Introduction
This section provides an extensive discussion of the methodology behind this experimental biological study and a detailed explanation of the procedures required to prepare the critical reagents. Combined with a detailed description of the study’s laboratory portion, this section sheds light on the origin of the chemicals and animals used for testing. In particular, The research project included the collection and preparation of plant materials, namely extracts of Punica granatum L. and Nerium oleander, a direct biological experiment on animals, and blood and tissue analyses. Thus, the section is a useful summary of various information and experimental facts together.
Chemicals
An essential part of the biological experiment was to determine the specific antibody properties of the test rats, so a highly sensitive radioimmunoprecipitation assay was chosen. The buffer solution and protease inhibitor were preliminarily purchased from Thermo Scientific (Massachusetts), an American scientific equipment supplier. Colorimetric kits were purchased from Elabscience Biotechnology inc., Texas. Finally, 2 mL test tubes with ceramic beads for homogenization of the working mixture were purchased from Omni International (Georgia), also the USA.
Collection of Plant Material, Punica granatum L.
To analyze the biochemical properties of Punica granatum L. peel samples, the fruit set was collected from local farms in three districts of Saudi Arabia: Khiasah, Baljurashi, and Al-Baha. Subsequently, the pomegranate specimens were subjected to organoleptic treatment and washing at the Medical Research Center of Umm Al-Qura University, Saudi Arabia, before being sent to the extraction step.
Preparation of Dried Pomegranate Peels
Preparation of Punica granatum L. peel extracts was carried out in five consecutive stages. At each of them, biological samples were carefully examined for the possibility of failure, while contaminated objects were removed from the collection. First, the fruit was sanitized under distilled water, after which the grains, pulp, and juicy pomegranate fragments were excluded. The peels were dried for ten hours at 45℃ and 60 mmHg in an SVAC4 vacuum oven (the USA made: Sheldon, Inc.). Then, the pomegranate peels pre-cooled at room temperature for 30 minutes were dispersed into a fine powder using a razor blade. For further use, the samples were placed in a sterile sealed container that excluded contamination.
Alcohol Extraction Step
The prolonged chemical extraction stage took ten full days, during which dried pomegranate peels (a sample of 6 g) were placed in 200 mL of 80% methyl alcohol solution (Sajjad et al.). Two measures were taken to avoid the formation of a natural precipitate and to make the extraction effective. First, the mixture was kept on a magnetic stirrer at 2,000 rpm for the entire period. Second, the methyl alcohol was isolated every 24 hours and placed in a glass sterile glass (Germany made: Duran). The liquid with the peel extract was filtered on Whatman paper, after which the excess moisture and alcohols were evaporated in an Eppendorf Concentrator Plus at 45℃ (Germany made).
Oleander Extraction
Another source of plant matter other than Punica granatum L. was the oleander tree, Nerium oleander, used in this biological study as toxin preparation for mice. The raw material was pre-purchased from a local seedling store and transported to the research laboratory. Since both the vegetative and generative parts of the plant are rich in beneficial molecules, it was decided to use green leaves and pink flowers for extraction. For this purpose, a preliminary sanitation step was carried out, including the secondary sampling of the best plant samples, washing with distilled water, and drying in a desiccator at 40℃. Utilizing a new razor blade, the dried plant fragments were finely crushed into a fine powder.
For extraction, 500 g of the resulting oleander powder was vigorously mixed with a significant amount of 90% ethyl alcohol and stirred vigorously by hand. A series of successive filtrations on Whatman paper was used to generate a liquid portion carrying Nerium oleander extracts. Before further application, the sample was transferred to a sterile, airtight storage container and placed in a refrigerator at 5℃ (Tittel and Wagner, 1981).
Preparation of Animal Material
The object of the biological study in this project was white albino rodent BALB/c, on which the effects of previously synthesized plant preparations and chemicals were investigated. For this purpose, the collection of mice ranging in weight from 19 to 21 g and 6 to 8 weeks of age were preliminarily collected. The mice were purchased from a local livestock market and transported to the research laboratory. Wide polycarbonate cages with wood bedding and ten mice each were prepared to house the rodents under laboratory conditions. Comfortable living conditions included the creation of light and food instructions. Thus, the cages had air ventilation and automatic light and dark cycle with 12-hour intervals at an average temperature of 23±2℃. The diet included classic chow food, which, coupled with an abundance of clean drinking water, was available to the rodents during the two weeks of rearing.
Separation of Mice and Biological Testing
A random sampling tool was used to divide the collection of reared rodents into four identical groups of ten animals. Each group of albino mice was offered a special diet that included previously prepared drugs and chemicals. The condition of each mouse and each group was monitored regularly for pathological clinical manifestations. A description of every group’s diet is given below:
- Group I. This is the control group, in which the mice were fed classic chow food for rodents and pure, potable water.
