiation of the experiment.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section5\"\u003e\u003ch2\u003e1.1.2.2.1. Ethics approval:\u003c/h2\u003e\u003cp\u003e All procedures of animal handling and sacrificing complied with the standard guidelines of the institutional ethics committee for animal care at the National Research Centre, Egypt (ethic No. 18/036).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e1.1.3. Chemicals:\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e1, 10-Phenanthroline (Panreac, Espain).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eABTS (Sigma-Aldrich, USA).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eAscorbic acid (Sigma-Aldrich, USA).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCarbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) (Sigma-Aldrich, USA).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDiethylnitrosamine (DENA) (Sigma-Aldrich, Japan).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eFerrozine (Sigma-Aldrich, USA).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eLinoleic acid (Fluka, Germany).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMethanol and ethanol (Piochem, Egypt).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThiobarbituric acid (Merck, Germany).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTrichloroacetic acid (SRL, India).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAll other chemicals and reagents used in this study were of analytical grade and purchased from Sigma-Aldrich Chemical Co. (Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e1.1.4. Instruments:\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eCooling centrifuge (SiGMA 3\u0026ndash;18 KS, Germany).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHomogenizer (Kinematica Polytron, PT 10\u0026ndash;35 GT, China).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIncubator shaker (Thermo Fisher Scientific, Model MAXQ 481R HP, USA).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eLaminar Flow: (heal force, hf safe 1200, China).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eRotary Evaporator (Heidolph, Germany).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eShaking water bath (DAIHAN Scientific, MAXturdy-18, Korea).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eSpectrophotometer (JASCO V-730, Japan).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eSterilizer (Tomy, SX-700, Japan).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e1.2. Methods:\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e1.2.1. Culture maintenance and inoculum preparation:\u003c/h2\u003e\u003cp\u003eThe yeast strain was grown and maintained according to Wickerham LJ [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The inoculum was prepared according to Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e1.2.2. Solid-state fermentation (SSF):\u003c/h2\u003e\u003cp\u003eThe protocol of Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] was adopted for SSF. Incubation was done in a static incubator for 48 hours at 45 ℃.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e1.2.3. Preparation of phenolic-rich extracts:\u003c/h2\u003e\u003cp\u003eBoth unfermented olive pomace methanolic extract (UFOP) and fermented olive pomace methanolic extract (FOP) were prepared according to Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBoth extracts were reconstituted to obtain different extract concentrations (2, 4, 6, 8, and 10 mg/ml) using methanol, distilled water, or phosphate-buffered saline (PBS) according to the instructions of each method applied in this study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e1.2.4. Assessment of \u003cem\u003ein vitro\u003c/em\u003e antioxidant activities of extracts:\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section4\"\u003e\u003ch2\u003e1.2.4.1. ABTS radical scavenging activity:\u003c/h2\u003e\u003cp\u003eABTS (2, 2\u0026prime;-azinobis (3-ethylbenzthiazolin-6-sulfonic acid) radical scavenging activity was assessed according to Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Both extracts were reconstituted in distilled water. The percentage inhibition was calculated using the formula:\u003c/p\u003e\u003cp\u003eScavenging activity (%)=[1-(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{a}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{a}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e) X 100%] (Eq.\u0026nbsp;1)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section4\"\u003e\u003ch2\u003e1.2.4.2. Reducing power:\u003c/h2\u003e\u003cp\u003eThe reducing power of both extracts was assessed using the reducing power method described by Oyaizu M [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Extracts were reconstituted in methanol. Ascorbic acid was used as a standard, and results were expressed as mg ascorbic acid equivalents per liter (mg\u003csub\u003eAscE\u003c/sub\u003e/L).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section4\"\u003e\u003ch2\u003e1.2.4.3. Metal chelating activity:\u003c/h2\u003e\u003cp\u003eThe chelation of ferrous ions was estimated using the method of Dinis TC, Madeira VM, Almeida LMJAob and biophysics [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Extracts were reconstituted in methanol. The metal chelating activity was calculated according to Eq.\u0026nbsp;(1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section4\"\u003e\u003ch2\u003e1.2.4.4. Assessment of antioxidant activities against reactive oxygen species:\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section5\"\u003e\u003ch2\u003e1.2.4.4.1. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) scavenging activity:\u003c/h2\u003e\u003cp\u003eThe method of Mukhopadhyay D, Dasgupta P, Roy DS, Palchoudhuri S, Chatterjee I, Ali S and Dastidar SG [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] was adopted for the assessment of the hydrogen peroxide scavenging activity of extracts. Extracts were reconstituted in methanol. Hydrogen peroxide scavenging activity was calculated according to Eq.\u0026nbsp;(1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section5\"\u003e\u003ch2\u003e1.2.4.4.2. Superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) scavenging activity:\u003c/h2\u003e\u003cp\u003eFor assessing superoxide anion radical scavenging activity, the method reported by Jiao Z, Liu J and Wang S [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] was followed. Extracts were reconstituted in methanol. The scavenging activity was calculated as a percentage inhibition of control according to Eq.\u0026nbsp;(1).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section4\"\u003e\u003ch2\u003e1.2.4.5. Assessment of protective activities:\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section5\"\u003e\u003ch2\u003e1.2.4.5.1. Lipid peroxidation inhibition:\u003c/h2\u003e\u003cp\u003eLipid peroxidation inhibition activity was assessed according to the method reported by Matkowski A and Piotrowska M [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Extracts were reconstituted in PBS. The percentage of peroxidation inhibition was calculated according to Eq.\u0026nbsp;(1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section5\"\u003e\u003ch2\u003e1.2.4.5.2. RBCs protecting activity:\u003c/h2\u003e\u003cp\u003eThe anti-hemolytic activity was assessed by following the spectrophotometric method of Yang Z-G, Sun H-X and Fang W-H [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Extracts were reconstituted in PBS. Protecting activity was calculated according to Eq.\u0026nbsp;(1).