MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, August, 2022 SKL 0 0 9 1 6 3 SUPERVISOR:
INTRODUCTION
Reasoning of the research
Obesity is a complex, multi-factorial disease that has seen a significant rise in prevalence globally since 1980, affecting individuals regardless of age, gender, race, or location Research indicates that senior citizens and women are particularly at risk The World Health Organization (WHO) classifies obesity as a global pandemic, with projections suggesting that the number of affected individuals could reach 300 million by 2025 This medical disorder involves the accumulation of excess body fat, which can adversely affect health, reduce life expectancy, and exacerbate existing health conditions The increase in obesity rates is linked to a rise in related diseases, including diabetes, stroke, cardiovascular issues, and certain cancers Effective prevention and treatment strategies are essential, with weight loss through diet and physical activity being the most common approaches However, weight loss medications can have side effects, prompting the need for alternative treatments with fewer adverse effects Consequently, the development of natural anti-obesity medications is gaining traction, leading to an evaluation of Olax imbricata leaf extracts for their potential in preventing overweight and obesity in in vivo studies.
Purposes of the research
This research aimed to assess the impact of the extraction on various health parameters in experimental animals, including weight management and energy consumption, glycemic control, blood lipid levels, and motor behavior regulation.
Limits and scope of the research
This study assessed the effectiveness of extracts in preventing and reducing disease conditions in experimental mouse models of overweight, obesity, and diabetes using the HFD-STZ-T2D paradigm The evaluation focused on key outcomes, including body weight, energy intake, blood lipid levels, fasting blood sugar, glucose tolerance, tissue microsurgery (liver, kidney, fat), and the locomotor activity of the experimental animals.
Scientific and practical significance
Medicinal plants have been essential for human health throughout history, and Vietnam is rich in herbal resources These plants have been utilized since ancient times to effectively treat various serious illnesses.
Research is being conducted to control and prevent diseases linked to the extraction of Olax imbricata leaves in experimental animals with overweight, obesity, impaired glycemic control, and elevated blood fat levels This study aims to establish a scientific basis for developing a natural extraction as a functional meal to address nutritional-related health issues Ultimately, Olax imbricata leaf extraction could provide an effective alternative to certain commercially available medications, contributing to a broader range of products for disease treatment and prevention.
Subjects, scope and limitations of the research
The research focused on Olax imbricata leaves and male white mice (Mus Musculus var albino) The leaves were collected in February 2022 from the Research and Production of Medicinal Materials Center in Phu Yen Province, Vietnam Meanwhile, the male white mice, averaging 13g±1g and 5 weeks old, were acquired in early April 2022 from the Pasteur Institute in Ho Chi Minh City, Vietnam.
This research aimed to assess the effectiveness of Olax imbricata leaf extracts in controlling and preventing diseases related to obesity in experimental mice The study specifically examined the physiological indicators of mice subjected to a high-fat diet and STZ injection, which induced irritation and overweight conditions.
The research investigates the effectiveness of Olax ỉmbricata leaf extracts in managing and preventing diseases related to body weight, blood lipids, and blood sugar levels Additionally, it assesses the microsurgical tissue structure and evaluates the behavior and activities of experimental animals.
Structure of report
Chapter 4: Experimental results and Discussion
OVERVIEW
Overview about Olax imbricata
Olax is the largest genus in the Olacaceae family, comprising 40 species that are widely utilized globally for health-related purposes This genus thrives in Africa, South Asia, and Australia, exhibiting a variety of biological functions Several species, particularly those native to the tropical forests of Asia and Africa, play significant roles in various biological processes Notably, Olax subscorpioidea, a shrub or tree found in Nigeria, can reach heights of at least 10 meters and possesses numerous therapeutic properties.
Olax imbricata, a member of the Olacaceae family, thrives in tropical regions, particularly in sandy soils and sunny, dry conditions like those in central Vietnam and Phu Yen province This shrub typically reaches a height of 5 meters and features slanting branches with oblong leaves.
Olax imbricata is a herb measuring 10 centimeters in length, with a peduncle of 6-8 millimeters It features small white calyxes in its leaf axils and blooms from December to January, producing fruit in August This plant is recognized for its antioxidant, anti-inflammatory, antibacterial, and anti-infective properties, making it beneficial for various gynecological disorders Additionally, it has been traditionally used in Vietnam as a treatment for diabetes.
The poplar tree contains various beneficial compounds, including polyphenols, flavonoids, glycosides, saponins, tannins, and alkaloids, which exhibit antibacterial and antioxidant properties In 2018, Vo Thi Nga et al successfully isolated two phenolic compounds, three phenol glycosidic compounds, and leonuriside A from Olax imbricata Their research also involved the isolation of triterpenoid glycosides and in vitro tests for α-glucosidase inhibition.
2019, findings were obtained by separating triterpenoid glycosides [118].
Overview about extration methods
Modern extraction techniques are utilized to obtain plant extracts rich in physiologically active compounds, with both traditional methods being refined and new methods being developed This process involves using solvents to extract active chemicals from natural materials, such as plants and animals, while removing unwanted components The solvent penetrates the material, dissolving polar compounds, and the primary aim is to isolate the soluble metabolites from the insoluble parts of the plant Additionally, the choice of extraction method significantly influences the quality and quantity of the resulting ingredients.
Maceration is a winemaking technique that has also been widely used in the study of plants with diverse biological activities This process involves soaking plant material, either in flakes or powder form, in a suitable solvent The mixture is stored at room temperature for a minimum of three to seven days, with intermittent stirring, ensuring that the plant matter is fully submerged in the solvent After the extraction period, the mixture is filtered to recover the solvent, which is then removed through drying or water heating.
