Barbigerone prevents scopolamine-induced memory impairment in rats by inhibiting oxidative stress and acetylcholinesterase levels

The current study was designed for the evaluation of barbigerone on memory loss. In this experimental study, 24 Wistar rats (n = 6) were used. Control rats and scopolamine (SCOP)-treated control group rats were orally administered with 3 ml of 0.5% sodium carboxymethyl cellulose (vehicle), whereas barbigerone was (10 and 20 mg kg−1) administered orally to the rats from the test group. During the 14-day treatment, control group rats were given 3 ml kg−1 day−1 saline, and all other groups were administered SCOP (1 mg kg−1 day−1, i.p.) 1 h after barbigerone p.o. treatment. The spontaneous alternation activities, learning capacities of a rat's memory were tested with Morris water maze and Y-maze. Reduced glutathione, malondialdehyde, acetylcholine esterase (AChE) and catalase (CAT) levels were measured in rat brain tissue as oxidative stress/antioxidant markers. Moreover, the levels of tumour necrosis factor, interleukin-6 (IL-6) and IL-1β were also estimated. Treatment with barbigerone in SCOP-administered rats dramatically reduced SCOP-induced neurobehavioural deficits, oxidative stress and neuroinflammatory markers, improved endogenous antioxidants, and restored AChE activity. By improving cholinergic function and reducing oxidative damage, barbigerone could mitigate the effects of SCOP-induced changes in the brain.


Animals
Male Wistar rats (180 ± 20g) were acclimated to the working environment. Laboratory conditions were used, with 12 h of light and darkness each, and the animals had free access to food and water. The procedure was permitted by the Institutional Animal Ethics Committee (IAEC-TRS/PT/021/006-021) and adhered to CPCSEA rules.

Acute toxicity study
Barbigerone was tested for acute oral toxicity (LD50) by applying the OECD's-423 criteria. Barbigerone was dissolved in a 0.5% solution of sodium carboxymethyl cellulose (Na-CMC) and administered orally at the maximum dosage as reported previously [41,42].

Behavioural parameters 2.5.1. Morris water maze paradigm test
Long-term memory impairment and spatial learning were determined using the MWM test. Aksoz et al.'s [46] methods were used to measure the MWM examination. The water in the tank was maintained at a constant temperature of 24 ± 10°C using creamy milk. The tank had four equally spaced quadrants. The platform was positioned in one quadrant and placed at 2.0 cm depth on the water surface. The platform was placed in the same quadrant throughout the experiment. On each day of the learning activity, one of royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230013 the animals had been placed in the tank's randomly selected positions (three trials for each event). For the commencement of the experiment, the animals were brought further into the tank. As soon as the animals were found and stepped out onto the platform the experiment terminated, and mean escape latency was calculated. Sixty seconds was the highest exposed time. Another 60 s escape latency was observed if the animal failed to reach the platform even after initial pushing in 60 s. Following sessions, the animal was kept on the stand for 20 s. In all three trials, the animals were gently cleaned, followed by being placed in cages. On the fifth day of the consolidation trial, the rat's memory of the place of the hidden platform for 60 s was evaluated. The platform was pulled out from the tank during this stage. Thus, the time spent in the target quadrants where the hidden platform was placed had been noted.

Y maze test
The Y-maze (45 × 12 × 35 cm) was composed of three identical arms that deviated at a 120°angle from one another, and a mid-symmetrical triangular area labelled A, B and C. During a 5 min session, the animal was positioned towards the end of one arm and had free access to movement throughout the maze. Animals must use working memory for spontaneous change, and the y-maze can be implemented for the assessment of spatial working memory. Therefore, they should trace the most recently visited arms, and this information should be renewed regularly. When the rat's hind paws were totally on the arm, the arm entry was considered complete. An alternate triplet set is characterized by successive entries into its three arms (i.e. ABC, BCA and CAB). Animals were evaluated for their memory loss as per the previously published protocol [47].
2.6. Biochemical parameters 2.6.1. Tissue homogenization Wash brain tissue was prepared in Isotonic saline (ice-cold), and phosphate buffer (0.1 M, pH 7.4, ice-cold) was used for the preparation of tissue homogenate whereas the biochemical analysis was performed on the supernatant [23,46,48,49].

Acetylcholin esterase activity
Acetylcholin esterase (AChE) activity (µM mg −1 protein) was calculated as per the protocol published by Ellman et al. [50]. Briefly, homogenized aliquoted brain tissue was centrifuged at 14 000 × g for 5 min after being dissolved in 0.1 M ( pH 7.5) PBS. The clear supernatants were separated to determine AChE activity and estimated using the kit as per the manufacturer's instructions. Briefly, a weighed amount of reagent was dissolved in the assay buffer to prepare the fresh working reagent.
In a 96-well plate, µl of sample and freshly prepared working reagent (190 µl) were combined by gently tapping the plate. As a reference, 200 µl of water (an assay blank) and 200 µl of calibrator were poured into separate wells. After incubating the samples at room temperature for about 2 min, the initial absorbance at 412 nm was measured. The plate was continuously incubated at room royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230013 temperature, and the final absorbance was noted after 10 min of reaction. The activity of AChE was determined and measured as U mg −1 protein.