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e1.2.5. Assessment of \u003cem\u003ein vivo\u003c/em\u003e antioxidant activities of extracts against HCC-associated oxidative stress:\u003c/h2\u003e\u003cdiv id=\"Sec25\" class=\"Section4\"\u003e\u003ch2\u003e1.2.5.1. Determination of median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) of extracts:\u003c/h2\u003e\u003cp\u003eThis study was designed to assess the acute oral toxicity produced when UFOPME and FOPME were administered by oral gavage to rats using the method described by Wilbrandt W [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section4\"\u003e\u003ch2\u003e1.2.5.2. Experimental design:\u003c/h2\u003e\u003cp\u003eAfter acclimatization for 1 week on based diet, the experimental animals were weighed and randomly divided into 8 groups, with 10 animals in each group for a study period of 16 weeks. The eight groups were designed as follows:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eGroup (1): Normal group: animals were fed on a standard diet and given the vehicle (saline) throughout the experiment.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (2): HCC group: in which HCC was induced in rats by intraperitoneal injection of a single dose of diethylnitrosamine (DENA) in saline at a dose of 200 mg/kg b.w., 2 weeks later rats received carbon tetrachloride in corn oil (1:1) (as a promoter for carcinogenesis) subcutaneously at a dose of 3 ml/kg/week for 10 weeks [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (3): UF control group: in which rats were daily orally treated with a safe dose of 283.3 mg UFOPME/kg b.w. (1/10 of the LD\u003csub\u003e50\u003c/sub\u003e) for 30 days, then the animals were given the vehicle till the end of the experiment.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (4): F control group: in which rats were daily orally treated with a safe dose of 283.3 mg FOPME /kg b.w. (1/10 of the LD\u003csub\u003e50\u003c/sub\u003e) for 30 days, then the animals were given the vehicle till the end of the experiment.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (5): UF pre-treated group (Pre-UF): in which rats were orally pre-treated daily with the safe dose of UFOPME for 30 days before HCC induction (as in group 2).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (6): F pre-treated group (Pre-F): in which rats were orally pre-treated daily with the safe dose of FOPME for 30 days before HCC induction (as in group 2).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (7): UF post-treated group (Post-UF): in which HCC-bearing rats were orally treated daily with the safe dose of UFOPME for 30 days.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup (8): F post-treated group (Post-F): in which HCC-bearing rats were orally treated daily with the safe dose of FOPME for 30 days.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAt the end of the experiment (16 weeks), the animals were anesthetized by injection of ketamine/xylazine mixture at a dose of 100 mg/kg-10 mg/kg BW, sacrificed by decapitation, and dissected, then their livers were excised and washed with saline. Biopsies of livers were immediately homogenized in ice-cold PBS (pH 7.4) using a tissue homogenizer. The homogenates were centrifuged at 1700 rpm for 30 minutes at 4 ℃ and the resulting supernatants were stored at-80 ℃ for assessment of hepatic oxidative stress markers.\u003c/p\u003e\u003cdiv id=\"Sec27\" class=\"Section5\"\u003e\u003ch2\u003e1.2.5.2.1. Hepatic oxidative stress markers:\u003c/h2\u003e\u003cdiv id=\"Sec28\" class=\"Section6\"\u003e\u003ch2\u003e1.2.5.2.1.1. Hepatic malondialdehyde (MDA) level:\u003c/h2\u003e\u003cp\u003eMalondialdehyde (MDA) level in liver homogenate, as lipid peroxidation end product, was determined by the endpoint colorimetric method of Satoh K [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and Ohkawa H, Ohishi N and Yagi K [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] using a commercial assay kit (Biodiagnostic, Egypt) where MDA reacts with thiobarbituric acid in an acidic medium at a temperature of 95\u0026deg;C for 30 minutes to form a pink product that can be measured at 534 nm. The assay was done according to the manufacturer\u0026rsquo;s guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCalculation of results\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eMDA concentration (nmol/g tissue used) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{s}\\text{t}\\text{a}\\text{n}\\text{d}\\text{a}\\text{r}\\text{d}}\\times\\:\\frac{10}{\\text{g}\\:\\text{t}\\text{i}\\text{s}\\text{s}\\text{u}\\text{e}\\:\\text{u}\\text{s}\\text{e}\\text{d}}\\)\u003c/span\u003e\u003c/span\u003e (Eq.\u0026nbsp;2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section6\"\u003e\u003ch2\u003e1.2.5.2.1.2. Hepatic nitric oxide (NO) level:\u003c/h2\u003e\u003cp\u003eNitric oxide (NO) level in liver homogenate was determined by the endpoint colorimetric method of Montogomery H and Dymock JF [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] using a commercial assay kit (Biodiagnostic, Egypt). The kit is based on the measurement of endogenous nitrite concentration (the final products of NO \u003cem\u003ein vivo\u003c/em\u003e) as an indicator of NO production. It depends on the addition of Griess reagent, which converts nitrite into a deep purple azo compound with a bright reddish-purple color that can be measured at 540 nm. The assay was done according to the manufacturer\u0026rsquo;s guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCalculation of results\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eNitrite concentration (μmol/L) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{s}\\text{t}\\text{a}\\text{n}\\text{d}\\text{a}\\text{r}\\text{d}}\\times\\:\\text{S}\\text{t}\\text{a}\\text{n}\\text{d}\\text{a}\\text{r}\\text{d}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\)\u003c/span\u003e\u003c/span\u003e (Eq.\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section6\"\u003e\u003ch2\u003e1.2.5.2.1.3. Hepatic total antioxidant capacity:\u003c/h2\u003e\u003cp\u003eThe total antioxidant capacity level in liver homogenate was determined by the endpoint colorimetric method of Koracevic D, Koracevic G, Djordjevic V, Andrejevic S and Cosic V [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] using a commercial assay kit (Biodiagnostic, Egypt) where the determination of the antioxidative capacity is performed by the reaction of antioxidants in the sample with a defined amount of exogenously provided H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The antioxidants in the sample eliminate a certain amount of the provided H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The residual H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is determined colorimetrically by an enzymatic reaction that involves the conversion of 3, 5, dichloro-2-hydroxybenzensulphonate to a colored product that can be measured at 505 nm. The assay was done according to the manufacturer\u0026rsquo;s guidelines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCalculation of results\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eTotal Antioxidant concentration (mM/L)\u0026thinsp;=\u0026thinsp;A \u003csub\u003eblank\u003c/sub\u003e-A \u003csub\u003esample\u003c/sub\u003e X 3.33 (Eq.\u0026nbsp;4).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e1.2.6. Statistical analysis:\u003c/h2\u003e\u003cp\u003eData are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was carried out using the SPSS 16.0 program according to IBM\u0026rsquo;s statistics guide [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Statistical results were reported according to APA-7 guidelines.