4 material will result in different sorts of compounds when treated with various solvents
Reflux is a process of continuous extraction that employs heat After pre-treatment (grinding, crushing, etc.), dried plant materials are added to the reflux boiler Then, add solvent while stirring
The ideal solvent to coarse residue ratio for extraction is 10:1 (v/w) Reflux heating, which involves submerging the mixture in a suitable solvent, accelerates the extraction process compared to traditional beam soaking due to temperature catalysis This method is particularly effective for extracting heat-stable compounds or resilient materials.
Soxhlet extraction is a highly efficient standard technique for solid/liquid extraction, surpassing conventional methods In this process, a finely powdered sample is placed in a filter paper bag within the Soxhlet device's sample tube The extraction solvent is heated in a flask at the bottom, causing it to evaporate and travel to the sample tube, where it interacts with the target compound The vapor then condenses in the condenser and returns to the sample tube Once the liquid reaches the siphon tube, it flows back into the flask, allowing the solvent to carry the extracted compounds downward This cycle of evaporation and condensation continues until all desired compounds are extracted from the sample.
This is the usual laboratory procedure for extractioning oils from diverse substances This method has the benefit of requiring a less amount of solvent than the beam immersion method
The solvent recovery process is effective and cost-efficient, making it easy to operate Additionally, there is no requirement to filter the residue post-extraction However, this method is not suitable for extracting heat-sensitive components like enzymes, alkaloids, and esters.
Microwave-assisted extraction (MAE) is an innovative technique that employs microwave energy to extract desired chemicals from raw materials Operating within a frequency range of 300 MHz to 300 GHz and a wavelength of 1 cm to 1 m, microwave radiation is a form of non-ionizing electromagnetic radiation This radiation interacts with the dipole bonds of polar materials, heating the sample's surface and facilitating conduction As the dipole linkages break hydrogen bonds, polar components are released, allowing solvents to penetrate more effectively Even dried plant materials retain some moisture, which, when heated by microwaves, increases internal pressure This pressure causes plant cell walls to expand and eventually disintegrate, enhancing the exposure of cellular components to the solvent.
The efficiency of Microwave-Assisted Extraction (MAE) is influenced by several key variables, including solvent selection, the solvent-to-raw material ratio, irradiation temperature, irradiation time, and microwave power This innovative extraction method significantly decreases both extraction time and solvent consumption when compared to traditional extraction techniques.
[87] Choosing the proper solvent will result in efficient extraction In this extraction method, solvents such as dichloromethane, methanol, acetone, petrol ether, etc are frequently employed
The dielectric properties of the solvent play a significant role in the extraction process, making the optimization of these parameters essential The MAE extraction method is specifically suited for small-molecule phenolic compounds, including stable phenolic acids like gallic acid and ellagic acid, as well as quercetin, which can withstand heating conditions of up to 100 °C for 20 minutes.
[87] Moreover, MAE is applicable only to extraction solvents with microwave absorption [130] Because tannins and anthocyanins are altered at high temperatures, they may not be suited for MAE [87]
Ultrasonic extraction (UAE) has been widely used in the food and pharmaceutical industries for decades, utilizing ultrasonic pulses ranging from 20kHz to 2000kHz This method operates on the principle of cavitation, where ultrasonic vibrations create small air bubbles in the solvent that grow until they reach a critical size and then collapse, releasing significant energy This process can generate extreme temperatures (up to 5000K) and pressures (up to 1000atm), enhancing the contact between the solvent and the sample The mechanical action of the sound waves alters the physical and chemical properties of the material, breaking down plant cell walls and facilitating the release of chemicals while improving solvent mobility into the cells In contrast, the microwave-assisted extraction (MAE) method transfers heat from the interior to the exterior of the plant cell, requiring the material to be dried, powdered, and sieved before extraction In UAE, high-frequency sound energy aids in the extraction process without the need for heat.
The effectiveness of the extraction process is influenced by factors such as frequency, intensity, temperature, and duration of ultrasound, along with sample properties like moisture and size, as well as solvent type, volume, and concentration The ultrasound-assisted extraction (UAE) method is advantageous due to its simplicity and low cost, making it suitable for both small and large-scale applications This technique significantly reduces extraction time and solvent usage However, it is important to note that using ultrasonic energy at frequencies above 20 kHz may lead to the generation of free radicals.
Current extraction methods include pressurized liquid extraction (PLE), supercritical fluid extraction (SFE), enzyme-assisted extraction (EAE), and supercritical water extraction (SWE), among others.
Pressurized liquid extraction (PLE) is an efficient technique for extracting solid or semi-solid samples using high temperatures (up to 200 °C) and pressures (around 1500 psi) This method allows the solvent to remain in a liquid state under these extreme conditions, resulting in a significantly faster extraction time of approximately 30 minutes and requiring less solvent compared to traditional methods The elevated temperature and pressure enhance the mass transfer rate, solubility of analyte chemicals, and solvent diffusion into the material being extracted Additionally, PLE can be automated, streamlining the quality control process.
Supercritical fluid extraction (SFE) utilizes supercritical fluids (SF), which possess solubility and diffusivity comparable to liquids and gases This unique property enables SF to effectively dissolve a diverse range of natural substances Notably, the solvation characteristics of SF change significantly at critical points with minor variations in pressure and temperature Carbon dioxide (S-CO2) is frequently used in SFE because of its benefits, including a low critical temperature, making it an ideal choice for solvent-free extraction.
