Protective Effect of Honey and Propolis against Gentamicin-Induced Oxidative Stress and Hepatorenal Damages

Bee products are a promising source of phenolic compounds with strong antioxidant activity. The present study was designed to explore the protective effect of honey, propolis, and their combination on gentamicin-induced oxidative stress and hepatorenal dysfunction. This study was conducted on male Wistar rats by intraperitoneal injections of gentamicin (120 mg/kg BW/day, i.p.) or normal saline (1 ml/kg BW/day, i.p.) for 10 consecutive days. Honey (2 g/kg BW), propolis (100 mg/kg BW), or their combination were given daily by gavage to normal and gentamicin groups. Honey and propolis samples were evaluated for their phytochemical composition and antioxidant capacity. The in vitro investigations showed that the evaluated samples especially propolis extract have high antioxidant power associated with the presence of several phenolic compounds such as flavonoids, flavan-3-ols, hydroxybenzoic acids, hydroxycinnamic acids, and stilbenes, while honey contains only hydroxybenzoic acids and hydroxycinnamic acids. It was also shown that simultaneous treatment with honey or propolis extract alone or in association prevented changes caused by gentamicin administration and improved hepatic and renal functions. Changes caused by gentamicin administration, observed by in vivo experiments, include significant elevation of uric acid, urea, creatinine, and hepatic enzyme levels (ALT, AST, and ALP) and kidney biochemical changes (an increase of urea, uric acid, and creatinine and a decrease of albumin and total protein) as well as remarkable changes of renal and liver oxidative stress markers (CAT, GPx, and GSH) and elevation of MDA levels. Overall, it can be concluded that honey and propolis might be useful in the management of liver and renal diseases induced by xenobiotics.


Introduction
Aminoglycoside antibiotics, especially gentamicin (GENT), are widely used in the treatment of human and animal bacterial infections, particularly aerobic Gram-negative bacteria. However, the clinical usefulness of gentamicin is limited by its serious side effects on liver and kidney functions [1]. Chronic treatment with GENT prompts tubular necrosis, reduced renal blood flow (RBF), and glomerular filtration rate (GFR) [2]. Previous studies suggest that GENTtoxicity is a complex process in which GENT triggers cellular responses involving various pathways that result in liver and renal injuries [3,4]. Until the moment, the precise mechanism of GENT hepatonephrotoxicity is not fully understood. Several hypotheses were proposed to explain the involved mechanism of toxicity pathways; oxidative stress is the most reinforced one. Reactive oxygen species (ROS) and other free radicals were considered to be one of the important mediators of GENT-toxicity [5]. In vivo and in vitro investigations have revealed that GENT stimulates the overproduction of ROS metabolites, leading to necrosis and cellular injury through different pathways, including protein oxidation, lipid peroxidation, and DNA damage. In addition, the excess of free radicals' generation enhances the activation of nitrosative tissue stress, inflammatory markers, modulation of the caspase family, and mitogenactivated protein kinases [6]. It has also been documented that nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and redox-sensitive transcription factors are associated with GENT-toxicity [7,8]. Therefore, many researchers are interested in finding new effective and safer antioxidant compounds able to prevent xenobiotic adverse effects.
Since ancient times, honey and propolis were used for the prevention and self-treatment of various illnesses and human disorders [9,10]. Honey is a functional food produced by bees on mixing plant nectar with its hypopharyngeal excretions [11]. Due to its diverse content of pollen and nectar from different melliferous plants, honey has a wide complex mixture of components. Generally, it contains more than 75% of sugars, around 17% of water, and 3.5% of other minor elements including phenolic compounds, organic acids, proteins, amino acids, enzymes minerals, and vitamins [12]. Owing to this multifaceted composition, honey plays a crucial function in the management of several pathologies such as cancer, inflammation, diabetes, and gastrointestinal dysfunction [13,14]. Bee glue or propolis is a sticky organic substance produced by honeybees from different exudates and plant buds mixed with wax, pollen, and saliva. It is applied by honeybees as a hive defensive material against various infections. Propolis has been used in folk medicine by different civilizations around the world as a remedy for various illnesses and health disorders [15]. Nowadays, it has become the objective of many scientific investigations. Several reports have documented that propolis extracts possess a broad spectrum of pharmacological actions such as antidiabetic, antimicrobial, and antitumoral effects [15,16]. Moreover, it was discovered that propolis has a potent protective action in kidney and liver damages [17] which makes it an ideal preventive candidate against GENT adverse effects. It is well documented that the antioxidant capacities of natural products, including beekeeping products, are mostly associated with in vivo activities [18]. The assessment of the antioxidant capacity and the quantification of bioactive constituents from honey and propolis by validated and recommended assays are crucial steps to predict their efficacy on oxidative stress and associated pathologies [19,20]. It has been reported that binary or multiple mixtures of food extracts or their individual bioactive components provide synergy with regard to antioxidant status, anti-inflammation, anticancer, and chemoprevention of several oxidative stress and metabolic disorders [21]. In this context, this study is aimed at assessing the antioxidant potential, identification, and quantification of polyphenols from honey and propolis as well as evaluating their protective effects alone or in combination on GENT-induced oxidative stress and hepatoreno toxicity in Wistar rats.
According to our previous survey [22], the predominant vegetation species present around the hives were Prunus cerasus L, Prunus domestica L, Ceratonia siliqua L, Rosa canina L, Olea europaea L, Rosmarinus officinalis L, Ruta graveolens L, Pinus halepensis Mill, and Quercus ilex L.
For experimental assays, propolis extract was obtained with ethanol (70%, v/v). For that one gram of raw propolis was macerated in 30 ml of ethanol-water , v/v) under mechanical stirring for one week. The final extracts were filtered (Whatman, n°1), and the supernatant was collected. One part of the resulting liquid extract was used for chemical analyses and in vitro antioxidant tests. Another part was subsequently concentrated in a rotary evaporator (Model R-200, Büchi labortechnik AG, Switzerland), and distilled water was added to prepare the chosen concentrations for in vivo tests. For honey, 2 g of material was dissolved in 10 ml of distilled water and subsequently diluted to prepare the desired concentration. Honey and propolis combination was also studied.