- Group II. This is the test group in which the mice were fed the classic chow food for rodents and clean, potable water. In addition, an injection of extracted Punica granatum L. was injected into the intragastric gavage of rodents using a long flexible syringe (obtained earlier) at a rate of 500 mg per kilogram of body weight.
- Group III. This was a test group in which the mice also received a normal diet in the form of chow food and drinking water. The biological intervention consisted of injecting extracted Nerium oleander (obtained earlier) into the intragastric gavage using a flexible long syringe at the rate of 200 mg per kilogram of rodent body weight twice a week. At the end of two weeks, an additional experiment was performed for this group. Twice a week after a twelve-hour overnight fast, the animals were injected with acetaminophen (Panadol baby drops 100 mg/mL) at a rate of 300 mg/kg and gentamicin (baby drops) at a rate of 50 mg/kg body weight. The choice of data for the experimental line was consistent with the studies of Kane et al. (2016) and Lebrun et al. (1999).
- Group IV. This is the test group of mice that underwent the most significant intervention. A diet of chow feed and drinking water was combined with an intragastric injection of Punica granatum L. at a rate of 500 mg/kg, Nerium oleander (200 mg/kg), and acetaminophen (300 mg/kg) mixed with gentamicin (50 mg/kg). Thus, the rodents in this group received all the drugs and chemicals that the representatives of the other three groups received in total.
Biochemical Blood Analysis and Organ Preparation
After the four groups’ 64-day course of unique nutrition and 12 injections of biological preparations, two blood tests, total and EDTA, were taken from each rodent representative, after which the animals were put to death.
Tissues from mouse hearts, livers, and kidneys were collected for extra histological analysis. Plain tubes were sent for general blood analysis, which included basic biochemical metrics: glucose, triglyceride, cholesterol, creatinine, and urea levels, as well as HDL-C, and LDL-C. Additionally, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), total creatinine kinase (CK), CK-MB, aspartate aminotransferase (AST), and troponin I was measured in samples. The analysis was performed using a Cobas Integra 400 plus analyzer (Roche, Switzerland). On the other hand, the EDTA blood samples were subjected to laboratory examination on a CBC Mindray BC-2800 (China made: Mindray) to obtain a complete biochemical profile.
Histological Analysis
2 mL silica beads were used to obtain a homogenized organ mixture. Pretreatment of the sample involved chemical degradation of the tissue while preserving unique cellular properties. For this purpose, 700 μL of radioimmunoprecipitation buffer combined with 7 μL of protease inhibitor was added to small organ pieces weighing approximately 1 mg. Subsequently, 2 mL microtubes containing the mixture were placed in a silica beads homogenizer three times, with each session lasting 99 seconds. Then, the homogenized mixture was placed in a SIGMA 1-14 k centrifuge (Germany made) at the following basic settings: -1℃ and 15,000 rpm for 30 minutes. The supernatant after centrifugation was transferred to an Eppendorf tube and subjected to the same centrifugation session. The sample was stored at -20℃ for further histological analysis.
Conducting Basic Biological Experiments
Lipid Peroxidation Products
A colorimetric setup with a base wavelength setting of 534 nm was utilized to monitor the synthesis of a lipid peroxidation product having a pinkish color. The chemical reaction defining this process occurred in an acidic environment between thiobarbituric acid and malondialdehyde. The following reaction conditions were used: 95°C for 30 minutes.
Carbonyl Carbon
The wavelength utilized for the detection of carbonyl carbon activity was 366 nm. Specifically, carbonyl carbon reacted with dinitrophenylhydrazine to initiate Schiff base formation, which could be detected colorimetrically.
Reduced Glutathione
The wavelength for the detection of the yellow complex salt from the interaction of glutathione with 3,5-dinitrobenzoic acid was 405 nm.
Catalase
The wavelength for the detection of the yellow complex from the enzymatic decomposition of hydrogen peroxide by catalase was 405 nm.
Glutathione Peroxidase
The wavelength to detect the yellowish complex from the interaction of the reduced form of glutathione peroxidase with 3,5-dinitrobenzoic acid was 412 nm.
Superoxide Dismutase
The central idea behind the qualitative identification of superoxide dismutase in the samples was the inverse proportionality between the substance and the formazan dye concentration. In particular, the action of oxygen on water-soluble tetrazolium 1 resulted in the formation of formazan, which was transformed into catalyzed xanthine oxidase. Since superoxide dismutase tends to inhibit the action of this enzyme, its identification was possible.