Graded effects of dry-feed added sodium benzoate/ascorbic acid combination on neurobehaviour, brain markers of oxidative stress, and markers of inflammation in mice

doi:10.21203/rs.3.rs-2480579/v1

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Graded effects of dry-feed added sodium benzoate/ascorbic acid combination on neurobehaviour, brain markers of oxidative stress, and markers of inflammation in mice

https://doi.org/10.21203/rs.3.rs-2480579/v1

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Background:Controversies concerning the safety of sodium benzoate/ascorbic acid combination have remained grounds for continued scientific investigations. However, there is a dearth of scientific literature on its effect on the brain. This study investigated the effects dry-food added sodium benzoate/ascorbic acid combination on neurobehaviour, brain oxidative stress and inflammatory cytokines in mice.

Methods: Adult male mice were assigned into ten groups of 10 mice each. The groups are: one group fed standard diet, three groups fed diet containing sodium benzoate (NaB) at 125 mg/kg with ascorbic acid (AA) at 100, 200 or 300 mg/kg of feed, another three groups fed NaB at 250 mg/kg with AA at 100, 200 or 300 mg/kg of feed respectively, and the last three groups fed NaB at 500 mg/kg with AA at 100, 200 or 300 mg/kg respectively. Behavioural tests were assessed following which animals were sacrificed and their brains homogenised for the assessment of biochemical parameters. Results: The result showed a decrease in body weight, self grooming, total antioxidant capacity and inflammatory cytokines, mixed response with food intake, locomotor activity, Y maze spatial working memory and anxiety related behaviours and an increase in rearing and radial arm maze spatial working memory.

Conclusion: Dry food added NaB/AA altered behavioural, oxidative and inflammatory markers in mice. While not indicating outright deleterious effects at the concentrations used, the results suggest the need for further investigations into the effects of their combination, especially, when we consider their wide range of applications in the food industry.

Toxicology

Food additives

Memory

Neurobehaviour

Neuroinflammation

Preservatives

Food additives are compounds that are added to foods or drinks in order to enhance appearance, and ensure freshness, texture, taste and flavour; they also increase safety profile of foods and drinks [1]. Salt and sugars are among the commonest, and for over two centuries, humans have preserved and/or enhanced food and fruit flavours with salt and sugars respectively [2]. In the last few decades or more the demand for, and use of food additives has skyrocketed largely due to; a) an exponential growth in human population (increasing global food demand), b) the threat of food scarcity (as a result of conflict, desertification and/or climate change) and; c) the need to enhance the quality, preserve and increase the shelf life of food and food products that has arisen from an increase in the global food production capacity as a result of improved agricultural and industrial practices [1, 3].

Several types of food additives exist to meet the growing demands of the food industry in the 21st century. These include chelating agents, curing agents, colour fixatives, flour improvers, and food preservatives. There are over three thousand different natural and/or chemical compounds available for use in that capacity [4]. Food preservatives such as sodium benzoate (NAB) are compounds known for their ability to prevent or delay enzymatic, microbiological, or chemical changes that predispose to or cause food spoilage [5, 6]. Sodium benzoate, which is the sodium salt of benzoic acid is a stable water-soluble food preservative with significant fungistatic and bacteriostatic effects [7]. It is beneficial in food preservation, as additives in the cosmetics industry, and as a potential drug in the management of schizophrenia and a number of other medical conditions [8–12]. Its detrimental effects have also been well documented [6, 7, 12–14].