\u003c/p\u003e\u003cp\u003eTwo-way ANOVA was conducted to examine the effect of fermentation and extract concentration on antioxidant activities. Residual analysis was performed to test for the assumptions of the two-way ANOVA. Outliers were assessed by inspection of boxplots; normality was assessed using Shapiro-Wilk\u0026rsquo;s normality test for each cell of the design; and homogeneity of variances was assessed by Levene\u0026rsquo;s test. Differences were considered significant at a significance level of \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. All pairwise comparisons were run for each simple main effect with reported 95% confidence intervals using statistical significance, receiving a Bonferroni adjustment, and being accepted at a significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.025 level [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The EC\u003csub\u003e50\u003c/sub\u003e of extracts was calculated using regression analysis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne-way ANOVA was conducted to detect statistically significant differences between different groups of the HCC animal model in all biochemical parameters. The test assumptions were checked out. For each group, outliers were assessed by inspection of boxplots; normality was assessed using Shapiro-Wilk\u0026rsquo;s normality test and homogeneity of variances was assessed by Levene\u0026rsquo;s test. For data sets with homogeneity of variances, one-way ANOVA/Tukey HSD post hoc analysis was used. Differences were considered significant at a significance level of \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For data sets that violate homogeneity of variance, one-way Welch ANOVA/Games-Howell post hoc analysis was used. Differences were considered significant at a significance level of \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. .\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eNotably, UFOPME and FOPME were previously subjected to GC/MS analysis by Mahmoud AE, Fathy SA, Ali MM, Ezz MK and Mohammed AT [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e] and HPLC analysis by Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previously published GC/MS analysis revealed that fermentation of OP using \u003cem\u003eK. marxianus\u003c/em\u003e led to the biosynthesis of carvacrol, thymol, eugenol, and caryophyllene oxide since they were detected only in the methanolic extract of FOP. In addition, the fatty acid esters (oleic acid methyl ester, oleic acid ethyl ester, and methyl palmitate) have been identified as major volatile compounds in both extracts [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the other hand, published HPLC results confirmed the alteration of the phenolic profile of OP after fermentation by reinforcing its content of rutin, vanillin, cinnamic acid, quercetin, catechin, and syringic acid by 69.22%, 39.35%, and 31.44%, 22.78%, 7.06%, and 5.81%, respectively. However, fermentation decreased OP gallic, caffeic, and \u003cem\u003ep\u003c/em\u003e-coumaric acid contents by 59.24%, 55.25%, and 53.96%, respectively [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eFigure (1) demonstrates the effect of extract concentration of both UFOPME and FOPME on ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity. EC\u003csub\u003e50\u003c/sub\u003e was found to equal 4.58 and 1.17 mg/ml for UFOPME and FOPME, respectively. Both UFOPME and FOPME demonstrated concentration-dependent ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity (Fig. 1). Two-way ANOVA (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.206 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that the two independent factors, fermentation and extract concentration, significantly affect ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity with high effect size, where, for the first factor, fermentation, \u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;592.246, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.967, and for the second factor, extract concentration, \u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;171.118, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.972. Also, the interaction between the two independent factors significantly affects ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;87.708, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.946). The mean difference in ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations except for concentration 10 mg/ml.\u003c/p\u003e\n\u003cp\u003eFermentation had a strong effect on OP\u0026rsquo;s scavenging activity, as revealed by the decreased EC\u003csub\u003e50\u003c/sub\u003e value after fermentation (EC\u003csub\u003e50\u003c/sub\u003e is 4.58 and 1.17 mg/ml for UFOPME and FOPME, respectively). An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in ABTS radical scavenging activity for UFOPME at different concentrations (F(4, 20)\u0026thinsp;=\u0026thinsp;246.134, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;2\u0026thinsp;=\u0026thinsp;0.98), as for FOPME (F(4, 20)\u0026thinsp;=\u0026thinsp;12.692, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;2\u0026thinsp;=\u0026thinsp;0.717). Pairwise comparisons demonstrated that for UFOPME, all concentrations were significantly different from each other, while for FOPME, there were no significant differences between concentrations 4, 6, 8, and 10 mg/ml.\u003c/p\u003e\n\u003cp\u003eThe results of UFOPME coincide with those of Tapia-Quir\u0026oacute;s P, Montenegro-Land\u0026iacute;var MF, Vecino X, Alvarino T, Cortina JL, Saurina J, Granados M and Reig M [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] and Albahari P, Jug M, Radić K, Jurmanović S, Brnčić M, Brnčić SR and Vitali Čepo D [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003ePhenolic compounds can scavenge ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e through hydrogen atom donation and/or electron transfer mechanisms. It is worth noting increasing the number of hydroxyl groups in the aromatic ring does not necessarily lead to an increase of ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity of the compound. Structure-activity relationship of individual phenolic compounds reported before by Nenadis N, Wang L-F, Tsimidou M and Zhang H-Y [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] can explain the increased ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity of FOPME despite the decrease of caffeic and \u003cem\u003ep\u003c/em\u003e-coumaric acid content after fermentation, as reported by Mohammed AT, Mahmoud AE, Ali MM, Ibrahim DM and Fathy SA [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. This is because quercetin possesses radical scavenging activity nearly 67.3 times more than caffeic acid and 34.1 times more than \u003cem\u003ep\u003c/em\u003e-coumaric acid, the increase of quercetin after fermentation probably compensated for the decrease of both compounds [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe enhanced ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e scavenging activity of FOPME can also be attributed to the microbial biosynthesis of the monoterpenoids carvacrol, thymol, and eugenol, since all these compounds can scavenge ABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e radicals [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eReducing agents play a key role in oxidative stress reliving and macromolecule injury repair [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Reducing power assays can show that the tested compounds are electron donors, and thus they can reduce the oxidized intermediates [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe reducing power of UFOPME ranged from 5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 mg\u003csub\u003eAscE\u003c/sub\u003e/L to 49.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22 mg\u003csub\u003eAscE\u003c/sub\u003e/L; while the reducing power of FOPME ranged from 10.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mg\u003csub\u003eAscE\u003c/sub\u003e/L to 90.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mg\u003csub\u003eAscE\u003c/sub\u003e/L with a concentration-dependent increasing pattern of reducing power for both extracts and FOPME expressing higher activity than UFOPME at all studied concentrations (Fig. 2).