Endogenous antioxidants
Reduced glutathione (GSH): GSH levels in the homogenates of brain tissue were measured following the method described by Ellman [51]. Proteins were precipitated by adding 100 µl (25%) TCA to 500 µl of homogenate, which was then centrifuged for 5 min at 4000 rpm, and the supernatant was collected. The supernatant (300 µl) was mixed with 500 µl of phosphate buffer (0.1 M, pH 7.4), and 200 µl of DTNB (10 mM) was added and incubated for 10 min. Absorbance was measured at a wavelength of 412 nm against a blank [36]. GSH activity was measured in U mg −1 protein.
Superoxide dismutase (SOD): SOD activity was assessed as per the methods given by Misra & Frodvich [52]. The activity of the SOD enzyme was carried out by a photochemical method that involved the addition of 1.5 ml of the supernatant to a reaction mixture comprising 100 mM Tris/HCl (pH 7.8), 75 mM NBT, 2 M riboflavin, 6 mM EDTA and 200 l of the reaction mixture. The detection of blue formazan was carried out by measuring the change in absorbance at 560 nm resulting from the formation of formazan. The SOD activity was quantified as U mg −1 . SOD activity was measured as U mg −1 protein.
Catalase (CAT): in the cuvette, 0.1 ml of the supernatant was added to 1.9 ml of phosphate buffer ( pH 7.0, 50 mM). Subsequently, the reaction was started by adding 1.0 ml of freshly made H2O2 (30 mM). CAT activity is calculated as U mg −1 protein [37].
Malondialdehyde: the level of malondialdehyde (MDA) in the homogenate of brain tissue was determined by the Wills method. After being placed in boiling water for 30 min, a test tube with 0.1 ml tissue homogenate, 1 ml TCA (10% w/v) and 1 ml TBA (0.67% w/v) was added. Subsequently, the test tubes were cooled in an ice bath for 10-15 min and centrifuged at 4000 × g for 10 min. After separating the clear, pink supernatant, the absorbance was measured at 532 nm [38].

Pro-inflammatory cytokines
Immunoassay kits were used to measure IL-1β, IL-6 and TNF-α levels in the homogenate of brain tissue samples. Marker concentrations expressed in pg ml −1 protein were calculated using standard curves.

Statistical analysis
As a result of using GraphPad, all results are presented as means with standard errors of the means (s.e.m.). ANOVA was used for behavioural and biochemical parameters followed by Tukey's post hoc analysis. A two-way ANOVA followed by a Bonferroni post hoc test was used to evaluate the MWM test. The significance level for the study was set at p < 0.05.

Acute oral toxicity studies
Barbigerone was tested for acute oral toxicity (LD50) as per the OECD's 423 criteria. During the 14-day timeframe, no mortality or adverse effects were recorded as shown in table 1. Based on safety data 10 and 20 mg kg −1 barbigerone doses were selected.

Morris water maze
In the MWM test, SCOP impaired rats' memory, as a greater ability to escape from swimming and step on the immovable platform was observed as a result of latency.
The MWM test evaluated cognitive performance. During learning trials in all groups, the trained rats' mean escape latency was reduced. SCOP-induced rats showed significantly increased latency time on the second, third and fourth days of training trials when compared to saline-control rats ( p < 0.0001). Twoway ANOVA by a Bonferroni post hoc test showed that barbigerone administration significant reduced latency time in SCOP-treated rats ( p < 0.0001). Additionally, the post hoc test showed that barbigerone royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. The consolidation phase evaluates how effectively the rats retained and learned the hidden platform position during the 4 days of the trial. One-way ANOVA showed that in SCOP-treated rats less time was spent in the target quadrant in comparison to the saline-control rats, and barbigerone treatment significantly influenced the same ( p < 0.0001). Additionally, the post hoc test showed that barbigerone (10 and 20 mg kg −1 ) enhanced time spent in the target quadrant in comparison to SCOP-treated rats [F 3, 20 = 34.26, ( p < 0.0001)] (figure 3b).

Y-maze test
Spontaneous change and total arm entries were lower in SCOP-treated animals compared to control animals. The Y-maze test results were statistically substantial ( p < 0.0001). Barbigerone-10 and 20 mg kg −1   Figure 4 represents the results of the Y-maze test (figure 4a,b).