Chemical Analyses of Extracts
2.3.1. Total Polyphenol Content (TPC). Total polyphenols were determined by the Folin-Ciocalteu method following the procedure described by Santos et al. [23]. Briefly, 15 μl of Folin-Ciocalteu reagent sodium tungstate (10 g) and sodium molybdate (2.5 g) was dissolved in 70 ml of distilled water; then, 5 ml of phosphoric acid (85%) and 10 ml of concentrated hydrochloric acid were added. After 10 hours, 15 g of lithium sulfate and 5 ml of distilled water were added, and 100 ml with distilled water and 60 μl of sodium carbonate (Na 2 CO 3 , 75 g/l) were added to 5 μl of honey or propolis extracts. The intensity of the produced color was read at 700 nm by a UV/Vis spectrophotometer (Synergy HT, Bio-Tek Instruments, Inc., USA) after incubating the mixture at 60°C for 5 min. Gallic acid (from Sigma Aldrich, St. Louis, MO, USA) was used as a standard to achieve the calibration curve (2,500-100 mg/l, R 2 = 0:996), and the results were expressed in milligram gallic acid equivalent (GAE) per gram of propolis or honey (mg GAE/g).

Total Flavonoid Content (TFC).
Total flavonoids were determined according to the procedure described by Kong et al. [24]. Briefly, 100 μl of honey or propolis extracts was 2 Oxidative Medicine and Cellular Longevity mixed with sodium nitrite (5%) and 150 μl of aluminum chloride (AlCl 3 ) (10%). After 5 min, 200 μl of sodium hydroxide (NaOH) (1%) was added after a 1 h incubation period in the dark. The intensity of the produced color was measured at 510 nm by a UV/Vis spectrophotometer (Synergy HT, BioTek Instruments, Inc., USA). A calibration curve (500-0 mg/l, R 2 = 0:998) was prepared using quercetin (from Sigma Aldrich, St. Louis, MO, USA), and the flavonoid content was expressed as milligrams of the quercetin equivalent per gram of the propolis or honey (mg QE/g).

