Animals

The protocol for all animal tests conducted in this work was approved by the animal experimentation committee of the Department of Biochemistry at Imo State University in Nigeria and was assigned the ethical number IMSU/EC/01/2023. The National Institute of Health Care Guide for the Care and Use of Laboratory Animals (NIH publication #85–23, amended in 1985) was followed in all procedures involving experimental animals. Every attempt was made to lessen the suffering of the animals and to employ fewer animals overall for the experiment. The investigations employed male and female Wistar rats and mice. Animals from the Veterinary Research Institute, Vom, Jos, Nigeria, were obtained since healthy animals were needed. Commercial carrier trucks were used to move the animals from Vom, Jos to Owerri, Imo State. After being transported in plastic cages for animal housing, the animals were put in secondary containers. To lessen the effects of transit, the animals were first kept in the animal house of the Department of Biochemistry at Imo State University in Owerri, Imo State, for two weeks before being transferred to the testing room. To ensure rigorous experimental control and minimize variation, animals were individually housed under standard colony conditions. These cages provided a specific pathogen-free environment and allowed unrestricted access to food and water throughout the acclimatization period. They were also kept in a 12-hour light/dark cycle (lights on at 6:00 a.m.), with a temperature of 25 to 27 0 C and a relative humidity of 40 to 60%, which was measured with a CEM hydrometer (DT-615, Shenzhen, China).

Onion husk preparation

Onion husk (Allium cepa L.) was collected from the local market within Owerri, Imo State. The Onion husk was characterised by neutral smell and taste, as well as without any signs of mould infection. The husks were washed again with distilled water and left open in a porous tray for 10 min to remove excess water, then kept in a drying oven at 500C for 48 h. The dried husk was grounded using a mixer-grinder for the formation of onion husk powder and stored at -300C in air-tight plastic containers for further use.

Flavonoid-rich fraction (FRF)

As previously reported by Ekeanyanwu and Njoku (2015), the flavonoid-rich fraction (FRF) of the grounded oven-dried onion husk was produced by solvent-solvent extraction with slight remodelling. Here, 1.65 kg of crushed onion husk was macerated for 72 h at room temperature in 10 L of methanol. A cotton cloth was used to filter the macerate, followed by Whatman filter paper No. 1. Liquid-liquid partitioning of the extract produced the FRF. Chloroform (1 L) was used as a solvent extractor for liquid-liquid extraction. The top layer (methanol residue) was then separated using a 1.5 L liquid-liquid extraction method using ethyl acetate to produce the FRF. After that, the top layer of the ethyl acetate fraction was condensed and used to create the FRF.

Total flavonoids determination

The calibration curve [0.04, 0.02, 0.0025, and 0.00125 mg/ml in 80% ethanol (v/v)] was made using standard quercetin. 75 µl of 5% sodium nitrite was combined with 0.5 ml of the standard solutions and the test sample at 1 g/ml of the FRF. 500 µl of 1 M sodium hydroxide was added after 6 min and after that 150 µl of a 10% aluminium chloride solution was added and let to stand for an additional 5 min. The blank was filled with the equivalent amount of distilled water in place of the 10% aluminium chloride. At 510 nm, the absorbance of the reagent was measured using a blank. The overall flavonoid content was calculated as Mean ± SEM (n = 3) and expressed as mg/g of onion husk extract’s quercetin equivalent using the formula below. The flavonoid content of the FRF was evaluated using the linear regression equation derived from the quercetin standard curve;

Where y = Absorbance of each fraction.

X = Concentration of quercetin from the calibration curve.

Flavonoid content = \(\frac\)

C = concentration of quercetin from calibration curve (mg/ml).

V = Volume of extract in ml and.

M = weight of extract in grams.

The mean of the three readings was used and the total flavonoids content was expressed in milligrams/gram of quercetin equivalent (The concentration of total flavonoids in the FRF was 316.39 ± 16.78 mg/g quercetin equivalent).

Qualitative and quantitative phytochemical analysis

Following established protocols as described by Sofowora 1993; Trease and Evans, 1989, and Ayoola et al. (2008), the onion skin husk powder was phytochemically screened for the presence and makeup of certain phytochemicals.

GC-MS analysis

The Perkin-Elmer Clarus 680 GC (Perkin-Elmer, Inc., USA) was utilized for the GC-MS analysis of FRF. It was outfitted with an Elite-5ms capillary column that was packed with a fused silica column (30 m in length, 0.25 mm in diameter, and 0.25 μm in thickness). The carrier gas was pure helium gas (99.99%) at a steady flow rate of 1 mL/min. An electron ionization energy approach was used for GC-MS spectral detection, with a high ionization energy of 70 eV (electron volts), 0.2s scan time, and fragments spanning from 40 to 600 m/z. A split ratio of 10:1 was employed with an injection quantity of 1µL, and a constant injector temperature of 2500C was maintained. The temperature of the column was even for 30 min at 500 C, then increased at a rate of 100 C per minute to 2800C. Finally, the temperature was elevated to 3000C for 10 min. By comparing the test sample’s retention time (min), peak values, peak height, and mass spectral patterns with those of authentic compounds kept in the National Institute of Standards and Technology (NIST) library, the content of bioactive compounds was found. The names, component composition, and molecular weight of the inquiry materials were found.