Supercritical carbon dioxide (S-CO2) is an effective and cost-efficient solvent for extracting non-polar natural substances like lipids and volatile oils, thanks to its low polarity and nontoxicity While S-CO2 has limited solubility for polar compounds, the extraction process can be enhanced by adding small amounts of ethanol or methanol, improving the extraction of polar molecules.
Enzyme-assisted extraction (EAE) utilizes enzymes to effectively break down cell walls, membranes, and intracellular macromolecules, enhancing the release of biological compounds from natural materials This method is influenced by the coagulation and denaturation of proteins at high temperatures Commonly used enzymes in EAE include cellulose, α-amylase, and pectinase, which can be sourced from bacteria, fungi, plants, and animal tissues Research has shown that EAE significantly improves the extraction efficiency of antioxidants such as phenolics, flavonoids, anthocyanins, and carotenoids.
Metabolism processes of nutritional compounds in humans
For normal growth and development, organisms require three essential dietary components: carbohydrates, lipids, and proteins Carbohydrates are particularly important, as they constitute a significant portion of the body's nutritional needs and serve as the primary energy source.
[64] Carbohydrates in the diet consist mostly of starch, fiber, and different sugars, such as monosaccharides (such as glucose, galactose, and fructose) and disaccharides (such as sucrose, lactose, and maltose) [111]
Carbohydrate metabolism primarily occurs in the mouth and small intestine, particularly in the duodenum The process begins when carbohydrates interact with salivary enzymes, mainly α-amylase, in the oral cavity This enzyme hydrolyzes carbohydrates by cleaving α-1,4 glycosidic linkages, producing maltose, maltotriose, and short-chain dextrins, while leaving α-1,6 bonds intact Once the food reaches the stomach, hydrochloric acid inactivates any remaining α-amylase due to the low pH As the partially digested food enters the duodenum, the pancreas releases α-amylase and bicarbonate, with the latter neutralizing the acidic gastric fluids to facilitate enzyme action Similar to the oral phase, pancreatic α-amylase only cleaves α-1,4 glycosidic bonds, resulting in partial digestion of amylopectin, while sucrose and lactose remain unaffected.
Disaccharides are indigestible but can be broken down into monosaccharides by the enzyme α-glucosidase, which plays a crucial role in starch digestion This enzyme hydrolyzes α-1,4 glycoside linkages in dextrins and oligosaccharides, allowing glucose to be absorbed by intestinal epithelial cells and enter the bloodstream Once in the bloodstream, monosaccharides are transported to various tissues, and the pancreas releases insulin to lower blood sugar levels and facilitate glucose uptake by cells and the liver Glucose is then utilized in glycolysis to produce adenosine triphosphate (ATP) for energy Insulin also activates the liver's GLUT-2 transporter for glucose absorption, and excess glucose is stored as glycogen through glycogenesis When the body requires energy, glycogen is converted back into glucose via glycolysis and gluconeogenesis.
After carbohydrates are digested, glucose enters the bloodstream, leading to elevated blood glucose levels The pancreas is essential for regulating these levels by secreting insulin However, when the body struggles to metabolize carbohydrates and lipids, insulin production and sensitivity decrease, contributing to the onset of type 2 diabetes This condition is marked by hyperglycemia, which can lead to chronic complications Research indicates that individuals with diabetes typically have a fasting blood glucose level of 8mmol/L (144.14mg/dL) and a 2-hour glucose tolerance level of 11mmol/L (198.2 mg/dL) In laboratory animals, normal fasting blood glucose levels are 199mg/dL, while pre-diabetes is indicated by levels between 200-249mg/dL, and diabetes is diagnosed at levels exceeding 250mg/dL.
Dietary lipids provide about 30% of daily energy intake, primarily consisting of 95% long-chain triglycerides (TG) and 5% phospholipids, cholesterol, sterols, and fat-soluble vitamins The majority of dietary fat is metabolized as triglycerides, with approximately 95% being efficiently absorbed in the intestine and only 5% excreted in stool Due to their insolubility in water, fats require emulsification for digestion, and TG must be hydrolyzed into fatty acids (FA) and monoglycerides (MG) for absorption by the body.
Most lipid digestion takes place in the small intestine, beginning in the oral cavity where food is mechanically broken down through chewing This process results in smaller food particles that are then transported to the stomach In the stomach, intestinal motility plays a crucial role in emulsifying lipid particles The digestion of triacylglycerols (TAG) starts with gastric lipase acting on the surface of these emulsified granules.
Lipase is a water-soluble enzyme that functions on the surface of fat particles in the acidic environment of the stomach, specifically at a pH of 3.0-6.0, where it hydrolyzes triglycerides (TAGs) into fatty acids (FAs) and glycerides Secreted continuously from the serous glands, lipase accumulates in the stomach between meals, but only 20 to 30% of fat is digested there, with no hydrolysis of phospholipids and cholesteryl esters occurring As food transitions from the stomach to the small intestine, the predominant fats present are TAGs and diacylglycerol (DAG), with the pancreas secreting additional enzymes, including lipase, to aid in further digestion.
The digestion of lipids begins in the duodenum, where bicarbonate and pancreatic enzymes, including lipase, are secreted to create a neutral environment that enhances lipase activity Bile salts released from the gallbladder emulsify large lipid particles, breaking them into smaller droplets to increase surface area for enzyme access The stable duodenal emulsion consists of triglycerides, cholesteryl esters, free fatty acids, monoglycerides, and bile salts Pancreatic lipase, aided by co-lipase, hydrolyzes triglycerides at specific sites, producing free fatty acids and monoglycerides, which are further broken down Phospholipids, primarily lecithin, are hydrolyzed by pancreatic phospholipase-A2, while cholesterol esterase converts cholesteryl esters into free cholesterol The products of lipid digestion form micelles that facilitate absorption in the jejunum through diffusion and protein transport Medium-chain fatty acids are readily absorbed, while short-chain fatty acids are primarily absorbed in the large intestine Inside mucosal cells, lipids are recombined and packaged into chylomicrons, which consist of triglycerides, cholesteryl esters, and phospholipids, enabling their transport in the bloodstream.