Oxidative Medicine and Cellular Longevity
Group DW (control group): received daily by gavage distilled water and injected with normal saline solution (1 ml/kg BW/day, i.p.).
The study lasted for 10 days. At the end of the experiment, rats underwent fasting for 12 hours after their last feeding; then, urine samples were collected using specific metabolic cages, and blood samples were taken into tubes from each rat by retroorbital bleeding under ether anesthesia, and then, plasma was separated by centrifugation (2000 g) during 10 min.
The treatment duration was chosen according to [25]. Honey and propolis extract doses were selected based on the studies of El-Haskoury et al. and El Menyiy et al. [26,27].
Honey and propolis were prepared every single day one hour before their administration at a concentration of 2 g of honey for 10 ml and 100 mg of dry propolis extract for 10 ml. The combination of both samples was performed by mixing 2 g of honey and 100 mg of propolis dry extract in 10 ml of distilled water. The combined mixture is vortexed throughout the gavage period.

Liver and Kidney Antioxidant Enzyme Activities.
Catalase (CAT) activity was calculated according to the method of [29]. A decrease in absorbance due to H 2 O 2 degradation was monitored spectrophotometrically at 240 nm for 1 min, and the activity was expressed as μmol H 2 O 2 /min/mg protein. Glutathione peroxidase (GPx) activity was estimated according to the method of [30]. The activity was expressed as moles of GSH oxidized/min/mg protein.
2.8. Reduced Glutathione (GSH). GSH levels were measured following the protocol described by Ellman [31]. Briefly, 3 ml of sulfosalicylic acid (4%) was added to 500 ml of homogenate liver and kidney tissues. The mixture was centrifuged at 2,500 × g for 15 min, and then prepared Ellman's reagent was added to 500 ml of supernatant. The absorbance was measured at 412 nm after 10 min. Total GSH content was expressed as micrograms per milligram of protein.

Lipid Peroxidation (TBARS).
The formation of products of lipid peroxidation was quantified in liver and kidney tissues using the thiobarbituric acid-reactive substances (TBARS) method, as reported previously by Kassan et al. [32], and absorbance was measured at 532 nm. Results were expressed as malondialdehyde (MDA) concentration (nmol/g tissue).
2.10. Statistical Analysis. All data are presented as mean ± SD ðstandard deviationÞ. Statistical comparisons between the groups were performed with a one-way analysis of variance (ANOVA) followed by the Tukey test. Chi-square was used to compare the difference in the proportion of the effect of gentamicin, honey, propolis, or both of them on oxidative parameters between kidney and liver. The GraphPad Prism® software (version 5.0; GraphPad Software, Inc., San Diego, USA) was used, and p < 0:05 was considered significant.

Effect of Honey and Propolis Extracts on Renal
Biochemical Changes. The data in Figure 1 indicates that gentamicin administration (120 mg/Kg/BW) for 10 consecutive days increased plasmatic levels of urea, uric acid, creatinine, and CRP as well as decreased plasma albumin and total protein levels as compared to the control group (DW). The cotreatment with propolis extract alone (100 mg/Kg/BW) or honey alone (2 g/Kg/BW) or both (propolis+honey) significantly prevented the increase of plasma urea, uric acid, creatinine, and CRP levels and increased plasma albumin and total protein levels in comparison with group Gent. In addition, GENT injection induced a significant elevation in urinary uric acid and total protein levels, while showed a decrease in urea and urinary creatinine compared to the control group (DW). However, its coadministration with propolis, honey, or their combination attenuated the deleterious effects of GENT showed a significant decrease in urinary uric acid and total protein levels accompanied by a higher level of urinary urea and creatinine comparatively to the Gent group ( Figure 2). Propolis and honey showed no (negative) effects on the kidney of normal rats.