\u003c/p\u003e\n\u003cp\u003eFermentation of OP with \u003cem\u003eK. marxianus\u003c/em\u003e strongly enhanced its reducing power (Fig. 2) with a high effect size (partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.992). Results align with Pasten A, Uribe E, Stucken K, Rodr\u0026iacute;guez A and Vega-G\u0026aacute;lvez A [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] who reported a reducing power of OP methanolic extract equivalent to 101.75\u0026thinsp;\u0026plusmn;\u0026thinsp;5.47 trolox equivalents/gds evaluated using ferric reducing antioxidant power assay. While Andr\u0026eacute;s AI, Petr\u0026oacute;n MJ, Ad\u0026aacute;mez JD, L\u0026oacute;pez M and Tim\u0026oacute;n ML [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e] reported a reducing power of 0.46 mg\u003csub\u003eAscE\u003c/sub\u003e/L at a concentration of 1 mg/ml for the aqueous extract of OP.\u003c/p\u003e\n\u003cp\u003eTwo-way ANOVA test (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.092 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) demonstrated that both independent factors, fermentation (\u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;2415.823, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.992) and extract concentration (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;1663.672, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.997) significantly affect the reducing power with high effect sizes. Moreover, the interaction between the two independent factors had a significant effect on reducing power (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;171.665, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.972). The mean difference in reducing power between UFOPME and FOPME was found to be statistically significant at all studied concentrations.\u003c/p\u003e\n\u003cp\u003eAn analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in reducing power for UFOPME at different concentrations (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;400.033, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.988), as for FOPME (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;1435.303, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.997). Pairwise comparisons demonstrated that for both extracts, all concentrations were significantly different from each other.\u003c/p\u003e\n\u003cp\u003eThe reducing power of phenolic compounds depends mainly on the degree of hydroxylation and conjugation. Generally, flavonoids with a catechol moiety and an enolic group in the C-ring show stronger reducing activity. Thus, the enhanced reducing power after fermentation can be attributed to the increase in quercetin and rutin concentrations rather than catechin. This is because favonols have higher reducing power than favanols due to the presence of the double bond between the 2-and 3-positions of the C-ring and also the double-bonded oxygen atom at the 4-position of the C-ring [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. On the other hand, the reducing power of phenolic acids depends on the number and position of hydroxyl groups. Regarding phenolic acids and aldehydes, Mathew S, Abraham TE and Zakaria ZA [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e] reported that they follow the order of gallic acid\u0026thinsp;\u0026gt;\u0026thinsp;caffeic acid\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e-coumaric acid\u0026thinsp;\u0026asymp;\u0026thinsp;vanillin in their reducing power. The lower reducing power of vanillin than caffeic acid is attributed to the methoxyl group, which greatly reduces the reducing power. Also, quercetin is a more potent reducing compound than caffeic and gallic acids [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, the decreased concentrations of phenolic acids (gallic, caffeic, and \u003cem\u003ep\u003c/em\u003e-coumaric acids) after fermentation did not adversely affect the reducing power of FOPME, as it was compensated for by the increase in quercetin.\u003c/p\u003e\n\u003cp\u003eOn the other hand, the enhanced reducing power of FOPME can also be attributed to the exclusive presence of carvacrol, thymol, and eugenol since the three volatile compounds were reported to exhibit reducing activity in the potassium ferricyanide-reducing power assay [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eTransition metals play a major role in the generation of ROS in living organisms. Chelating agents, such as phenolic compounds, may inactivate metal ions and potentially inhibit metal-dependent oxidative processes [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eFermentation of OP with \u003cem\u003eK. marxianus\u003c/em\u003e positively affected its chelating activity (Fig. 3). This was approved by decreasing EC\u003csub\u003e50\u003c/sub\u003e value from 4.76 mg/ml for UFOPME to 1.765 mg/ml after fermentation. Two-way ANOVA (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.079 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that fermentation (\u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;1537.728, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.987) as well as extract concentration (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;881.879, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.994) significantly affected metal chelating activity with high effect sizes. Also, the interaction between the two factors had a significant effect on metal chelating activity (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;204.086, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.976). The mean difference in metal chelating activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in metal chelating activity for UFOPME at different concentrations (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;966.406, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.995), as for FOPME (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;119.560, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.96). Pairwise comparisons demonstrated that all concentrations were significantly different from each other for both extracts.\u003c/p\u003e\n\u003cp\u003eThe value of EC\u003csub\u003e50\u003c/sub\u003e of UFOPME is lower than that reported by Moudache M, Silva F, Ner\u0026iacute;n C and Zaidi F [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e] who reported a value of 8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 mg/ml for the methanolic extract of OP. On the other hand, Andr\u0026eacute;s AI, Petr\u0026oacute;n MJ, Ad\u0026aacute;mez JD, L\u0026oacute;pez M and Tim\u0026oacute;n ML [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e] reported a very low EC\u003csub\u003e50\u003c/sub\u003e value (0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/ml) for the aqueous extract of OP.