In the last decade or more, there have also been reports that combining NaB (or other benzoates) and ascorbic acid (a food antioxidant) especially in drinks resulted in the formation of benzene. This was confirmed in a number of drinks tested by the United States Food and Drug Administration in 2006 that observed benzene levels in some drinks above the 5ppb maximum contaminant levels set by the Environmental protection agency for drinking water [15].While a number of other factors including shelf life, heat and light have been posited as possible culprits, a number of companies have erred on the side of caution by removing NaB from the composition of their products. More recently however, reports from a study that administered NAB orally revealed that although the administration of NAB was associated with alterations in brain function and chemistry especially at the highest dose studied (600 mg/kg body weight), however, the co-administration of NAB at this dose with ascorbic (100 mg/kg) was associated with a reversal of the behavioural and biochemical changes [16]. While there is some evidence (from our laboratory) that the consumption of dry feed containing NaB had deleterious effects on the brain of mice, there is however a dearth of scientific information regarding the possible effects of combining sodium benzoate with ascorbic acid in dry foods. This study investigated the effect of co-administering NaB at concentrations considered safe in humans with ascorbic acid also at three different doses with known therapeutic benefits on parameters such as body weight, neurobehaviour, antioxidant status and inflammatory response in mice. This was with a view to determining the effects (if any) on the brain as well as determining which NAB/ascorbic acid combinations have possible health benefits when combined with NAB.

2.1 Materials

Sodium benzoate and ascorbic acid were purchased from the Open market in the Osogbo metropolis, Osun State, Nigeria. Kits for inflammatory markers were from ENZO Life Sciences, U.S.A, while Caspase-3, acetylcholinesterase, lipid peroxidation and antioxidant tests were purchased from BioVision Inc. USA

2.2 Animals

Swiss mice, weighing 20–25 g were obtained from Empire farms in Osun state, Nigeria and used for this study. Animals were kept in cages (plastic) located in temperature-controlled quarters (23–26 degree Celsius) with 12 hours dark-light cycle. Animals had easy access to food and water ad libitum. Experiments complied strictly with the ARRIVE guidelines, as well as approved protocols of the Ladoke Akintola University of Technology and the provisions of the European Council Directive (EU2010/63) for the use and care of laboratory animals.

2.3 Feed

Animals were fed commercially available rodent diet (Standard diet) sourced from Top Feeds Ltd, Ibadan Nigeria. Sodium benzoate and ascorbic acid were incorporated into standard rodent diet at 125/100, 125/200 and 125/300 mg/kg of feed, 250/100, 250/200 and 250/300 mg/kg of feed and 500/100, 500/200 and 500/300 mg/kg of feed respectively.

2.4 Experimental methodology

Hundred adult male mice were assigned at random into ten groups of 10 mice each. The groups include one group fed standard diet (SD), three groups fed NaB at 125 mg/kg co-administered with ascorbic acid (AA) at 100, 200 or 300 mg/kg of feed, another three groups fed NaB at 250 mg/kg co-administered with AA at 100, 200 or 300 mg/kg of feed and the last three groups fed NaB at 500 mg/kg co-administered with AA also at 100, 200 or 300 mg/kg). Sodium benzoate/ascorbic acid combinations and/or standard diets were administered for eight weeks. Body weight and food intake was measured weekly. Open field, elevated plus maze (EPM), Y-maze, and radial-arm maze behaviours were assessed on day 57, following which animals sacrificed (24 hours after the last behavioural test) by cervical dislocation. The brains were excised and homogenised for the assessment of lipid peroxidation using malondialdehyde (MDA) levels, superoxide dismutase, total antioxidant capacity, tumour necrosis factor (TNF)-α, interleukin (IL)-10, Caspase-3 and acetylcholinesterase activity.

2.4.1 Determination of body weight and food intake

Weekly body weight and daily food intake measurements were carried out using an electronic weighing balance as described previously [17–20]. Relative change in body weight or food intake was calculated for each animal using the equation below, following which results for all animals were computed to find the statistical mean

$$\frac{\text{F}\text{i}\text{n}\text{a}\text{l} \text{b}\text{o}\text{d}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{r} \text{f}\text{o}\text{o}\text{d} \text{i}\text{n}\text{t}\text{a}\text{k}\text{e} – \text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{b}\text{o}\text{d}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{r} \text{f}\text{o}\text{o}\text{d} \text{i}\text{n}\text{t}\text{a}\text{k}\text{e} }{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{b}\text{o}\text{d}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{r} \text{f}\text{o}\text{o}\text{d} \text{i}\text{n}\text{t}\text{a}\text{k}\text{e} } \left(\text{g}\right)$$

2.4.2 Neurobehavioural tests

At the end of the experiment, animals were exposed to the neurobehavioural paradigms (elevated plus maze, Y-maze, Open field and radial arm maze). Protocols for the care of animals before and during the test period are as previously described [19, 21].