9 lipids to tissues and the liver [39]
Lipoproteins are categorized into chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) Chylomicrons transport digested fats from the intestines to the bloodstream via lymphatic vessels, where the enzyme lipoprotein lipase hydrolyzes triglycerides into fatty acids for energy use in muscle and heart tissues or storage in adipose tissue Over 90% of triglycerides in chylomicrons are absorbed by tissues during circulation, with the remaining cholesterol absorbed by the liver to form VLDL, which is the main transporter of endogenous fat from the liver to tissues As triglycerides are depleted, VLDL converts to LDL, which primarily carries cholesterol esters and cholesterol, accounting for about 70% of plasma cholesterol and contributing to atherosclerosis, particularly in the aorta and coronary arteries HDL is essential for reverse cholesterol transport, moving cholesterol and phospholipids from peripheral tissues to the liver for degradation and bile acid synthesis.
Research indicates that high levels of HDL in plasma can significantly lower the risk of atherosclerosis and cardiovascular diseases HDL is known for its antioxidant, anti-inflammatory, antithrombotic, and vasodilator properties, which contribute to improved arterial health.
Protein is a crucial nutrient for maintaining nutritional balance in humans Upon entering the body, proteins undergo complex breakdown processes to form simpler molecules for absorption Digestion begins in the stomach, where acidic pH denatures proteins and activates pepsinogen into pepsin, which breaks down polypeptides into amino acids and oligopeptides As the acidic mixture moves into the small intestine, it triggers hormone release that stimulates the pancreas to secrete bicarbonate, raising the pH to around 7.0 While the gastric phase of digestion is less significant, the pancreatic phase is vital In the duodenum, hormones like cholecystokinin and secretin prompt the release of inactive enzyme precursors from the pancreas, which are activated by enteropeptidase This activation leads to the formation of active enzymes such as trypsin and chymotrypsin, which further convert peptides into free amino acids and oligopeptides Oligopeptides then diffuse to the villi epithelium, where peptidase enzymes facilitate their absorption.
The border membrane cleaves proteins to produce dipeptides, tripeptides, and free amino acids, which are then absorbed by the small intestine Amino acid absorption is facilitated by Na\(^+\) co-transport, while peptide absorption involves proton co-transport These compounds are transported into the epithelial cells of the small intestine, entering the villous capillaries and traveling to the liver In the liver, aminotransferase converts amino acids into keto acids and amino groups Keto acids can be oxidized to CO\(_2\) and H\(_2\)O or converted into glucose for energy The amino groups are recycled to synthesize new amino acids or other nitrogenous compounds, while excess nitrogen is excreted through the urea cycle.
Combination model of high-fat diet and low-dose streptozotocin (HFD-STZ-T2D)
Numerous high-fat diets for rodents differ in fat content and source, with 40 to 60% of calories from fat potentially leading to metabolic issues, hypertension, obesity, and inflammatory cytokine production A diet with 10% of calories from fat is classified as a low-fat diet (LFD), while 30 to 50% is considered a high-fat diet (HFD), and over 50% is categorized as a very high-fat diet (VHFD) Both HFD and VHFD can induce diabetes in rodents.
High-fat diets disrupt the PI3K/AKT signaling pathway, leading to insulin resistance and an increased risk of type 2 diabetes In mice on a high-fat diet, insulin-stimulated tyrosine phosphorylation of insulin receptors and IRS is significantly reduced This dietary pattern activates c-Jun N-terminal Kinase (JNK), which further suppresses IRS and triggers inflammatory responses that inhibit insulin signaling Additionally, high-fat diets result in lower GLUT4 levels at the cell surface, a key factor in the development of insulin resistance.
Streptomycetes achromogenes synthesizes streptozotocin (STZ), a free radical that can damage DNA and cells STZ is typically administered in a single large dose or in repeated modest doses It contains nitro radicals that harm β-cells, while its deoxyglucose component facilitates the transfer of molecules across these cells STZ enters pancreatic β-cells through glucose transporter 2 (GLUT2), affecting other organs like the kidneys, liver, and intestines that also express this transporter The alkylation or breakage of DNA increases poly-ADP-ribose synthase activity, leading to cell damage At low dosages, STZ causes rapid β-cell necrosis, contributing to the development of Type 1 diabetes (T1D) In contrast, multiple modest doses result in partial β-cell destruction, inflammation, and progressive loss of activation, resembling the pathophysiology of Type 2 diabetes (T2D) Metabolic alterations associated with STZ exposure are typically observed within the first 2–8 weeks.
To induce insulin resistance and trigger the onset of diabetes in animals, a high-fat diet (HFD) must be consumed for a specific duration, which varies across different conversion studies.
In the majority of trials, a two-week diet followed by a low dosage of streptozotocin (STZ) resulted in elevated blood glucose levels within 3-7 days post-injection The effects on blood glucose levels were influenced by the amount of STZ administered and whether the subjects were fasting Notably, blood glucose levels exhibited less variation during fasting compared to non-fasting conditions Additionally, other studies employed intraperitoneal STZ injections of 30–40 mg/kg alongside a diet comprising 40–60% of calories from fat to induce high-fat diet (HFD)-STZ-induced type 2 diabetes (T2D).