Endogenous antioxidants status
The number of antioxidants (GSH, SOD and CAT) was reduced in SCOP-administered groups. The SCOP control group had considerably ( p < 0.0001) less SOD, GSH and CAT than the control rats. Barbigerone

Pro-inflammatory cytokines
The IL-1β, IL-6 and TNF-α were significantly ( p < 0.05) elevated in SCOP-treated rats in comparison to control rats. Administration of barbigerone (10 and 20 mg kg

Discussion
The effect of barbigerone on memory impairment induced by SCOP was studied using exteroceptive behavioural tasks, including the MWM and Y-maze tests. These paradigms have mostly been used for assessing animal learning and memory, most notably rodent behavioural influences [39]. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230013 In animal models of neurodegenerative illnesses, SCOP-induced memory impairment is extensively used to examine the causes and therapies of cognitive impairment [53].
Working memory is evaluated using the total arm entries and spontaneous change score in the Y-maze test [39]. In this study, total arm entries and spontaneous change decreased more in the SCOP group than in the control group. This shows that SCOP-treated animals have poor working memory. Barbigerone administration to SCOP-treated rats enhanced total arm entries and spontaneous change. These findings suggest that barbigerone protects treated rats against memory impairment caused by SCOP. Learning ability and long-term spatial memory can be assessed by the MWM test [1,2,12]. SCOP treatment delayed the escape latency indicating decreased learning ability and spatial memory in rats as well as reduced time spent in the target quadrant on the fifth day of the trial. Barbigerone treatment to SCOP-treated animals improved escape latency in the MWM paradigm. These results point to barbigerone's ability to protect animals from SCOP-induced memory deficits.
An ACh receptor antagonist, SCOP interrupts cholinergic neurotransmission and can cause learning and memory issues [45,54]. Learning and memory are both aided by the central cholinergic system [1], and ACh is a key neurotransmitter that modulates intellectual performance and learning processes [1,55]. Enzyme AChE metabolizes ACh in the synaptic cleft to acetic acid and choline [56]. On the other hand, AChE activity might lead to a lack of ACh and cognitive deficit [57]. In the current study, SCOP-induced animals improved AChE levels compared to control rats. However, barbigerone treatment with 10 and 20 mg doses to SCOP-treated animals significantly decreased AChE activity. These findings suggest that barbigerone can protect treated rats from SCOP-induced AChE activity.
Along with cognitive function, ACh is a key component of the cholinergic anti-inflammatory system [58]. Cholinergic anti-inflammatory mechanisms regulate IL-6, IL-1β and TNF-α, which promote inflammation and facilitate TNF-α separation, resulting in an inflammatory response. It also triggers and causes the death of nerve cells and synapses that contribute to neurodegeneration [58,59]. SCOP treatment increased IL-1β, IL-6 and TNF-α levels in the treated animals. Barbigerone therapy of SCOPtreated rats restored IL-6, TNF-α and IL-1β levels. The cytokines are proteins that are involved in the regulation of inflammation and the immune system, and when they are increased due to SCOP, this suggests that inflammation occurred. Barbigerone helped to restore the balance of the immune system by reducing the production of these cytokines, i.e. IL-6, TNF-α and IL-1β levels.
Along with the cholinergic theory, oxidative stress has been considered a probable cause of neurocognitive illness [60]. SCOP dramatically exacerbated oxidative stress in the rats, as demonstrated by greater MDA levels and lower levels of CAT, GSH and SOD [61,62]. Oxidative stress-driven illness occurs when levels of peroxides and ROS surpassed endogenous antioxidant defences [20,22]. In the brain, polyunsaturated fatty acids are damaged by lipid peroxidation [21,49]. Furthermore, the brain is especially prone to oxidative damage due to its lack of antioxidant defence mechanisms [21,49]. The use of barbigerone restores antioxidants and reduces MDA levels. Barbigerone's antioxidant properties may account for its neuroprotective effects in rats suffering from SCOP-induced neurotoxicity. These findings also suggest that barbigerone may have neuroprotective effects on the brain by reducing oxidative stress and improving cholinergic function. The limitations of the study are of short duration with the use of minimum animals. Based on the available evidence, this is the first study to show that barbigerone can royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230013 protect against SCOP-induced memory loss in rats. This mechanism needs to be confirmed by further studies including immunohistochemistry and molecular studies, including western blotting, RT-PCR, tissue immunohistochemistry also with other model amyloid oligomers injections, streptozotocin and other genetic modes.

Conclusion
During the 14-days treatment schedule we observed that barbigerone could mitigate these SCOP-induced alterations in the brains of SCOP-treated rats by improving cholinergic function and decreasing oxidative damage. The current investigation reveals that SCOP-induced behavioural and physiological alterations in rats are reduced by the barbigerone, a flavonoid mainly lowering levels of the inflammatory response. However, more studies need to be performed to determine the impact of barbigerone on persons with memory problems.
Ethical. The protocol used in this study was approved by the TRS, India-Institutional Animal Ethics Committee -IAEC-TRS/PT/021/006-021, as per CPCSEA and were reported in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) requirements [63]. Institutional Animal Ethics Committee (IAEC/TRS/PT/021/006-021).
Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material https://10.6084/m9.figshare.21961622 [64]. The data are provided in the electronic supplementary material [65].