Effect of Honey and Propolis Extracts on Liver
Biochemical Changes. Regarding hepatic function, GENT treatment (group Gent) induced a significant increase in enzymatic levels of AST, ALT, and ALP as compared to the normal group (DW), while no significant change was observed in plasma levels of LDH between studied groups (Figure 3), whereas the cotreatment with propolis (Gent +PR), honey (Gent+H), or their combination (Gent+PR +H) showed a preventive effect in the increase of plasmatic AST, ALT, and ALP levels promoted by GENT administration. Propolis and honey did not show negative effects on liver normal rats.
3.5. Effect on Kidney Oxidative Stress. GENT administration induced marker disorganization of oxidant status and proteins in the kidney tissue, as characterized by a significant reduction in CAT, GSH, GPx, and proteins with a concomitant elevation in MDA concentration compared to the DW group (Table 3). However, the coadministration of propolis alone, honey alone, or their association significantly lowered the MDA levels and prevented the decrease of CAT, GSH, and GPx, activities, and protein levels as compared to the GENT group. These results demonstrated that propolis and honey administrations especially their combination improve the kidney state and exhibit a significant nephroprotective effect by restoring the antioxidant capacity and attenuating the oxidative stress in the kidney.
3.6. Effect on Liver Oxidative Stress. Regarding the liver tissue, GENT treatment (group Gent) caused a remarkable reduction in CAT, GSH, and GPx activities (p < 0:01) and induced a decrease in protein content (p < 0:05) associated with a high elevation in MDA (Table 4) in comparison with the nontreated group (group DW), while the cotreatment with propolis alone, honey alone, or their combination significantly prevented the increase of MDA levels and promotes beneficial effects on antioxidant enzyme activities, as well in protein levels as compared to the GENT group.

Comparison between Kidney and Liver Oxidative Stress.
Regarding the effect of interventions on the kidney and liver tissues, GENT caused a remarkable decrease in the CAT ( (Table 5).
3.8. The Effect of Propolis, Honey, or Their Combination on the Gentamicin-Induced Body and Organ Weight Changes. As shown in Table 6, gentamicin injection induced a marked reduction in body weight gain accompanied by a significant increase in the relative kidneys and liver weights. However, the treatment with propolis (Gent+Pr), honey (Gent+H), or their mixture (Gent+Pr+H) prevented the body-weight loss and improved the relative organs' weights.