\u003c/p\u003e\n\u003cp\u003eThe metal-chelating activity of phenolic compounds depends on the number of catechol groups, galloyl groups, or pyrone oxygens present in the compound [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Stability constants for polyphenol-iron interactions provide insight into their antioxidant behavior. The stability constant for different polyphenol-iron interactions was reported to follow the order of gallic acid\u0026thinsp;\u0026lt;\u0026thinsp;rutin\u0026thinsp;\u0026asymp;\u0026thinsp;quercetin\u0026thinsp;\u0026lt;\u0026thinsp;catechin [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBased on these outlined reports, the enhanced metal chelating activity after fermentation can be attributed to the increased concentration of catechin (one metal-binding site), rutin (has two metal-binding sites), and quercetin (has three metal-binding sites) [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e] rather than the increase in syringic content since syringic acid does not complex with metal ions as its structure lacks catechol and galloyl groups [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe decreased concentrations of \u003cem\u003ep\u003c/em\u003e-coumaric (has no metal binding site), caffeic (one metal binding site), and gallic (two overlapped metal binding sites) acids after fermentation did not negatively affect the iron chelating activity of FOPME. This is probably because phenolic acids seem to play a weaker role in iron binding than other polyphenolic compounds since they either do not bear catechol or galloyl moieties; or bear small numbers of metal chelating groups [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) scavenging activity results demonstrated that fermentation greatly enhanced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity of OP since EC\u003csub\u003e50\u003c/sub\u003e was reduced from 4.89 mg/ml for UFOPME to 2.6 mg/ml for FOPME (Fig. 4). Two-way ANOVA (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.642 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that fermentation (\u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;4988.684, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.996) as well as extract concentration (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;8244.717, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999) significantly affected H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity with high effect size. Also, the interaction between the two factors had a significant effect on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;425.839, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.988).\u003c/p\u003e\n\u003cp\u003eThe mean difference of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations except for concentration 10 mg/ml. An analysis of simple main effects for extract concentration was performed. There was a statistically significant difference in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity for UFOPME at different concentrations (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;5080.027, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999), as for FOPME (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;3590.529, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999). Pairwise comparisons demonstrated that for UFOPME, all concentrations were significantly different from each other, while for FOPME, there were no significant differences between concentrations 6, 8, and 10 mg/ml.\u003c/p\u003e\n\u003cp\u003eThe finding that OP extract can scavenge H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in a concentration-dependent manner align with that reported by Morsi MKE-S, Galal SM, Alabdulla OJCJoFS and Technology [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003ePreviously published structure-activity relationship (SAR) studies confirmed that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity of phenolic acids depends mainly on the available number of hydroxyl groups, and the presence of these groups in ortho position to each other enhances their activity more than para positions [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. The enhanced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity after fermentation can be attributed to the increased concentration of quercetin. These findings align with previously reported SAR results showing quercetin is a stronger H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger than gallic acid. Both compounds also show a concentration-dependent response [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDespite being a weak oxidant, superoxide anion can combine with molecules such as nitric oxide to generate powerful oxidative species that are harmful to human health. Some antioxidants can inhibit the chain propagation reaction caused by superoxide radicals through hydrogen donation [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eResults of the superoxide anion scavenging assay demonstrated that fermentation of OP with \u003cem\u003eK. marxianus\u003c/em\u003e demonstrated an adverse, moderate effect on superoxide anion scavenging activity at all studied concentrations (Fig. 5). EC\u003csub\u003e50\u003c/sub\u003e was found to equal 7.368 mg/ml for UFOPME and 8 mg/ml for FOPME. Two-way ANOVA (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.096 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that fermentation (\u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;63.498, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.760), as well as extract concentration (F(4, 20)\u0026thinsp;=\u0026thinsp;1522.577, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;2\u0026thinsp;=\u0026thinsp;0.997), significantly affected superoxide anion radical scavenging activity with high effect sizes. Also, the interaction between the two factors had a significant effect on superoxide anion radical scavenging activity (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;7.598, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.603). The mean difference in superoxide anion radical scavenging activity between UFOPME and FOPME was found to be statistically significant at all studied concentrations except for concentration 2 mg/ml. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in superoxide anion radical scavenging activity for UFOPME at different concentrations (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;864.784, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.994), as for FOPME (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;665.391, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.993). Pairwise comparisons demonstrated that for both extracts, all concentrations were significantly different from each other.\u003c/p\u003e\n\u003cp\u003eThe results of the superoxide anion scavenging assay align with those reported by Squillaci G, Marchetti A, Petillo O, Bosetti M, La Cara F, Peluso G and Morana A [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e] who declared that the superoxide anion scavenging activity of OP can range between 23.33 (\u0026plusmn;\u0026thinsp;0.01) and 75.72 (\u0026plusmn;\u0026thinsp;1.78)% according to the extracting solvent and extraction temperature.\u003c/p\u003e\n\u003cp\u003ePreviously reported SAR studies can explain the slightly increased EC\u003csub\u003e50\u003c/sub\u003e (decreased activity) after fermentation. It was reported that phenolic compounds with more than one hydroxyl group in their aromatic ring have stronger inhibitory potency against superoxide anion than monohydroxy substituents due to increased hydrogen donating ability, thus, gallic and caffeic acids are stronger superoxide anion scavengers than \u003cem\u003ep\u003c/em\u003e-coumaric acid. It was also stated that the phenolic compounds follow the order of gallic acid\u0026thinsp;\u0026gt;\u0026thinsp;caffeic acid\u0026thinsp;\u0026gt;\u0026thinsp;vanillin, then \u003cem\u003ep\u003c/em\u003e-coumaric acid in superoxide anion scavenging activity [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Vanillin was also reported to have weak superoxide anion scavenging activity by Bezerra DP, Soares AKN and de Sousa DP [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. On the other hand, the flavonoid quercetin has no superoxide anion scavenging activity, unlike the phenolic acid, and gallic acid as Abdullah MZ, Mohd Ali J, Abolmaesoomi M, Abdul-Rahman PS and Hashim OH [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e] reported. Also, regarding the flavonoid, rutin, Kong KW, Mat-Junit S, Aminudin N, Ismail A and Abdul-Aziz A [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e] reported that its superoxide scavenging activity is surpassed by gallic acid. Meanwhile, catechin was reported to possess a prooxidant activity in superoxide anion-generating systems that leads to induced radical formation rather than scavenging [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eIn the light of these SAR studies, the slightly increased EC\u003csub\u003e50\u003c/sub\u003e after fermentation can be explained. This adverse effect can be attributed to the decreased concentration of phenolic acids (gallic, caffeic, and \u003cem\u003ep\u003c/em\u003e-coumaric acids) since quercetin has no activity toward superoxide anions, and rutin, as well as vanillin, are less active than phenolic acids. Moreover, the adverse effect can be attributed not only to decreased phenolic acids concentration but also to increased catechin concentration after fermentation which induces superoxide anion radical generation rather than scavenging.