2.4.2.1 Open field test

In the open-field arena, animals were allowed to explore for 10 minutes, during which locomotor activity, number of rearing, and grooming episodes were observed and scored. The open-field box used for this study was a rectangular box made of a white painted wood measuring 36 x 36 x 26 cm as previously described [20, 22, 23].

2.4.2.2 Memory tests (Y-maze and radial arm maze)

Mice were exposed to the Y-maze and radial arm maze for 5 minutes each to assess spatial working memory. Arm entry sequences in the Y-maze were observed and recorded as described previously [24–26]. Working-memory in the radial arm maze is scored as previously described [24–26].

2.4.2.3 Anxiety Model: Elevated plus-maze

The elevated plus-maze which is a plus-shaped apparatus with four arms arranged at right angles to one other was used to measure anxiety-related behaviours. Anxiety behaviours were scored as previously described [27–29].

2.4.3 Lipid peroxidation (Malondialdehyde)

The lipid peroxidation kit was used to determine malondialdehyde (MDA) levels following the manufacturer‘s instructions.

2.4.4 Antioxidant activity

Superoxide dismutase activity was assayed using the method previously described by Nikishimi et al [30]. Superoxide dismutase activity was expressed as units/mg protein. Total antioxidant capacity measures the number of free radicals scavenged by the test solution in any biological sample [31–33]. Total antioxidant capacity was based on the principle of the Trolox equivalent antioxidant capacity.

2.4.5 Tumour necrosis factor-α and Interleukin (IL) -10

Tumour necrosis factor-α and interleukin (IL)-10 were measured using enzyme-linked immunosorbent assay (ELISA) techniques with commercially available kits (Enzo Life Sciences Inc. NY, USA) designed to measure the ‘total’ (bound and unbound) amount of the respective cytokines.

2.4.6 Acetylcholinesterase activity and Caspase 3 levels

Brain acetylcholinesterase activity and caspase-3 levels (Biovision, USA) were determined using commercially-available assay kits following the manufacturer‘s instructions.

2.5 Brain homogenization

The brain of five animals in each group were removed from the skulls of the animals, weighed and homogenised as described previously [34].

2.6 Statistical analysis

Statistical analysis was carried out using Chris Rorden’s ezANOVA for windows. Data obtained were subjected to analysis of variance (ANOVA) and post-hoc tests (Tukey HSD). Results are expressed as Mean ± S.E.M., p < 0.05 was taken as the accepted level of significant difference from control.

3.1 Effect of sodium benzoate/ascorbic acid co-ingestion on body weight and food intake

The effect of sodium benzoate/ascorbic acid (NaB/AA) co-ingestion on change in body weight (upper panel) and change in food intake (lower panel) is depicted in Fig. 1. There was a significant [F = 8.98, p < 0.001] decrease in body weight in all the groups coadministered NAB and AA compared to control diet. Food intake decreased significantly [F (9, 90) = 1121, p < 0.001] with NaB/AA at 125/100, 250/100, 5O0/100,500/200 and 500/300 mg/kg of feed and increased with 125/200, 250/200, 500/100, mg/kg of feed compared to control diet.

3.1.1 Effect of sodium benzoate/ascorbic acid co-ingestion on locomotor activity, rearing and self-grooming behaviours

The effect of NaB/AA co-ingestion on locomotor (upper panel) and rearing (lower panel) activity is shown in Fig. 2. There was a significant [F (9, 90) = 30.4, p < 0.001] decrease in locomotor activity with NaB/AA at 125/100, 250/300 and 500/300 and an increase at 250/100, 500/100 and 250/200 mg/kg of feed compared to control diet. Rearing activity increased significantly [F (9, 90) = 19.40, p < 0.001] with NaB/AA at 250/100 and 500/100 compared to control diet.