FT-IR spectroscopy analysis

The FT-IR analysis was carried out using the Cary 630 FT-IR Spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). Using a high-pressure vacuum pump and Whatman No. 1 filter paper, the FRF was sifted via FT-IR analysis after being centrifuged for 10 min at 3000 rpm. The test sample was diluted to a ratio of 1:10 using the same solvent. A Cary 630 FT-IR Spectrometer operating in the 4000–650 nm wavelength range was used to analyze the extract. The peaks were found and their values were recorded.

Single oral dose toxicity study

An acute oral toxicity study of the FRF was performed using mice according to the Organisation for Economic Cooperation and Development (OECD) guideline 423 (OECD, 2001). Single-dose studies often utilize mice because they require a smaller amount of test compound compared to larger animals. Nine mice were randomly divided into 3 groups of 3 mice each. The FRF and distilled water were orally administered to the mice after overnight fasting at a volume of 10 ml/kg body weight (Ekeanyanwu and Njoku 2014). The mice in group I were administered 300 mg/kg body weight of the FRF dissolved in distilled water. The mice were observed for general behaviour changes; symptoms of toxicity and mortality after treatment for the first 4 h, then over 48 h. Group 2 was administered sequentially at 48-hour intervals with the next higher dose of 2000 mg/kg body weight of the FRF in distilled water when there were no signs of toxicity or mortality showed in group 1 after 48 h of treatment. In parallel, group 3 mice were treated with vehicle (distilled water) to establish a comparative negative control group. All animals were observed at least once during the first 30 min in the first 4 h following vehicle or FRF administration and then once a day for 14 days. According to OECD recommendations (OECD, 2001), this observation was made to determine when clinical or toxicological signs started to appear. Every observation, including alterations to the eyes, mucous membranes, skin, and fur, as well as behavioural tendencies, was methodically documented and kept up to date with a personal file. Observations of convulsions, tremors, diarrhoea, salivation, lethargy, sleep, coma, and mortality were also taken into consideration. The mice in each group were decapitated using a rodent guillotine (Harvard Apparatus, USA) after the study. Their kidney, liver, and heart were promptly removed, and they were then washed in ice-cold saline (0.9% NaCl). Following acute oral toxicity testing, samples from the heart, kidney, and liver were examined histopathologically.

14-Day repeated oral dose toxicity study

For a 14-day repeat-dose toxicity study, healthy male Wistar rats were randomly assigned to four groups (5/sex/group). Vehicle (distilled water) or graded doses of the FRF (500, 1000 and 2000 mg/kg of body weight) were administered to rats by oral gavage once daily for 14 days at a dose of 10 ml/kg of body weight. The food composition and water intake are recorded daily. The body weights of animals were recorded shortly before the administration of the tested substance and at the end of each week. The percentage of body weight change is calculated according to the following equation:

$$\eqalign}\,}\,}\,}\, = \cr & \over }\, \times \,100 \cr}$$

On the 15th day, about 5 ml of blood was collected from the retro-orbital plexus without the use of topical anaesthesia after overnight fasting and sera were prepared from 4 ml of the collected blood by centrifugation at 640 g for 10 min and then stored at 4 °C for different biochemical parameters (Urea, Creatinine, Albumin, Globulin, Total bilirubin, Conjugated bilirubin, Alkaline phosphatase, Alanine transaminase and Aspartate aminotransferase). Analysis of haematological markers, including haemoglobin, white blood cells, neutrophils, lymphocytes, monocytes, Eosinophils, and basophils, was done on the remaining uncoagulated blood from the 5 ml of blood that was collected. Following the collection of blood samples from the rats, the hearts, livers, and kidneys of each group of rats were promptly dissected out and washed in ice-cold saline (0.9% NaCl) after the rats were killed by beheading with a rodent guillotine (Harvard apparatus, USA). The liver, kidney, and heart were removed, fat was removed, and the organs were promptly weighed and blotted with clean tissue paper. Using the following equation, the relative organ weight (ROW) was determined and noted about the body weight:

$$ROW\, = \, \over }\,}\,100$$

Samples from the vital organs (liver, kidney and heart) of acute oral toxicity tests were subjected to histopathological evaluation. They were fixed in 10% buffered formalin, routinely processed and embedded in paraffin wax. Paraffin Sect. (5 μm) were cut on glass slides and stained with haematoxylin and eosin. An experienced pathologist who was unaware of the experimental groups to which each section belonged conducted the analysis. The slides were examined under a light microscope as earlier stated by Ekeanyanwu and Njoku (2014).