Blood glucose levels and diabetes severity are closely linked to STZ dosage Previous studies indicate that blood insulin levels in the HFD-STZ-T2D model can vary compared to the control group, with most findings showing an increase in insulin levels after a high-fat diet (HFD) and a decrease following STZ treatment The dosage of STZ is crucial, as higher amounts lead to significant cell death and a rapid decline in insulin levels While blood insulin levels serve as a reliable marker for the stage of T2D, further research has also highlighted the importance of oxidative and antioxidative indicators, islet area, islet insulin content, and T2D-positive cells.
2.4.4 Advantages and disadvantages of the HFD-STZ-T2D paradigm
Rodents, particularly mice, are preferred for inducing high-fat diet-streptozotocin type 2 diabetes (HFD-STZ-T2D) due to their omnivorous nature, small size, quiet behavior, and cost-effectiveness While genetic diabetes models offer deeper insights into disease progression, they are often expensive and impractical for routine screening Other diabetes models, like STZ-nicotinamide, require high doses of STZ, leading to hyperglycemia primarily from pancreatic cell loss The HFD-STZ-T2D model closely mimics human type 2 diabetes in metabolic characteristics and progression, producing sustained hyperglycemia, as evidenced by studies showing increased blood glucose levels in mice after HFD and STZ treatment However, this model's lengthy testing duration can escalate costs, and improper management of STZ doses may result in the death of the mice.
In vivo testing on laboratory animals
In the drug development process, the safety clinical trial of a new medicine relies on in silico (virtual simulations), in vitro (test-tube experiments), and in vivo (animal testing) methods Currently, in vivo testing serves as a vital connection between the success of in vitro testing and ensuring human safety Historically, in vivo testing was a requirement before a medicine could proceed to human clinical trials.
The use of animals in research remains a common practice, with various species such as mice, rabbits, fish, guinea pigs, amphibians, primates, dogs, and cats being utilized These studies primarily aim to evaluate potential treatments for both infectious and noncommunicable diseases, focusing on toxicity and medication efficacy Animals serve as essential experimental tools in the development of pharmaceutical products, including vaccines and antibiotics The growth of medical technology has led to an increase in the number of animals used in research.
2.5.1 Origin and classification of mice
Mice are among the most often utilized animals in scientific research [32] They are prized
Mice are favored in research due to their small size, short breeding period, ease of reproduction, and superior genetics, sharing significant morphological, physiological, and genetic similarities with humans This enhances the reliability of research outcomes They are particularly well-suited for studies related to sepsis, obesity, diabetes, cancer, and organ transplants Future advancements in mouse models aim to develop "humanized" mice that incorporate human genes, cells, tissues, and organs, facilitating the creation of therapies for human diseases.
2.5.2 The advantages and disadvantages of utilizing laboratory animals
Mice are the preferred species for scientific research, especially in medical and epidemiological studies, due to their omnivorous diet, small size, calm nature, and long lifespan They are quick to breed, easily accessible, and cost-effective Furthermore, the similarities in metabolic characteristics and natural history between mice and humans make them a suitable choice for experiments instead of using healthy human subjects.
In the United States, over 100 million animals, including dogs, cats, mice, and rabbits, suffer and die each year due to laboratory chemical testing These animals endure severe mistreatment, such as being burned, poisoned, and incapacitated for extended periods, as well as experiencing organ removal and other forms of abuse.
Animal corpses release various bacteria, viruses, and parasites during decomposition, necessitating extensive treatment with specific chemicals and advanced equipment Without proper management, these carcasses can emit harmful compounds into the environment, affecting water, soil, and air quality The long-term accumulation of these chemicals poses direct risks to human health For decades, efforts have been made to reduce chemical use in animal testing to mitigate its environmental impact.
2.5.3 Conditions for raising laboratory mice
Accurate in vivo testing requires strict regulation of laboratory animal care, as unreliable results can arise from stressed or ill animals To maintain optimal physiological conditions during research, it is essential to adhere to the Guidelines for the Care and Use of Laboratory Animals Key factors such as housing, temperature, humidity, ventilation, lighting, noise, and nutrition must be carefully developed and monitored.
To ensure effective cleaning and maintenance, housing must remain clean, pest-free, and well-maintained Mice should be housed in cages that meet their physical, physiological, and behavioral needs; otherwise, inadequate environments can adversely affect brain development, health, and scientific outcomes Cage materials must be durable, safe, escape-proof, non-toxic, and facilitate easy cleaning and organization.
Adding litter to the cage is essential for insulating animals, absorbing moisture, and significantly minimizing odors and pollutants like ammonia in the barn Various types of litter can be utilized for these purposes.
Thirteen materials, including sawdust, wood shavings, maize cobs, and rice husks, are commonly used for animal bedding Maintaining cage cleanliness is essential for optimal animal productivity, requiring regular cleaning and timely replacement of litter to ensure animals remain clean, dry, and comfortable However, excessive cleaning can inadvertently lead to cannibalism among livestock It is recommended to change the bedding 3 to 4 times a week and to use scented wax around the breeding area to help mitigate odors.
The comfort, health, and performance of laboratory mice are significantly affected by their environmental temperature and humidity While no specific optimal temperature has been established, research indicates that mice perform best in temperatures ranging from 20 to 26 °C and relative humidity levels between 30% and 70% It is crucial to maintain temperature and humidity levels within 1°C of each other to ensure that the metabolic rate of the mice remains stable.