The effect of food-added sodium benzoate/ascorbic acid co-ingestion (NaB/AA) on self-grooming behaviour is shown in Fig. 3. There was a significant [F (9, 90) = 17.70, p < 0.001] decrease in self-grooming in all groups co administered sodium benzoate with ascorbic acid compared to control diet.

3.1.2 Effect of sodium benzoate/ascorbic acid co-ingestion on working memory in the Y- and Radial-arm maze

The effect of NaB/AA on working memory in the Y-(upper panel) and radial arm (lower panel) maze is shown in Fig. 4. There was a significant [F (9, 90) = 8.71, p < 0.001] increase in spatial working memory in the Y-maze with NaB/AA at 500/200 and 500/300 and a decrease at all other concentrations of NAB and ascorbic acid compared to control diet. Radial arm maze spatial working memory increased significantly [F (9, 90) = 5.98, p < 0.002] with NaB/AA at 500/100 and 500/200 mg/kg of feed compared to control diet.

3.1.3 Effect of sodium benzoate/ascorbic acid co-ingestion on anxiety-related behaviour

The effect of NaB/AA on time spent in the open (upper panel) and closed (lower panel) arm of the elevated plus maze (EPM) is shown in Fig. 5. There was a significant [F (9, 90) = 6.75, p < 0.024] increase in time spent in the open arm of the EPM with NaB/AA at 250/100, 250/200 and 250/300 and a decrease with NAB at 125/100, 125/200, 125/300 and 500/300 compared to control diet. Closed arm time did not differ significantly at any of the doses of NaB/AA compared to control

3.1.4 Effect of sodium benzoate/ascorbic acid co-ingestion on oxidative stress parameters and antioxidant status

The effect of NaB/AA on oxidative stress parameters and antioxidant status is shown in Table 4. Brain malondialdehyde levels increased significantly [F (9, 90) = 1034, p < 0.001] with NaB/AA at 125/100, 250/100, 500/100, 125/200. 250/200, 500/200 and 500/300 mg/kg of feed compared to control diet. Brain superoxide dismutase activity decreased significantly [F (9, 90) = 210, p < 0.001] with NAB/AA at 250/300 and increased at all other concentrations of NAB/AA compared to control diet, while total antioxidant capacity decreased significantly [F (9, 90) = 1150, p < 0.001] at all concentrations of NaB/AA compared to control diet.

Table 4

Oxidative stress parameters and antioxidant status
Groups	MDA nmol/g	SOD U/mg/protein	TAC (TE mg/protein)
Control	7.87 ± 0.10	1.50 ± 0.06	7.10 ± 0.01
NaB 125/AA 100	16.22 ± 0.20*	1.76 ± 0.06*	1.68 ± 0.01*
NaB 125/AA 200	15.23 ± 0.10*	2.15 ± 0.02*	2.50 ± 0.01*
NaB 125/AA 300	7.95 ± 0.25	1.60 ± 0.02*	2.69 ± 0.03*
NaB 250/AA 100	22.21 ± 0.12*	1.65 ± 0.01*	4.44 ± 0.21*
NaB 250/AA 200	18.23 ± 0.28*	1.62 ± 0.04*	3.95 ± 0.02*
NaB 250/AA 300	9.25 ± 0.22	1.20 ± 0.01*	6.40 ± 0.05*
NaB 500/AA 100	20.13 ± 0.25*	2.10 ± 0.02*	2.75 ± 0.02*
NaB 500/AA 200	20.10 ± 0.19*	1.77 ± 0.01*	2.55 ± 0.03*
NaB 500/AA 300	18.55 ± 0.24*	1.92 ± 0.02*	1.76 ± 0.01*

Values presented as Mean ± S.E.M, ^*p < 0.05 vs. control, number of mice/group = 10, NaB: Sodium Benzoate, MDA: Malondialdehyde, SOD: superoxide dismutase, TAC: Total antioxidant capacity, TE: Trolox equivalent.