\u003c/p\u003e\n\u003cp\u003ePrimary products of lipid peroxidation, derived from oxidative stress, can contribute to cancer by forming harmful adducts with macromolecules including proteins, lipids, and DNA [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. Phenolic compounds can inhibit lipid peroxidation by several mechanisms, including radical scavenging (both radicals initiating and produced by the lipid peroxidation process), metal chelation, and interaction with biological lipid membranes [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eFermentation of OP using \u003cem\u003eK. marxianus\u003c/em\u003e strongly increased its protective activity against lipid peroxidation (Fig. 6). EC\u003csub\u003e50\u003c/sub\u003e was found to be 4.76 mg/ml for UFOPME and 1.76 mg/ml for FOPME. Two-way ANOVA (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.230 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that both fermentation (\u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;703.819, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.972) and extract concentration (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;19803.747, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.000) can significantly affect lipid peroxidation inhibition activity. Also, the interaction between the two factors had a significant effect on lipid peroxidation inhibition activity (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;40.391962, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.89).\u003c/p\u003e\n\u003cp\u003eThe mean difference in lipid peroxidation inhibition activity between UFOPME and FOPME was found statistically significant at all studied concentrations. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in lipid peroxidation inhibition activity for UFOPME at different concentrations (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;10579.694, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.000), as for FOPME (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;9264.445, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999). Pairwise comparisons demonstrated that for FOPME, all concentrations were significantly different from each other, while for UFOPME, there was no significant difference between concentrations 6 and 10 mg/ml.\u003c/p\u003e\n\u003cp\u003eThe lipid peroxidation inhibiting activity of phenolic compounds depends on the combination of their polarity and portioning in mediums, the degree of hydroxylation (or radical scavenging groups), and the number of metal chelating sites. Therefore, structure alone cannot predict the lipid peroxidation inhibition behavior [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eQuercetin is more efficient than catechin in lipid peroxidation inhibition since its apolarity (lipophilicity) is higher and it has a higher number of metal chelating sites and hydroxyl groups. On the other hand, despite the fact that \u003cem\u003ep\u003c/em\u003e-coumaric acid is apolar more than caffeic acid, its protecting activity is lower. This is attributed to the increased number of hydroxyl groups in caffeic acid. For the same reason (increased hydroxyl groups), catechin is superior to caffeic acid regarding its protecting activity, although the former is a mono-chelator and weaker apolar. Therefore, the combination of the three ruling factors makes the order of compounds in lipid-protecting activity as quercetin\u0026thinsp;\u0026gt;\u0026thinsp;catechin\u0026thinsp;\u0026gt;\u0026thinsp;caffeic acid\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ep\u003c/em\u003e-coumaric acid [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. This explanation align with the finding of increased lipid-protecting activity in FOPME.\u003c/p\u003e\n\u003cp\u003eRegarding other phytochemical classes, the enhanced lipid protective effect of FOPME can also be attributed to the exclusive presence of the monoterpenoids carvacrol and thymol [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e], the phenylpropanoid, eugenol [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], and the terpenoid caryophyllene oxide [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eRBCs are a good model for studying antioxidant effects since they are susceptible to endogenous and exogenous oxidative damage because of their specific role as oxygen carriers. Oxidative stress negatively affects RBCs, as they are anucleated cells with poor repair and biosynthetic mechanisms [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. Oxidation of the RBC membrane\u0026rsquo;s lipids and proteins by oxidants and free radicals may lead to loss of membrane stability and subsequent cell hemolysis [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. Phenolic compounds can protect RBCs against radical-induced hemolysis [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBoth extracts demonstrated good antihemolytic activity, with EC\u003csub\u003e50\u003c/sub\u003e values of 3.27 mg/ml and 3.61 mg/ml for UFOPME and FOPME, respectively (Fig. 7). The difference in EC\u003csub\u003e50\u003c/sub\u003e values was not statistically significant. Two-way ANOVA (no outliers, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.057 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that the two independent factors, fermentation and extract concentration, significantly affect RBCs protective activity, where, for the first factor, fermentation, \u003cem\u003eF\u003c/em\u003e(1, 20)\u0026thinsp;=\u0026thinsp;28.543, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.588, and for the second factor, extract concentration, \u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;47.988, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.906. Also, the interaction between the two independent factors significantly affects RBCs protective activity (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;5.062, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.503).\u003c/p\u003e\n\u003cp\u003eThe mean difference of RBCs protecting activity between UFOPME and FOPME was found to be statistically significant at concentrations 6, 8, and 10 mg/ml only. An analysis of simple main effects of extract concentration was performed. There was a statistically significant difference in RBCs protecting activity for UFOPME at different concentrations (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;30.866, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.861), as for FOPME (\u003cem\u003eF\u003c/em\u003e(4, 20)\u0026thinsp;=\u0026thinsp;22.184, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, partial \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.816). Pairwise comparisons demonstrated that for UFOPME, there were no significant differences between concentrations of 4 and 8 mg/ml; 4 and 10 mg/ml, 6 and 8 mg/ml, and 8 and 10 mg/ml. While for FOPME, there were no significant differences between concentrations 2 and 4 mg/ml; 2 and 8 mg/ml; 2 and 10 mg/ml; and 4 and 8 mg/ml.\u003c/p\u003e\n\u003cp\u003eAlso, two-way ANOVA results demonstrated that the concentration of OP extracts is the ruling factor in their antihemolytic activities. However, the decreased protective activity of both extracts at high concentrations was not significantly different from low concentrations, as revealed by multiple pairwise comparisons. The EC\u003csub\u003e50\u003c/sub\u003e of UFOPME is higher than that reported by Madureira J, Dias MI, Pinela J, Calhelha RC, Barros L, Santos-Buelga C, Marga\u0026ccedil;a FMA, Ferreira ICFR and Cabo Verde S [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e] who reported an EC\u003csub\u003e50\u003c/sub\u003e for OP extract of 32.