Phytochemical Constituents and Antioxidant Activities of
Honey and Propolis Extracts. The identification and quantification of bioactive components of natural products are crucial tests to better understand the pharmacological effects and their effectiveness. Flavonoids and other phenolic compounds are powerful antioxidant molecules with a stronger ability to fight free radicals that induce oxidative stress [33]. DPPH, ABTS, and FRAP are the most commonly used tests to evaluate the antioxidant activities of various biologi-cal matrices, evaluating different mechanisms of action. The present study illustrates that propolis has a more affluent profile of antioxidant phenolic compounds when compared to honey alone or mixed with propolis extract. Polyphenol content in propolis extract was 76:00 ± 2:15 mg GAE/g, and the value obtained was higher than the data reported by Touzani et al. [34] for propolis sample harvested from the same locality where our evaluated sample was collected (Sefrou). The flavonoid content (42:22 ± 0:96 mg QE/g) was higher than those obtained by Miguel et al. [35] for Moroccan propolis, where the values ranged from 0.2 to 34.3 mg QE/g. The amount of antioxidant (flavonoids and nonflavonoids) compounds in propolis depends strongly on the botanical origin of resin and pedoclimatic characteristics of the collecting region, which explain the wide fluctuation between the evaluated sample and other propolis samples from different localities [18,36]. Regarding antioxidant activity, the propolis extract concentration required to inhibit 50% of DPPH was 0:15 ± 0:01 mg/ml. Data are in line with those reported by Miguel et al. [35] who studied fourteen propolis samples harvested from different ecoregional origins in Morocco, with IC 50 values oscillated between 0.025 mg/ml and 1.813 mg/ml. ABTS and FRAP activities were 0:08 ± 0:02 mg/ml and 2:15 ± 0:04 mmol Fe2+/g, respectively. The FRAP value was higher than those signaled by Svečnjak et al. [37] for Adriatic Sea islands propolis, ranging between 0.1 and 0.8 mmol Fe 2+ /g.
Concerning honey, the polyphenol content was 2:08 ± 0:04 mg GAE/g, which was higher than those reported by Petretto et al. [38] for teen Moroccan monofloral honey. Regarding antioxidant activities, the concentration of honey necessary to inhibit 50% of DPPH radical was 13:53 ± 1:3 mg/ml, and this value was similar to the results of Laaroussi and coworkers [11] reported on eight Moroccan honey samples, varied from 13.54 to 45.34 mg/ml and higher than those signaled for the Malaysian honey ranging from 6.12 to 11.56 mg/ml [39]. There is a strong correlation between the pollen profile of different honey samples and their amount in phenolic and flavonoid compounds [11]. In addition to the floral origin, the wide variation seen between the antioxidant compounds of examined sample and those reported by other investigations is attributed to many other factors including harvest season and geographical and environmental characteristics of the areas where it is produced [40]. ABTS and FRAP activities were 4:91 ± 0:12 mg/ml and 0:025 ± 0:00 mmol Fe 2+ /g, respectively. The ABTS and FRAP values of analyzed honey were found to be lower than those reported by Kıvrak and Kıvrak [41] for Turkish honey, where ABTS values are oscillating from 8.22 to 41.20 mg/ml and by Mahmoodi-Khaledi et al. [42] where FRAP values are ranging between 0.027 and 0.182 mmol Fe 2+ /g. It is well documented that in vitro interactions between phytobioactive components induce changes in overall antioxidant capacities [43]. The mixture of honey and propolis extract showed a higher amount of phenolics and flavonoids and displayed strong DPPH radical scavenging capacity, ABTS scavenging capacity, and high reducing power antioxidant activity (FRAP) as compared to honey alone and revealed higher antioxidant activities (DPPH and FRAP) as compared to  Oxidative Medicine and Cellular Longevity propolis extract alone (Table 1)  Oxidative Medicine and Cellular Longevity effect of gallic acid, caffeic acid, quercetin, catechin alone, or in combination with resveratrol using three different antioxidant assays (DPPH, FRAP, and Briggs-Rauscher reaction) [44]. The obtained results showed better antioxidant capacities of evaluated compounds when tested in the mixture form as compared with the activities displayed when testing individual compounds, which proves the possible interaction between these bioactive components leading to synergistic antioxidant effects.

Effect on Kidney Biochemical
Changes. Nowadays, infectious diseases are a challenging public health problem worldwide, and face to bacterial resistance and antibiotics overuse including GENT may prompt side effects such as hepatorenal toxicities [45].
The present data showed that GENT (120 mg/kg BW) caused kidney dysfunction demonstrated by a significant elevation in blood levels of urea, uric acid, and creatinine and a remarkable decrease in plasmatic albumin and total protein levels. In addition, GENT administration increased urinary uric acid and protein levels and decreased urea and creatinuria levels, which are classified as a major sign of renal tubular necrosis and kidney damage [46]. These results are similar to the data reported by Marinho et al. [47]. A study published by Al Za'abi et al. [48] showed that GENT reduces the ability of adenosine triphosphate (ATP) in the renal  10 Oxidative Medicine and Cellular Longevity tubular cells by inhibiting its phosphorylation process, which leads to low membrane integrity and thus the sensitivity to the overproduction of free radicals [49]. The animals submitted to GENT injection and cotreated with propolis alone (Gent+PR), honey (Gent+H), or their association (Gent+PR+H) restored all investigated plasmatic and urinary biomarkers. Similar data have been reported by Aldahmash and coworkers [50]. The pharmacological effects of functional organic products are most often linked to their phenolic compound composition and their ability to counteract free radicals [51]. In vitro investigations showed that honey and propolis extracts contain several bioactive molecules including rosmarinic acid, resveratrol, hesperidin, and gallic acid. Tavafi and coworkers have reported that rosmarinic acid exerts its renoprotective action by inhibiting lipid peroxidation and upregulating renal GSH, GPx, and CAT activities as well as by decreasing renal MDA levels [52]. Moreover, rosmarinic acid was found to protect kidney damage caused by cisplatin through the downregulation of tumor suppressor p53 and p-p53 and reduction of cleaved caspase-3, signifying the inhibition of tubular apoptosis [53]. As a part of its nephroprotective action, resveratrol has been revealed to inhibit the expression of transforming growth factor-beta (TGF-β), transforming growth factorbeta receptor1 (TGF-βR1), and Smad3 that play a key role in kidney fibrosis [54].