3.1.5 Effect of sodium benzoate/ascorbic acid co-ingestion on inflammatory markers, brain acetylcholinesterase and caspase-3 levels

The effect of NaB/AA combination on brain levels of tumour necrosis factor (TNF)-α, interleukin (IL-10), acetylcholinesterase and caspase-3 levels are shown in Table 5. Brain TNF-α levels decreased significantly [F (9, 90) = 189, p < 0.001] at all concentrations of NaB/AA compared to control diet, and brain IL-10 levels decreased significantly [F (3, 36) = 50.54, p < 0.001] at all concentrations of NaB/AA compared to control diet. Brain acetylcholinesterase (ACHe) activity and caspase-3 levels showed no significant difference in any of the groups compared to control diet.

Table 5

Tumour necrosis factor-α, Interleukin-10, acetylcholinesterase and caspase-3
Groups	TNF-α ng/g/protein	IL-10 pg/mg/protein		ACHe nmol/mg	Caspase 3 (ng/mg)
Control	44.11 ± 0.18	25.10 ± 0.22		36.11 ± 1.01	0.31 ± 0.12
NaB 125/AA 100	32.13 ± 0.08^*	21.10 ± 0.10^*		35.20 ± 1.10	0.28 ± 0.10
NaB 125/AA 200	33.25 ± 0.30*	22.20 ± 0.20		36.35 ± 1.60	0.23 ± 0.12
NaB 125/AA 300	40.33 ± 0.11^*	20.32 ± 0.11^*	35.30 ± 1.60			0.18 ± 0.13
NaB 250/AA 100	36.221 ± 0.20^*	20.60 ± 0.18^*		34.35 ± 1.60	0.27 ± 0.14
NaB 250/AA 200	38.11 ± 0.20^*	22.25 ± 0.30^*	35.20 ± 1.10			0.22 ± 0.11
NaB 250/AA 300	37.32 ± 0.14*	19.12 ± 0.20*	36.35 ± 1.60			0.17 ± 0.11
NaB 500/AA 100	37.53 ± 0.18^*	20.28 ± 0.11^*		35.30 ± 1.60	0.27 ± 0.11
NaB 500/AA 200	34.22 ± 0.20^*	18.10 ± 0.10^*	34.35 ± 1.60			0.21 ± 0.14
NaB 500/AA 300	29.11 ± 0.20^*	20.10 ± 0.10^*	35.20 ± 1.10			0.19 ± 0.12
Values presented as Mean ± S.E.M, ^*p < 0.05 vs. control, number of mice/group = 10, NaB: Sodium Benzoate, TNF α: Tumour necrosis factor –alpha, IL-10: Interleukin − 10: ACHe: Acetylcholinesterase

This study examined the effects of co-administering sodium benzoate at doses considered safe in humans with ascorbic acid also at three different concentrations (with known therapeutic benefits) on body weight, neurobehaviour, antioxidant status and inflammatory response in mice. The result showed an overall decrease in body weight, self grooming, total antioxidant capacity and inflammatory cytokines, mixed response with food intake, locomotor activity, Y maze spatial working memory and anxiety related behaviours and an increase in rearing and radial arm maze spatial working memory.

The ability of diet or dietary composition to positively or negatively impact body weight and/or feed intake has been reported severally [35-39]. In this study, overall the co-ingestion of NAB with ascorbic acid was associated with a decrease in body weight and a mixed response with regards to food intake. In previous studies from our laboratory, the addition of NAB to food at similar concentrations for 8 weeks was associated with an increase in body weight and food intake at the lowest concentration [7, 13], while the fortification of food with ascorbic acid at the same concentrations was associated with a decrease in body weight at the 100 and an increase at 200 mg/kg of feed despite an increase in food intake across all AA concentrations [40]. The result of this study suggests that the combination of NAB and AA could possibly reduce the palatability of food or cause early satiety at most of the combinations resulting in a decrease in food intake and a resultant decrease in body weight. However, the increase in food intake observed when NAB (at 125 and 250) was coadministered with AA at 200 could be due to an improvement in palatability. Therefore, it suffices to say that combining NAB with AA reverses the body weight changes that had been reported to occur with NAB alone [7, 13]. Widely reported is the weight reduction ability of AA [41-43], with studies in humans [43]\ and guinea pigs [42] reporting the weight reduction effects of an AA-chitosan diet, supporting our observations in this study.

