Chapter 3: Materials & Methods
Equipment Used
The following equipment was utilized throughout the study:
- Animal Housing Unit: Standard laboratory animal housing facilities with adequate ventilation and controlled temperature were used for housing the Swiss mice.
- Scales: Electronic weighing balances (Sartorius, Germany) were employed for the accurate measurement of plant materials and dosages.
- Rotarod Apparatus: For assessing the locomotor activity of mice, a rotarod apparatus (Ugo Basile, Italy) was utilized.
- Elevated Plus Maze: Used to evaluate exploratory behaviors and anxiety levels in the animals (Harvard Apparatus, USA).
- Microcentrifuge: A microcentrifuge (Eppendorf, Germany) was used for the centrifugation of brain homogenates.
- Homogenizer: A tissue homogenizer was used to prepare brain tissue samples.
- Spectrophotometer: A UV-Vis spectrophotometer (Shimadzu, Japan) was used for assessing the concentration of phytochemicals in the extracts.
Chemicals and Reagents Used
The chemicals and reagents employed in this study included:
- Aluminum Chloride (AlCl3): A neurotoxin used to induce neurotoxicity in the animal model (Sigma Aldrich, USA).
- Ethanol: Used for extracting the active phytochemical compounds from plant materials (Merck, Germany).
- Dimethyl Sulfoxide (DMSO): Utilized as a solvent for the administration of certain compounds (Sigma Aldrich, USA).
- Phosphate Buffered Saline (PBS): Used for diluting and preparing the solutions (Thermo Fisher Scientific, USA).
Collection of Plant Materials
Kolaviron from Garcinia kola
Kolaviron was sourced from locally purchased Garcinia kola seeds, obtained from Okuku Market, located in the vicinity of the school campus. The seeds were verified for authenticity and quality before proceeding with extraction.
Bryophyllum
Leaves of Bryophyllum, a member of the Crassulaceae family, were harvested from the school botanical garden. Collection was conducted ensuring that plants were free from any visible diseases or external damage. Plant materials were properly identified and authenticated with the assistance of a botanist.
Methods: Aqueous Extraction of Plant Phytochemicals
The extracts of Kolaviron and Bryophyllum were prepared using a cold aqueous extraction method. The steps are outlined as follows:
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Preparation: Freshly collected plant materials were washed thoroughly with distilled water to remove contaminants. They were then air-dried for seven days in the shade to prevent degradation of active compounds.
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Grinding: The dried materials were ground into powder using a mortar and pestle.
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Extraction: Thirty grams of the powdered plant material were mixed with 300 mL of distilled water and allowed to soak for 72 hours at room temperature. The mixture was then filtered through a muslin cloth followed by Whatman filter paper to obtain the clear extract.
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Concentration: The filtered extract was evaporated at room temperature to concentrate the solution. The final volume was adjusted to obtain the desired concentrations for treatment.
Experimental Animals
Swiss mice (Mus musculus) of both sexes, aged 6-8 weeks, were obtained from the animal house of the institution. A total of 70 mice were used for the experiments, with all necessary ethical considerations and protocols approved by the Institutional Animal Ethics Committee. The mice were allowed to acclimatize for one week before experimentation in standard laboratory conditions (temperature 22±2°C, humidity 50±5%).
Duration of Research
The research was conducted over a period of six weeks, starting from January 31st and concluding on March 10th. Acute studies were carried out over the first two weeks of this period.
Experimental Design
The experimental design was structured to assess the comparative effects of Kolaviron and ethanolic leaf extract of Bryophyllum on learning, memory, locomotion, and exploratory behavior following AlCl3-induced neurotoxicity.
Animal Grouping and Treatment
The Swiss mice were divided into seven groups (n=10 for groups 2-7 and n=9 for group 1) as follows:
- Group 1: Control (saline solution) (9 mice)
- Group 2: Aluminum Chloride only (10 mice)
- Group 3: Kolaviron only (10 mice)
- Group 4: Crassulaceae only (10 mice)
- Group 5: Aluminum Chloride + Crassulaceae (10 mice)
- Group 6: Aluminum Chloride + Kolaviron (10 mice)
- Group 7: Aluminum Chloride + Kolaviron + Crassulaceae (10 mice)
Induction of AlCl3
Neurotoxicity was induced in the experimental groups (Group 2 to Group 7) with an intraperitoneal injection of Aluminum Chloride at a dosage of 50 mg/kg body weight, administered once daily for fourteen days.
Sacrifice of Test Animals
At the end of the treatment period, animals were humanely sacrificed using cervical dislocation after anesthesia with isoflurane.
Homogenization and Centrifugation of Brain
Brain tissue samples were collected and immediately rinsed with ice-cold PBS. Each brain was homogenized in 10% W/V phosphate buffer at 4°C, followed by centrifugation at 3,000 rpm for 15 minutes at 4°C. The supernatants were stored at -20°C for subsequent biochemical analyses.
Statistical Analysis
Results were analyzed using Statistical Package for Social Sciences (SPSS) version 22.0. Data were expressed as mean ± standard error of the mean (SEM). One-Way ANOVA was employed to compare means among different treatment groups, followed by Post Hoc Tukey’s test for pairwise comparisons. A p-value of less than 0.05 was considered statistically significant.
References
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