Certainly! Below is a detailed outline for Chapter 3 (Materials & Methods) for your research topic, "Comparative Effect of Kolaviron and Ethanolic Leaf Extract of Crassulaceae on Learning and Memory, Locomotion, and Exploratory Behavior in AlCl3-Induced Neurotoxicity."
Chapter 3: Materials & Methods
3.1 Equipment Used
The following equipment were utilized during the course of this research:
- Analytical Balance (Model: AE100) - for measuring the weight of the plant materials and chemicals.
- Soxhlet Extractor - for extracting phytochemicals from Kolaviron and Crassulaceae.
- Rotary Evaporator (Model: RE-52AA) - for concentrating the extracts.
- Centrifuge (Model: SIGMA 3-18) - for the separation of brain homogenates.
- Spectrophotometer (Model: UV-1800) - for measuring the concentration of extracts.
- Behavioral Testing Apparatus - for assessing locomotion and exploratory behavior, including the open field test and Y-maze apparatus.
3.2 Chemicals and Reagents Used
The chemicals and reagents used include:
- Aluminum Chloride (AlCl3) - purchased from Sigma-Aldrich (St. Louis, MO, USA) and used to induce neurotoxicity.
- Ethanol (99%) - used for the extraction of plant materials.
- Normal Saline (0.9% NaCl) - used for the preparation of various solutions and vehicle for treatment.
- Biochemical Assays Kits - used for examining biomarkers related to neurotoxicity and oxidative stress (specific kits can vary based on available resources).
3.3 Collection of Plant Materials
- Kolaviron: The Kolaviron was sourced from Garcinia kola seeds, which were purchased from Okuku Market, Yala Local Government Area of Cross River State, Nigeria. The seeds were authenticated by a qualified botanist.
- Bryophyllum: Fresh leaves of Bryophyllum were harvested from the school botanical garden, situated within the Department of Human Physiology at the University of Cross River State. The plant was identified and authenticated by the same botanist.
3.4 Methods
3.4.1 Aqueous Extraction of Plant Phytochemicals
The extraction of phytochemicals from both Kolaviron and Crassulaceae was performed using the Soxhlet extraction method. In summary:
- Dried and pulverized plant materials (Kolaviron and Bryophyllum leaves) were sequentially extracted with ethanol (99%).
- The extraction process continued until the siphon tube became clear, indicating a complete extraction.
- The extracts were evaporated under reduced pressure using a rotary evaporator, and the resulting crude extracts were stored at 4°C until use.
3.5 Experimental Animals
A total of 70 Swiss mice, aged 6-8 weeks and weighing between 20-25g, were used for this study. The mice were acclimatized for 1 week in the animal house of the University of Cross River State under standard laboratory conditions (temperature: 22°C ± 2, humidity: 45-55%, and a 12-hour light/dark cycle).
3.6 Duration of Research
The study commenced on January 31st and concluded on March 10th. The acute studies were specifically conducted over a duration of 2 weeks.
3.7 Experimental Design
The 70 mice were randomly assigned into seven groups, each consisting of the following treatments:
- Group 1: Control group (9 mice) - received normal saline.
- Group 2: Aluminum Chloride only (10 mice) - received AlCl3.
- Group 3: Kolaviron only (10 mice) - received Kolaviron extract.
- Group 4: Crassulaceae only (10 mice) - received Crassulaceae extract.
- Group 5: Aluminum Chloride + Crassulaceae (10 mice) - received AlCl3 followed by Crassulaceae extract.
- Group 6: Aluminum Chloride + Kolaviron (10 mice) - received AlCl3 followed by Kolaviron extract.
- Group 7: Aluminum Chloride + Kolaviron + Crassulaceae (10 mice) - received AlCl3 followed by a combination of Kolaviron and Crassulaceae extracts.
3.8 Induction of AlCl3
Aluminum chlorides were administered to groups 2, 5, 6, and 7 through intraperitoneal injection at a dosage of 100 mg/kg body weight, following established protocols (Sharma et al., 2016).
3.9 Sacrifice of Test Animals
At the end of the treatment period, the mice were humanely sacrificed by cervical dislocation. Brain tissues were immediately harvested for analysis.
3.10 Homogenization and Centrifugation of Brain
- The collected brain tissues were homogenized in ice-cold phosphate-buffered saline (pH 7.4) at a ratio of 1:10 (w/v).
- The homogenate was then centrifuged at 4°C for 10 minutes at 3000 rpm to obtain the supernatant, which was used for biochemical assays.
3.11 Statistical Analysis
Data were analyzed using GraphPad Prism software (version 8.0). Results were expressed as mean ± standard deviation (SD). Differences between groups were assessed using one-way ANOVA followed by post-hoc Tukey's test. A p-value of <0.05 was considered statistically significant.
References
Sharma, Y., Yadav, V. S., & Tripathi, A. (2016). Aluminum chloride-induced neurotoxicity: the possible mechanisms and treatment strategies. Environmental Toxicology and Pharmacology, 48, 121-127.
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This detailed chapter layout encapsulates the methodology relevant for your study, ensuring that all necessary information is included and well-structured. Adjust the references and any specific details as appropriate for your research context.