The diet and/or dietary constituents have been reported to have the ability to exert either central excitatory or inhibitory effects on open-field behaviours including horizontal locomotion, rearing and grooming (21-23]. In this study, an evaluation of the three parameters measured in the open field paradigm revealed a concentration-dependent decrease in locomotor activity, self grooming and an increase in rearing. The incorporation of sodium benzoate alone into diet in an earlier study had revealed a concentration dependent decrease in locomotor activity, rearing and grooming behaviours [13], while another study reported that dietary AA fortification decreased horizontal locomotion/rearing and increased self-grooming at these same concentrations [40]. The result of this study shows that the combination of NAB and AA modulated the individual effects of NAB diet or dietary fortification of AA on the brain. While NAB diet caused a central depressant effect in mice, NAB/AA combination resulted in a mixed response with a central excitatory effect on rearing and a depressant effect on horizontal locomotion and grooming. While the induction of either a central excitatory or depressant effect is contingent upon the ability of a compound such as NAB and/or AA to cross the blood-brain-barrier and elicit neurotransmitter responses, the totality of the effect on locomotion (horizontal or vertical) and/or stereotypy (grooming) is dependent on the region of the brain in which the effects of the compound are maximal. Reports on the possible effects of NAB on the brain vary considerably with some studies reporting that NAB does not alter brain monoamine levels [44, 45], although a few other studies have demonstrated NAB’s ability to alter levels and/or activities of dopamine and tyrosine hydroxylase [46]. The excitatory or depressant effects of NAB/AA combination on open field behaviours observed in this study could be linked to a summation of the neurotransmitter interactions between NAB and AA.

The results from an earlier study from our laboratory had also revealed the memory impairing effects of NAB at 125 mg/kg of feed in the Y-maze model and at 125 and 500 mg/kg of feed in the radial-arm maze model [13] The results of another study from our laboratory that also administered ascorbic acid added to dry food at similar concentrations as used in this study revealed that the consumption of ascorbic acid at 200 mg/kg of feed was associated with a consistent improvement in memory scores in both the Y and radial arm maze paradigms The response to the combination of NAB and AA in diet was dual. In the radial arm-maze improvement in memory scores was observed at a combination that consisted of the highest concentration of NAB and two lower concentration of AA, although in the Y maze improvement in memory scores was observed at the same concentration of NAB/AA memory feed as in the radial arm maze; memory impairment was observed at all other combinations of NAB/AA in the Y maze. The overall result shows that the effect of combining AA with NAB was both maze dependent and concentration dependent..

The administration of sodium benzoate has been reported to have anxiogenic effects in rodents [45, 47]. In a recently published study from our laboratory the consumption of sodium benzoate at 125 mg/kg of feed was associated with an anxiogenic response in rodents [13]. The effect of dietary fortification with ascorbic acid on anxiety has been studied severally [48-51].. In another study carried out in our laboratory, an anxiolytic effect was observed in animals that were fed ascorbic acid at 300 mg/kg of feed [40]. In this study however the co-administration of ascorbic acid with sodium benzoate resulted in both an anxiogenic and an anxiolytic effect. In animals fed sodium benzoate at the lowest concentration (125 mg/kg of feed, a concentration that had previously been reported to cause anxiety) the co administration of ascorbic acid (at all three concentrations) did not reverse the anxiogenic behaviour even when ascorbic acid was added at a concentration that had previously been reported to have an anxiolytic effect. However, when sodium benzoate at a concentration of 250 mg/kg of feed was combined with ascorbic acid at all three concentrations of AA, significant anxiolysis was observed, meanwhile at 500 mg/kg of sodium benzoate there was no significant difference from control was observed when co administered with AA at 100 and 200, however with AA at the highest concentration anxiety was observed