An automated system should monitor temperature and humidity to ensure that variations remain within permissible parameters [89]
The primary objective of ventilation is to provide adequate air supply and maintain a stable experimental environment This technology effectively delivers sufficient oxygen while minimizing air pollution from gases like CO2 and NH3 Additionally, it helps dissipate heat, aiding in the removal of body heat from animals, humans, and lighting, thus regulating temperature and humidity within the cage Proper design of the ventilation system is crucial, taking into account the size of the facility and the number of test animals, with an efficient ventilation rate typically defined by the frequency of fresh air introduction per hour.
Mouse' physiology, morphology, and behavior can be affected by light [89] The light level in the room should be suitable for human experimentation and testing of mouse, but not excessive
Mice, being nocturnal, have eyes adapted to low light conditions, and exposure to light levels above 100 lux for more than 16 hours can lead to blindness High light intensity not only increases aggression and cannibalism among mice but also affects their physiological processes The maximum permissible light intensity in culture rooms is 350 lux, with fluorescent lighting commonly used Uniform light distribution is essential, and the photoperiod significantly influences various physiological parameters Mice require time to adjust to light changes to maintain normal growth, metabolism, reproduction, and behavior The most common light-dark cycles in in vivo testing are 12:12 and 14:10, and maintaining consistency in these cycles is vital for accurate research outcomes.
Noise exposure can adversely affect both humans and laboratory mice, impacting various systems including auditory, digestive, immune, reproductive, neurological, and cardiovascular functions, while also leading to metabolic and behavioral issues Levels exceeding 85dB are considered potentially harmful, and even short bursts of noise above 100dB or 160dB can cause serious harm, such as eardrum damage and convulsions in rodents Additionally, ultrasonic waves contribute to these negative effects.
Laboratory mice are highly sensitive to ambient noise, particularly ultrasonic waves, which should be kept at or below 50dB to protect their health These mice utilize ultrasonic vibrations for communication, making it crucial to minimize environmental noise Proper nutrition is essential for their health and performance, as dietary needs vary with age, species, health status, and research objectives Mice typically consume 20% of their body weight daily, necessitating adjustments in dietary content to meet their nutritional requirements A balanced diet must include three essential macronutrients: protein, carbohydrates, and lipids, along with small amounts of fiber, vitamins, and minerals Many contemporary studies utilize NRC diets for rat meals, while the American Academy of Nutrition has established the AIN-93 diet, which meets most NRC nutritional recommendations However, the AIN-93 diet contains vitamin B12 levels that are 50% below NRC standards, requiring supplementation to ensure adequate intake.
2.5.4 Animal Testing Ethics and the 3Rs rule
Previous relevant research
Pharmacologists are currently prioritizing the synthesis of effective medications for disease prevention and treatment using natural herbs In our country, numerous studies have explored the medicinal effects of plant extracts tested on animal models, consistently yielding positive results.
According to [26], their findings on the biological activity of the methanol extraction of
In 2020, research on Hedytis diffusa Willd revealed that its extraction is rich in total polyphenols and flavonoids, showcasing strong antioxidant and anti-inflammatory effects Furthermore, experimental mice treated with this extraction displayed notable hepatoprotective properties.
Extensive global research has focused on the in vivo effects of plant-derived substances A 2015 study examined the anti-obesity effects of an ethanol extract from Terminalia paniculata bark in mice on a high-fat diet The treated mice showed reduced food intake and significant weight management compared to untreated mice Additionally, the extract improved blood lipid profiles, evidenced by decreased triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL), alongside an increase in high-density lipoprotein (HDL) Furthermore, the findings indicated anti-lipase activity and modulation of lipid absorption and transport, as shown by increased fecal lipids and decreased blood TG levels.
Research indicates that the root bark extract of Olax mannii effectively regulates blood glucose levels and significantly lowers blood sugar in male mice with diabetes induced by streptozotocin injections.
The author of [79] evaluated the hypoglycemic, anti-dyslipidemic, and oxidative effects of
In a 2021 study on Vitellaria paradoxa bark extraction in mice with streptozotocin-induced Type 2 Diabetes (T2D), the analysis identified secondary metabolites such as polyphenols and flavonoids Treatment over four weeks with doses of 125, 250, and 500 mg/kg body weight resulted in significant reductions in body weight and food consumption, alongside decreased blood glucose levels, particularly at the 250 and 500 mg/kg doses Additionally, the extraction group showed a reduction in triglycerides (TG) and total cholesterol (TC), while high-density lipoprotein cholesterol (HDL-C) levels increased markedly.
MATERIALS AND METHODS
Materials
Fresh and green leaves of Olax imbricata were collected from the Central Center for Research and Production of Medicinal Materials in Dong Hoa District, Phu Yen Province, Vietnam, ensuring they were free from wilting, yellowing, or insect infestation A 96% ethanol extraction process was employed to obtain chemical compounds from these leaves.
Streptozotocin (Macklin) supplied from the BioLab chemical distribution company in Hanoi in March 2022, storing it at -40 O C to preserve its infectious potential when injected into mice
The study utilized two dietary components for in vivo testing: a normal diet (ND) and a high-fat diet (HFD) The ND, produced by Jolly Pet Products, mirrors the nutritional profile of the AIN-93 standard rat diet In contrast, the HFD consists of a mixture of finely ground Fullvit JP70 and liquid beef fat, prepared by washing beef belly fat to remove impurities, cooking it until liquefied, and then straining and storing it in a sealed jar at -5 °C The final high-fat diet was created by blending the commercial feed and beef fat in a 1:1 ratio.