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u0026micro;g/ml. the difference may be due to the different solvents used for the extraction of phenolic compounds.\u003c/p\u003e\n\u003cp\u003eBoth quercetin and rutin were reported to have dose-dependent antihemolytic activity, with higher protective activity associated with rutin [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. Moreover, vanillin was reported to have antihemolytic activity [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. Also, rutin, catechin, quercetin, caffeic acid, and gallic acid have the same activity in descending order, which contradicts the finding that UFOPME has higher antihemolytic activity than FOPME, while the latter has higher contents of rutin, catechin, and quercetin [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) required to kill 50% of both UFOPME-administered rats and FOPME-administered rats was found to equal 2833.3 mg/kg b.w. (Table\u0026nbsp;1 and Table\u0026nbsp;2 for UFOPME and FOPME, respectively). Consequently, animals were administered a safe dose of 283.3 mg/kg b.w. (which represents 1/10 of LD\u003csub\u003e50\u003c/sub\u003e) throughout the experiment.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;(1): Calculation of median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) for unfermented olive pomace methanolic extract (UFOPME)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003cp\u003e(mg/ kg b.w.)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of\u003c/p\u003e\n \u003cp\u003eanimals\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of dead animals\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ed\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZ \u0026times; d\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;(2): Calculation of median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) for fermented olive pomace methanolic extract (FOPME)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003cp\u003e(mg/ kg b.w.)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of\u003c/p\u003e\n \u003cp\u003eanimals\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of dead animals\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ed\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZ \u0026times; d\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eLevels of nitric oxide (NO) and malondialdehyde (MDA) in rats\u0026rsquo; liver tissue were assessed as markers for oxidative stress that occurs in association with DENA/CCl\u003csub\u003e4\u003c/sub\u003e \u0026ndash; induced HCC, and results are reported in Figure (8). For MDA, one-way ANOVA test (no outliers, \u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.545 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) demonstrated that MDA levels were statistically significantly different between different groups, \u003cem\u003eF\u003c/em\u003e(7, 25)\u0026thinsp;=\u0026thinsp;51.019, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001.\u003c/p\u003e\n\u003cp\u003eTukey HSD post hoc analysis revealed that the increase in tissue MDA level from the normal group to the HCC group was statistically significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001), Also, the decreases from the HCC group to the pre-UF group, pre-F group, post-UF group, and post-F group were statistically significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). On the other hand, the decrease from the normal group to the UF control group, F control group, and post-F group, as well as the increase from the normal group to the pre-UF group, pre-F group, and post-UF group, did not demonstrate statistical significance (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eFor NO, one-way ANOVA test (no outliers, \u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.618 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that NO level was statistically significantly different between different groups, \u003cem\u003eF\u003c/em\u003e(7, 25)\u0026thinsp;=\u0026thinsp;25.022, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001. Tukey HSD post hoc analysis revealed that the increase in tissue NO level from the normal group to the HCC group was statistically significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001), Also, the decreases from the HCC group to the pre-UF group, pre-F group, post-UF group, and post-F group were statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001). On the other hand, the decrease from the normal group to the UF control group, F control group, post-UF group, and post-F group, as well as the increase from the normal group to the pre-F group and pre-UF group, did not demonstrate statistical significance (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eResults demonstrate a significant increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of NO and MDA (the end product of lipid peroxidation) levels in liver tissue after DENA/CCl\u003csub\u003e4\u003c/sub\u003e administration compared to the normal group, indicating the occurrence of oxidative stress in the liver as a result of increased both reactive species production and lipid peroxidation. Similar results were reported by Hassan HA, Ghareb NE and Azhari GF [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e], Shawki AK, El-Desouky MA, Fouad SM, Ahmed AFM, Aboulhoda BE and Ahmed WA [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e] and Abdel-Hamid NM, Hassan MK, Ahmed AAM, Abd Allah SG and Anber NH [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e] for DENA/CCl\u003csub\u003e4\u003c/sub\u003e-induced HCC in rats.\u003c/p\u003e\n\u003cp\u003eOn the other hand, the administration of UFOPME or FOPME alone did not have any marked impact on NO or MDA levels in liver tissue, compared to the normal group, (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) reflecting no deleterious effects for both extracts on the oxidative homeostasis in liver tissue.\u003c/p\u003e\n\u003cp\u003eOral gavage of UFOPME and FOPME either pre- or post-DENA/CCl\u003csub\u003e4\u003c/sub\u003e administration restored NO and MDA levels to normal, as revealed by insignificant differences with the normal group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) suggesting potential protective and therapeutic effects, respectively, against DENA/CCl\u003csub\u003e4\u003c/sub\u003e-induced oxidative stress in liver tissue. This can be partially attributed to the enhanced antioxidant defensive system as measured by total antioxidant capacity (Fig.\u0026nbsp;9).\u003c/p\u003e\n\u003cp\u003eTotal antioxidant capacity was evaluated in liver tissue and considered as a collective representation of the antioxidant defense system (Fig. 9). One-way Welch ANOVA test (no outliers, \u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018 for Shapiro-Wilk\u0026rsquo;s test and Levene\u0026rsquo;s test, respectively) results demonstrated that total antioxidant capacity was statistically significantly different between different model\u0026rsquo;s groups, Welch\u0026apos;s \u003cem\u003eF\u003c/em\u003e(7, 11.118)\u0026thinsp;=\u0026thinsp;11.588, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). Games-Howell post hoc analysis revealed that the decrease in tissue total antioxidant capacity from the normal group to the HCC group was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027). Also, the increase from the HCC group to the pre-UF group, pre-F group, post-UF group, and post-F group was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049, 0.024, 0.029, and 0.023, respectively). On the other hand, the difference between the normal group and UF control, F control, pre-UF, pre-F, post-UF, and post-F was not statistically significant (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eResults demonstrate a significant decrease in total antioxidant capacity in liver tissue after DENA/CCl\u003csub\u003e4\u003c/sub\u003e administration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) compared to the normal group, indicating an impaired liver antioxidant defense system. Decreased total antioxidant capacity of liver tissue after DENA/CCl\u003csub\u003e4\u003c/sub\u003e induction of HCC was also reported by Hassan HA, Ghareb NE and Azhari GF [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eOn the other hand, administration of UFOPME or FOPME alone resulted in enhanced total antioxidant capacity in liver tissue. This enhancement was not statistically different from the normal group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) indicating that the enhanced total antioxidant capacity remains in the normal range.