The results of oxidative stress parameters revealed that co-administration of sodium benzoate/ascorbic acid was associated with an increase in lipid peroxidation and superoxide dismutase activity and a decrease in total antioxidant capacity at all concentrations of NAB/AA (although at NAB125/AA300 the increase in malondialdehyde levels was only visual). In two studies from our laboratory examining the effects of sodium benzoate (125, 250 and 500 mg/kg of feed) and ascorbic acid (100, 200 and 300 mg/kg of feed ) administered alone, we observed that sodium benzoate diet was associated with an increase in lipid peroxidation, superoxide dismutase activity; and a decrease in total antioxidant capacity [7]. While ascorbic acid supplementation was associated with a decrease in lipid peroxidation, an increase in superoxide dismutase and total antioxidant capacity at 200 and 300 mg/kg of feed [40]. There have been reports of the potential of sodium benzoate and ascorbic acid combination to result in oxidative stress in the testis [52]. In this study, it was observed that at the higher concentrations of ascorbic acid (200 and 300 mg/kg of feed) oxidative stress was lower compared to that observed at the lowest concentration of AA (100 mg.kg) suggesting that ascorbic acid at these concentrations possibly attempts to dampen the pro-oxidant effects of NAB. The proinflammatory effects of sodium benzoate had been reported previously, however in this study the combination of ascorbic acid with sodium benzoate in dry feed was associated with a decrease in both pro and anti-inflammatory markers.

The study shows the ability of dry food added NaB/AA to affect neurobehaviour, oxidative stress, and markers of inflammation in the brain of mice. It demonstrates that both beneficial and deleterious effects might be possible, depending on the concentrations ingested in food. While it would seem that increasing the concentration of ascorbic acid and sodium benzoates for the most part is associated with consistent deleterious effects, overall these findings suggest the need for more efforts in investigating the combination. A widening application of the compounds also drives home the imperativeness of such endeavour; as more questions might need to be answered in the journey towards absolute reassurance regarding their safety.

Ethics Approval and Consent to Participate:

All procedures performed on the animals were in accordance with approved protocols of the Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria and within the provisions for animal care and use as prescribed by the scientific procedures on living animals, European Council Directive (EU2010/63).

Human and Animal Rights:

No humans were used in this study. All procedures performed on the animals were in accordance with approved protocols of the Ladoke Akintola University of Technology

Consent for Publication:

Not Applicable.

Availability of Data and Materials:

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflict of interest:

All authors of this paper declare that there is no conflict of interest related to the content of this manuscript.

Source of funding:

This research did not receive any specific grant from agencies in the public, commercial, or not-for-profit sectors.

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Graded effects of dry-feed added sodium benzoate/ascorbic acid combination on neurobehaviour, brain markers of oxidative stress, and markers of inflammation in mice

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Abstract

Figures

1.0 Introduction

2.0 Materials And Methods

2.1 Materials

2.2 Animals

2.3 Feed

2.4 Experimental methodology

2.4.1 Determination of body weight and food intake

2.4.2 Neurobehavioural tests

2.4.2.1 Open field test

2.4.2.2 Memory tests (Y-maze and radial arm maze)

2.4.2.3 Anxiety Model: Elevated plus-maze

2.4.3 Lipid peroxidation (Malondialdehyde)

2.4.4 Antioxidant activity

2.4.5 Tumour necrosis factor-α and Interleukin (IL) -10

2.4.6 Acetylcholinesterase activity and Caspase 3 levels

2.5 Brain homogenization

2.6 Statistical analysis

3.0 Results

3.1 Effect of sodium benzoate/ascorbic acid co-ingestion on body weight and food intake

3.1.1 Effect of sodium benzoate/ascorbic acid co-ingestion on locomotor activity, rearing and self-grooming behaviours

3.1.2 Effect of sodium benzoate/ascorbic acid co-ingestion on working memory in the Y- and Radial-arm maze

3.1.3 Effect of sodium benzoate/ascorbic acid co-ingestion on anxiety-related behaviour

3.1.4 Effect of sodium benzoate/ascorbic acid co-ingestion on oxidative stress parameters and antioxidant status

3.1.5 Effect of sodium benzoate/ascorbic acid co-ingestion on inflammatory markers, brain acetylcholinesterase and caspase-3 levels

Discussion

Conclusion

Declarations

References

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