Table 3.1 Nutrient compositons in ND and HFD
Figure 1 Olax imbricata leaves Figure 3.1 Olax imbricata leaves
To prepare the high-fat diet (HFD) for experimental mice, melt 50g of beef fat and mix in 50g of finely powdered commercial feed while stirring continuously to ensure a thorough combination Enhance the diet's aroma and stimulate the mice's appetite by adding three drops of vanilla essential oil Store the HFD at 5°C and prepare it fresh daily to prevent mold growth that could harm the laboratory animals, ensuring it is consumed within 24 hours.
In this in vivo study, 5-week-old male white mice (Mus Musculus var albino) weighing 13±1g were sourced from the Pasteur Institute in Ho Chi Minh City, Vietnam The mice underwent a 5-day acclimatization period to reach a weight of 15±1g each Throughout the experiment, they were kept in conventional conditions with humidity levels between 60% and 70% and a temperature of 30±2°C, while a 12-hour light/dark cycle was maintained To promote optimal growth, the mice were provided with standard food and ample Aquafina-purified water daily.
Figure 3.2 White mice (Mus Musculus var albino)
Figure 3.3 In vivo experimental design
18 were given a few drops of vitamin Hagebuttentrunk from Beaphar company, Netherlands.
In vivo experimental design
Thirty adaptive feeding mice, each reaching a target weight of 15g, will be randomly assigned to six groups (n = 5 per group) and placed in three separate environments Groups A (Normal Diet) and B (High Fat Diet) will receive their respective diets throughout the study, while Group B and the remaining Group C will be on the High Fat Diet for the initial two weeks In weeks three and four, both groups will receive two doses of STZ, spaced one week apart, to induce type 2 diabetes at a dosage of 40 mg/kg body weight before the extraction administration Starting in week five, Group C will also be given the High Fat Diet, along with three subgroups receiving extracts from Olax imbricata leaves at doses of 50, 100, and 200 mg/kg body weight/day, respectively, divided into four meals per day Additionally, one group will receive Acarbose at a dose of 100 mg/kg body weight daily The experimental groups will receive the extracts and Acarbose in two sessions (morning and afternoon), with each dose split into two equal portions (0.5 mL per time).
After two weeks on a high-fat diet, mice exhibited significant increases in body weight and insulin resistance To achieve this, we fed the mice a high-fat diet, followed by the administration of extraction and acarbose for comparison with a control group The in vivo testing spanned 12 weeks, excluding a 5-day acclimatization period after retrieval from the Pasteur Institute in Ho Chi Minh City Throughout the experiment, mice had unlimited access to food and water to ensure accurate results, revealing significant variations among the groups Mice were weighed daily to assess calorie intake and monthly to monitor growth rates Evaluations for glucose tolerance, behavior, and movement were conducted at weeks 0, 2, 4, 7, 10, and 12 At the end of the 12th week, the mice were anatomized to collect histological samples from the liver, kidney, and fat, as well as blood for analysis of lipid indicators, including triglycerides, cholesterol, LDL-cholesterol, HDL-cholesterol, and insulin.
(A) Experimental group with standard normal diet (ND)
The study involved an experimental group subjected to a high-fat diet and administered two doses of streptozotocin (HFD) Additionally, it included two groups: one that followed the same high-fat diet and received two doses of streptozotocin, while also utilizing three doses of 96% ethanol extraction from Olax imbricata leaves (HFE).
The study involved three groups: a control group receiving HFE-50, HFE-100, and HFE-200 with extraction doses of 50, 100, and 200 mg/kg body weight/day, respectively; an experimental group on a high-fat diet; and a treatment group that received two doses of streptozotocin along with Acarbose at a dosage of 100 milligrams per kilogram of body weight per day.
Determine sample size
Before conducting in vivo testing on experimental animals, it is essential to determine the appropriate sample size An inadequate number of animals can lead to an inability to detect biologically significant reactions, ultimately resulting in misleading outcomes.
Excessive waste of time, chemicals, reagents, and laboratory animals is a significant concern in research The resource equation approach is commonly used for estimating sample size in animal studies, with a permissible range of degrees of freedom (DF) between 10 and 20 For one-way ANOVA, the degrees of freedom can be calculated using a specific formula.
DF = N – k = kn – k = k(n -1) (1) With N is the total number of experimental animals; k represents the total number of experimental groups; and n represents the number of experimental animals within each group
To determine the sample size for each experimental group, we deduce from the formula \( n = \frac{DF}{k} + 1 \) Given the permissible degrees of freedom (DF) range of 10 to 20, we calculate the minimum and maximum number of animals per group as follows: the minimum \( n_{min} = \frac{10}{k} + 1 = \frac{10}{5} + 1 = 3 \) and the maximum \( n_{max} = \frac{20}{k} + 1 = \frac{20}{5} + 1 = 5 \) Consequently, a sample size of 5 animals per group was selected to ensure reliability while adhering to ethical standards in in vivo testing.
Extraction receiving method
After harvesting Olax imbricata leaves, they are dried in airflow within a shaded area for about 14 days, avoiding direct sunlight The leaves are then convectively dried at 55°C until the moisture content reaches 8% Once dried, the leaves are hand-crushed into a powder with a particle size of approximately 5x5 mm A mixture of 3 kg of crushed leaves is soaked in 20 liters of 96% ethanol for three days at room temperature (30°C), with frequent stirring The extraction process involves a solvent-to-residue ratio of 10:1 (v/w), followed by heating the mixture at 78°C for 10 hours, cooling, and sealing the extraction in a glass jar This process is repeated four times, combining the extractions, filtering to remove residue, and concentrating at 78°C to separate the ethanol The concentrated ethanol is then used to extract the dried leaf powder After further evaporation under vacuum at 50°C and heating in a water bath at 80°C to eliminate residual solvent, approximately 500 grams of 96% EtOH concentrated extraction is obtained from 47 kilograms of starting material The final extraction is sealed in a glass flask and refrigerated at 10°C.