Effect on Kidney Oxidative
Parameters. It has been proven that overproduced ROS induces oxidative stress and organ damage. In this study, GENT administration deteriorates the renal antioxidant defense system and increases oxidative biomarkers. Rats injected with 120 mg/kg of GENT without cotreatment for 10 consecutive days expressed low CAT, GSH, and GPx activities and high levels of renal MDA. Results obtained from the current study are consistent with the findings of Edeogu et al. [55] in which GENT administration (100 mg/kg BW) markedly increased renal MDA levels and significantly depressed renal activities of GSH, SOD, CAT, and GPx levels in kidney tissues. It was suggested that GENT may trigger the release of renal mitochondrial iron to form a Fe-GENT complex which promotes the overproduction of ROS and other free radicals such as H 2 O 2 [56]. The generated H 2 O 2 enhances the production of superoxide anions (O 2 •-) in the renal mitochondrial cortex [57]. In the presence of redox metal ions, hydrogen peroxide (H 2 O 2 ) and superoxide ion (O 2 •-) generate hydroxyl radical (HO • ) which is considered as one of the most damaging ROS [58]. Moreover, [59] found that GENT enhances the overexpression of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) which largely contributes to tissue injury and leads to the development of various complications including nephrotoxicity. Likewise, GENT overuse leads to mitochondrial dysfunction and promotes the excessive generation of free radicals, which are considered as potent oxidizing agents that elicit lipid proteins and DNA oxidative damage via the upregulation of NADPH oxidase 4 (NOX4) and the suppression of adenine nucleotide translocase 2 (ANT2) [60][61][62][63]. Accordingly, the chronic accumulation of oxidative DNA injuries may enhance mutagenesis and human pathogenesis [64]. Interestingly, the coadministration of honey, propolis, or their association had significantly increased the activities of antioxidant enzymes and prevents the increased level of MDA in the kidney, which confirms their nephroprotective ability. These results were in agreement with the findings of [26], in which Moroccan carob honey increased CAT, GSH, SOD, and GPx activities and    The results were presented as mean ± SD of 6 rats; DW: distilled water; Pr: propolis; H: honey; Gent: gentamicin. a Comparison between the DW group and all groups; b comparison between the Gent group and gentamicin cotreated groups; c comparison between the Gent+Pr group and the Gent+Pr+H group; d comparison between the Gent+H group and the Gent +Pr+H group. * p < 0:05, * * p < 0:01, and * * * p < 0:001.

12
Oxidative Medicine and Cellular Longevity ascorbic acid as well decreased MDA levels in renal and liver tissues of rats treated with carbon tetrachloride (CCL4). In the same context, the results of Shi et al. showed that propolis improves streptozotocin-induced renal dysfunction and oxidative stress by inhibiting lipid peroxidation and ameliorating antioxidant system defenses [65]. Owing to its healthpromoting compounds, honey inhibits the production of proinflammatory cytokines NOx, IL-6, TNF-α, MCP-1, IL-12p70, IFN-γ, and IL-10 and attenuates nuclear factorkappa B (NF-κB) translocation to the nucleus [66]. Due to their rich composition in bioactive molecules, honey and propolis might have renoprotective action through one or different signaling pathways ( Figure 4). Indeed, gallic acid, a hydroxybenzoic acid present at a high concentration in honey, displays its nephroprotective effects via the modulation of oxidative stress and inhibition of inflammatory response [67]. Moreover, hesperidin, a flavonoid present in bee products, prevents kidney damage by inhibiting the cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) signaling pathway [68]. Overall, the nephroprotective effect of investigated honey and propolis extracts could be due to the interaction between their bioactive components through diverse signaling pathways (Figure 4).