Animal model of bipolar mania and extract and/or lithium administration

Considering the rigorous nature of the investigative procedures and the need for ample blood and brain tissue samples for analysis, Wistar rats were chosen as the animal model for this study. Thirty animals were divided into six groups of 5 rats each:

Group I (Normal saline, vehicle),

Group II (Ketamine, 25 mg/kg),

Group III (Ketamine 25 mg/kg + Lithium 45 mg/kg),

Group IV (Ketamine 25 mg/kg + FRF 500 mg/kg),

Group V (Ketamine 25 mg/kg + Lithium 45 mg/kg + FRF 500 mg/kg),

Group VI (Ketamine 25 mg/kg + Lithium 22.5 mg/kg + FRF 500 mg/kg).

The animals in Groups IV, V, and VI were given an oral dose of FRF, whereas the animals in Groups III, V, and VI were given Lithium (twice daily). From the eighth to the fourteenth day, the animals in Groups I and II received the same volume of saline solution (10 ml/kg b.wt) orally. Animals in Groups II, III, IV, V, and VI were also given ketamine, while those in Group I were given a vehicle intraperitoneally. The animals were given a single injection of ketamine or saline on the 15th day of treatment. The Locomotor activity was assessed in an open field apparatus thirty minutes later. The FRF, lithium chloride, and ketamine dosages and treatment times were determined using previous research (Spohr et al. 2019) and the findings of the current study.

Open field test

An open-field device was used to measure locomotor activity (Gazal et al. 2015; Debom et al. 2016). We applied the model that Wang et al. (2017) described to the animals. A thorough explanation of the improved method was previously given by Ekeanyanwu et al. (2021). Briefly, the study uses an open field box, which is a rectangular space with a hard floor that is 60 cm by 60 cm by 40 cm and is composed of painted white wood. Permanent read markers were used at the bottom to divide the floor into 16 equal squares. Every rat was put in a different part of the field, and we timed how much it moved overall every ten minutes. After the assay, 70% alcohol was used to clean the area and allow it to dry completely before adding a new rat to eliminate olfactory bias.

Brain sample preparation and biochemical analysesBrain samples

Following behavioural assessments, rats were euthanized by decapitation with a rodent guillotine (Harvard Apparatus, USA) and the cerebral cortex and hippocampus were dissected on ice. Samples were immediately frozen at -80 °C until further processing.

Homogenization

Frozen brain regions were thawed on ice and homogenized in ice-cold KClKH2PO4 buffer (12 mM KCl, 0.038 mM KH2PO4, pH 7.4) using a Polytron PT 3100 D homogenizer (Thomas Scientific, Thomas Scientific). Homogenates were centrifuged at 10,000 g for 10 min at 4 °C, and the supernatants were collected for subsequent analyses.

Thiobarbituric acid reactive species (TBARS) levels

Lipid peroxidation was assessed by measuring TBARS using a modified version of the Wilbur et al. (1949) method. Protein precipitation was performed with 10% trichloroacetic acid (TCA), and the supernatants were reacted with thiobarbituric acid (TBA) reagent at 95 °C for 20 min. Absorbance at 532 nm was measured in a spectrophotometer, and TBARS levels were calculated as nanomoles of malondialdehyde (MDA) equivalents per milligram of protein using a standard curve.

Superoxide dismutase (SOD) activity

SOD activity was determined based on its ability to inhibit pyrogallol autoxidation, as described by Misra and Fridovich (1977) with modifications. The reaction mixture containing pyrogallol, carbonate buffer, and homogenate was initiated with hydrogen peroxide (H2O2). The decrease in absorbance at 420 nm due to inhibition of autoxidation was monitored spectrophotometrically. SOD activity was expressed in units per milligram of protein based on the amount of enzyme required for 50% inhibition and a standard curve.

Catalase (CAT) activity

The enzymatic decomposition of H2O2 was measured to assess CAT activity using the method of Aebi (1984). The reaction mixture containing phosphate buffer, homogenate, and H2O2 was monitored for oxygen evolution by measuring the change in absorbance at 240 nm over time. CAT activity was expressed in units per milligram of protein based on the initial rate of change in absorbance.

Glutathione peroxidase (GPx) activity

GPx activity was determined according to Pagha and Valentine (1967) with modifications. The reaction mixture containing reduced glutathione (GSH), sodium azide, NADPH, glutathione reductase, and homogenate was initiated with H2O2. The decrease in absorbance at 340 nm due to NADPH oxidation was monitored spectrophotometrically. GPx activity was expressed in units per milligram of protein based on the rate of NADPH oxidation and a standard curve.

Acetylcholinesterase (AChE) activity

AChE activity was measured using the Ellman et al. (1961) method with modifications. The reaction mixture containing homogenate, 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB), and acetylthiocholine iodide was monitored for the formation of the yellow 5-thio-2-nitrobenzoate anion at 412 nm. AChE activity was expressed in units per milligram of protein based on the initial rate of change in absorbance.

Statistical analysis

Data were reported as mean ± SEM, with p < 0.05 considered significant. Group differences were assessed by one-way ANOVA followed by Bonferroni post-hoc tests where appropriate. Interactions between control groups (saline, ketamine) and treatment groups (ketamine + lithium, ketamine + FRF, combined regimens) were evaluated using one-way ANOVA with Bonferroni post-hoc for all measured outcomes (p < 0.001).