\u003c/p\u003e\n\u003cp\u003eOral gavage of UFOPME and FOPME either pre- or post-DENA/CCl\u003csub\u003e4\u003c/sub\u003e administration restored liver total antioxidant capacity to normal, as revealed by the insignificant difference with the normal group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) suggesting potential protective and therapeutic effects, respectively, against DENA/CCl\u003csub\u003e4\u003c/sub\u003e -induced oxidative stress in liver tissue.\u003c/p\u003e\n\u003cp\u003eThe insignificant differences between the four treated groups and the normal group make it impossible to choose between UFOPME and FOPME or between pre- and post-treatment. So, it can be concluded that at the studied concentration, both OP extracts are good exogenous antioxidant agents that exert comparable protective and therapeutic activities, which can relieve DENA/CCl\u003csub\u003e4\u003c/sub\u003e-induced oxidative stress in liver tissue, restoring the oxidative state to normal.\u003c/p\u003e\n\u003cp\u003eThe decreased oxidative stress markers after administration of different OP extracts may be attributed to the presence of quercetin, as it was experimentally proven to lower NO and MDA levels in liver homogenate when administered before or after HCC induction by thioacetamide in rats [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]. The same observation was augmented by Seufi AM, Ibrahim SS, Elmaghraby TK and Hafez EE [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e] and Vasquez-Garzon VR, Macias-Perez JR, Jimenez-Garcia MN, Villegas V, Fattel-Fazenta S and Villa-Trevino S [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e], who reported the ability of quercetin to lower MDA in liver homogenate in HCC in rats induced by DENA and DENA/2-acetylaminofluorene, respectively. Also, rutin was proven to reduce MDA levels in the liver homogenate of DENA-induced HCC in rats [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]. Independently of phenolic compounds, the carvacrol present in FOPME was reported to lower MDA levels and improve the overall liver antioxidant state in DENA-induced HCC in rats [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e], and the same effect was reported for thymol in acetaminophen-induced toxicity in HepG-2 cell lines [\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e]. Also, thymol was reported to lower MDA and enhance liver antioxidant status in doxorubicin-induced hepatotoxicity in rats [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSSF of olive oil pomace by the Generally Regarded As Safe \u003cem\u003eKluyveromyces marxianus\u0026nbsp;\u003c/em\u003eNRRL Y-8281 yeast strain is a precious eco-friendly and applicable technique\u0026nbsp;that not only allows sustainable olive oil production but also valorizes the environmental pollutant olive pomace\u0026nbsp;into\u0026nbsp;pomace enriched with potent \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antioxidant compounds that can find their applications in different industrial sectors, including food, chemical, pharmaceutical, and medical sectors. Moreover, the present study can contribute to attaining the 2030 global sustainability goals of the United Nations by considering environment preservation, competing climate change, and industrial and agricultural sustainability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.E. , M.M., S.A. , AT. and D.M. contributed equally to the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest :\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen access funding provided by The Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the National Research Centre funding program (13010603), for supporting this research. 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\u003cstrong\u003e2022:\u003c/strong\u003e6702773.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Olive pomace, solid-state fermentation, Kluyveromyces marxianus, phenolic compounds, antioxidant activity, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-7255027/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7255027/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study investigated the potential role of solid-state fermentation (SSF) to enhance the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antioxidant activities of olive pomace (OP, the environmentally polluting solid residue produced after olive oil extraction) using the GRAS (Generally Recognized As Safe) yeast \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e. \u0026nbsp;Fermented OP (FOP) was produced by SSF of OP by \u003cem\u003eK. marxianus\u003c/em\u003e. Both unfermented OP (UFOP) and FOP were extracted using methanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFOP methanolic extract (FOPME) demonstrated significantly higher antioxidant activity than UFOP methanolic extract (UFOPME) in ABTS radical scavenging, metal chelating, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003escavenging, lipid peroxidation inhibition, reducing power, and total antioxidant capacity. On the other hand, UFOPME demonstrated slightly higher superoxide anion radical scavenging and RBCs-protecting activities than FOPME. Prophylactic and therapeutic antioxidant activities of UFOPME and FOPME were assessed \u003cem\u003ein vivo\u003c/em\u003e against oxidative stress associated with diethylnitrosamine (DENA)-induced hepatocellular carcinoma (HCC) in rats. Relative to the normal group, the administration of DENA/CCl\u003csub\u003e4\u003c/sub\u003e in the HCC group significantly increased hepatic nitric oxide and malondialdehyde and significantly decreased total antioxidant concentration. Both UFOPME and FOPME protected against and relieved the oxidative stress associated with DENA/CCl\u003csub\u003e4\u003c/sub\u003e-induced HCC in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSSF of OP using the GRAS yeast \u003cem\u003eK. marxianus \u003c/em\u003esignificantly enhanced most of its \u003cem\u003ein vitro\u003c/em\u003e antioxidant activities alongside demonstrating simultaneous protective/therapeutic effects against oxidative stress associated with DENA/CCl\u003csub\u003e4\u003c/sub\u003e-induced HCC in rats.\u003c/p\u003e","manuscriptTitle":"Enhanced in vitro and in vivo antioxidant activities of olive pomace by solid- state fermentation using the yeast Kluyveromyces marxianus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 12:15:18","doi":"10.21203/rs.3.rs-7255027/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b534bf9-c378-4536-ac80-d4de44a50788","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-20T18:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 12:15:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7255027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7255027","identity":"rs-7255027","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}