Effect of Interventions on Liver Function
4.3.1. Effect on Liver Biochemical Changes. The current findings showed that GENT-treated rats exhibited extreme liver damage manifested by a remarkable increase in plasma levels of ALT, AST, and ALP ( Figure 3). These results were similar to those reported by Hegazy et al. [69]. The high plasmatic activity of the examined enzymes reflects hepatic cell necrosis and liver structural changes, which leads to their secretion into the bloodstream from the cytosol [70]. However, GENT animals cotreated with propolis, honey, or their association expressed a significant decrease in plasmatic AST, ALT, and ALP levels, which indicate that honey and propolis extract especially their combination blocked the enzyme leakage and prevented GENT-caused kidney dysfunction. These results go on hand with the findings of El-Haskoury et al. [26] who reported that honey exhibited strong hepatoprotective action against CCL4-induced toxicity. Likewise, propolis extract (150 mg/kg BW) was found able to prevent kidney injury and renal dysfunction induced by aluminum chloride in rats [17].

Effect on Liver Oxidative
Parameters. In addition to the biochemical investigations, the assessment of liver oxidative stress showed that GENT treatment decreased significantly CAT, GSH, and GPx activities, as well as protein levels with a concomitant increase in liver MDA level, a major sign that the organ is functionally compromised, with an imbalance of its redox state. It is well reported that liver tissue is rich in polyunsaturated fatty acids (PUFAs), which are sensitive to peroxidative damage [71]. Alterations in gene expression profiles resulting from chronic exposure to xenobiotic substances have a negative impact on human health [72]. A previous study showed that CCL4 treatment downregulated the mRNA expression of SOD, GPx, and CAT, which was reversed by Actiniopteris radiata active compounds in a dose-dependent manner [73]. The suppressed activity of these antioxidant enzymes may be due to the interruption of their synthesis by the overproduced ROS and other free radicals, which could be the same mechanism involved following the GENT treatment. Additionally, it has been 13 Oxidative Medicine and Cellular Longevity

14
Oxidative Medicine and Cellular Longevity documented that total protein and globulin levels were correlated positively with SOD and GPx activities of rats treated with curcumin against sodium salicylate-induced oxidative liver and kidney damages [74]. However, rats receiving GENT and simultaneously treated with honey, propolis, or their combination expressed lower MDA levels and higher CAT, GSH, GPx activities and protein contents. Similar data have been documented by other researchers [75,76]. In a human trial study, Diniz et al. have reported that the administration of microencapsulated standardized Brazilian green propolis extract (375 and 750 mg/day) during 7 days rose the endogenous enzymatic and nonenzymatic antioxidants and reduced the biomarkers associated with the cellular membrane and DNA damage in healthy young participants [77]. Similarly, Ebeid and coworkers have demonstrated that propolis supplementation (400 mg, 3 times daily) for 10 consecutive days before and 10 days after the radiotherapy session had been able to reduce significantly the DNA oxidative damage of breast cancer patients [78]. This effect was expected since several propolis components could promote the DNA repair processes, which in turn scavenge the free radicals generated by chemotherapeutic agents and counteract the impairment caused by infrared radiations [79]. In vitro, similar results were documented by Haza and Morales [80] in which polyfloral honey improved the antioxidant defense system and protected HepG2 cells against different food mutageninduced DNA oxidative damage.
The hepatoprotective action of these bioactive products could be attributed to their antioxidant content which assists in the preservation of membrane integrity. Honey and propolis extracts contain a variety of natural antioxidants, such as flavonoids, flavan-3-ols, hydroxybenzoic acids, hydroxycinnamic acids, and stilbenes (Table 2), which make them functional foods with a broad spectrum of pharmacological activities. The antioxidative properties of phenolic components are related to several mechanisms of action including, singlet oxygen quenching, free radical scavenging, metal ion chelation, and hydrogen donation [81]. Previous works documented that phenolic compounds possess several biological activities and numerous beneficial health effects. The outcome of Mahmoud and coworkers [82] showed that ferulic acid prevents oxidative stress and exerts its hepatoprotective activity against methotrexate-induced hepatotoxicity through the upregulation of nuclear factor erythroid 2related factor/heme oxygenase-1 (Nrf2/HO-1) signaling pathway. Gallic and hydroxybenzoic acids present in our studied samples prevent liver injury by modulating the expression and phosphorylation of epidermal growth factor receptor (EGFR) [83]. A previous study reports that kaempferol has been revealed to exert a potent hepatoprotective effect by inhibiting cytochrome P-450 2E1 (CYP2E1) and thus reducing ROS production [84]. In the same context, chlorogenic acid, an active phenolic compound found in bee products, has been already documented to have potent antiradical action and high preventive ability on arsenicinduced oxidative stress and apoptosis [85]. The food synergy notion, which assumes that different dietary constituents have additive or even synergistic effects on human health, has been previously pointed out [21]. As found in nature, bioactive compounds are usually present in mixture form, which increases the possibility of interactions between them. The combination of food extracts or their bioactive compounds may enhance the in vitro antioxidant status and promote the in vivo antioxidants/ROS ratio [86]. For instance, a binary combination of α-tocopherol with kaempferol or myricetin showed a better preventive effect on free radicals inducing oxidative stress and lipid peroxidation than each component alone [87]. Likewise, rats fed a diet supplemented with a mixture of quercetin (30 mg/kg/day) and resveratrol (15 mg/kg/day) synergistically reduced the fat accumulation in the white adipose tissue and improve triacylglycerol metabolism [88]. Moreover, Baranowska and coworkers have reported that quercetin combined with naringenin improves synergistically cellular redox status and reduces global DNA methylation [89].
Overall, the hepatoprotective action documented by our examined samples could be related to molecular interactions between their individual bioactive constituents through one or several signaling pathways (Figure 4).

Effect of Interventions on Body and Organ Weight
Changes. The rats' weights were measured at the beginning and the end of the experiment. Gentamicin treatment (120 mg/kg BW/day, i.p.) exhibited a significant reduction in body weight gain as compared to the normal untreated rats (DW). The documented body weight loss was in agreement with the findings of Sahu et al. [90]. This could be attributed to the skeletal muscle protein and lipid breakdown in adipose tissue [91]. Hence, the coadministration of propolis (Gent+Pr), honey (Gent+H), and especially their combination (Gent+Pr+H) prevents markedly the final body weight loss. Both the control, propolis, and honey groups showed similar weights. The body weight change was accompanied by the alteration of the liver and kidney weights. The data ( Table 6) showed that the daily administration of propolis or honey to normal rats does not affect the organs' relative weight. The data illustrates that the intraperitoneal injections of gentamicin (120 mg/kg BW/day, i.p.) for 10 consecutive days lead to a significant increase of organs' relative weight in comparison with the normal group, and this goes in hand with the findings of Laaroussi et al. [92]. The relative organ weight increase might be related to the inflammatory and oxidative stress effects of gentamicin overuse. GENT-rats simultaneously cotreated with honey alone, propolis alone, or both showed a significant decrease in the liver and kidney relative weights, with the best data displayed by the combined coadministration (Gent+Pr+H). This is consistent with the result of Nassar and coworkers, in which propolis, honey, and their mixture prevent the liver, kidney, heart, and lung relative weight decrease [93].

Conclusion
Phytochemical analysis of bee honey and propolis extracts showed the presence of several natural antioxidants belonging to different chemical groups: flavonoids, phenolic acids, flavonols, and stilbenes. These may be responsible for the documented efficacy of bee honey and propolis extracts in protecting biochemical characteristics and enzymatic activities of kidneys and liver tissues from alterations induced by gentamicin. Overall, daily intake of propolis and/or honey could offer promising protective effects on hepatic and renal functions, as well as maintaining the redox homeostasis. Further investigations would be needed to evaluate and understand the exact mechanism by which these extracts, possibly phenolic compounds, improve gentamicin-cause oxidative stress and hepatorenal injuries.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.