3.5 In vitro testing to analysis α-glucosidase enzyme inhibitory activity of 96% EtOH extraction from Olax imbricata leaves and acarbose
The α-glucosidase inhibitory activity of extraction and acarbose samples was evaluated at the Biochemistry laboratory of the Institute of Applied Materials Science, Vietnam Academy of Science and Technology, located in Ho Chi Minh City The assessment followed a previously established method, where materials were dissolved in dimethyl sulfoxide (DMSO) at varying concentrations The reaction mixture included 40μl of phosphate buffer (0.1M, pH 6.8), 10μl of the test samples, and 25μl of α-glucosidase (0.2 IU/mL) on a 96-well ELISA plate, incubated at 37°C for 10 minutes Subsequently, 25μl of a 2.5 mM p-nitrophenyl-α-D-glucopyranoside solution in phosphate buffer was added to the mixture.
Incubate the reaction mixture at 37°C for 30 minutes, then add 100 μl of 0.2M sodium carbonate solution Measure the absorbance of p-nitrophenol (pNP) at 410 nm using an ELISA reader (ELX800, BIOTEX).
The test measures α-glucosidase activity by hydrolyzing the substrate 1-nitro-4-hydroxybenzene-α-D-glucopyranoside (pNPG) into α-D-glucose and p-nitrophenol (pNP) The inhibitory activity of α-glucosidase is assessed by analyzing the absorbance of the produced p-nitrophenol.
Formula to determine % inhibition (%I): %I = Acontrol − Asample × 100 With: Acontrol is absorbance of the standard sample (no inhibitors), Asample is the absorbance of the test sample.
Method for determining the chemical components in Olax imbricata leaves
The method for determining medicinal herbs involves systematic analytical techniques to identify the chemical components present in medicinal plant samples This identification is essential for ensuring the quality control and assurance of both medicinal plants and pharmaceuticals However, many therapeutic herbs remain under-researched regarding their chemical composition, despite their significant efficacy.
When collecting 5 samples of 96% EtOH extraction extraction, our team utilized Soxhlet
Table 3.2 Equipments in determining the chemical components method
Soxhlet refluxing equipment Single refluxing device
22 reflux equipment to conserve solvents and compounds, hence minimizing solvent loss of the extractions
Mayer reagent HgCl2 (XiLong, China)
Dragendorff reagent Bi(NO3)2 (XiLong, China)
CH3COOH (XiLong, China) Bertrand reagent Acid Silicowolframic (XiLong, China)
Hager reagent NaOH (XiLong, China)
Acid Picric (Kanto, Japan) Bouchardat reagent I2 (XiLong, China)
The extraction process involves isolating a mixture of compounds from plant material into three fractions based on increasing polarity: less polar, medium polar, and strongly polar This is achieved by sequentially using diethyl ether, ethanol, and water as solvents Subsequently, the chemicals present in the raw materials are identified through characteristic reactions.
To extract Olax imbricata leaves, prepare 15g of the leaves and use diethyl ether in a Soxhlet extractor or shake in a conical flask for 30 minutes Ensure that after evaporation, the ether extraction does not leave a translucent film on the watch glass.
HCl (XiLong, China) Ethanol (Chemsol, Vietnam)
H2SO4 (XiLong, China) Diethyl Ether (Chemsol, Vietnam)
Chloroform (Chemsol, Vietnam) Na2CO3 (XiLong, China)
Acetic anhydride (XiLong, China) Gelatin (XiLong, China)
KOH (XiLong, China) NaCl (XiLong, China)
Figure 3.6 Procedure for preparation of extractions
Combine, filter, and concentrate the extraction until approximately 50mL of the ether extraction remains
After the ether extraction of Olax imbricata leaves, the residue should be extracted with strong alcohol for 30 minutes, repeating this process 2-3 times The combined extractions are then filtered and concentrated to yield approximately 50 mL of ethanol extraction This extraction is divided into two portions: one for the direct identification of compound classes and the other for hydrolysis to identify aglycones Hydrolysis involves adding 10 mL of 10% HCl to 15 mL of the ethanol extraction and refluxing for 30 minutes, followed by cooling in a decantation flask and extracting three times with 15 mL of diethyl ether.
After the ethanol extraction of Olax imbricata leaves, the residue is further extracted with water, resulting in approximately 50mL of aqueous extraction This extraction is then divided into two portions: one for direct identification of substance groups and the other for hydrolysis to identify aglycones The hydrolysis process involves adding 10mL of 10% HCl to 15mL of the aqueous extraction and refluxing for 30 minutes, followed by cooling in a decantation flask and extracting three times with 15mL of diethyl ether.
3.6.3.2 Determination of compounds in ether extraction
The ether extraction used to determine the following groups of compounds:
1 Fatty acids 4 Free Triterpenoid 7 Anthraquinone
Describe the qualivative analysis for ether extraction:
To test the extraction, apply a few drops onto a thin piece of paper and allow it to evaporate; if the fluid contains essential oils, the scent will fade A faint patch on the paper indicates the presence of fat.
Figure 3.7 Qualivative analysis chemical compounds in ether extraction
To determine the presence of essential oil, 5 mL of the extraction is placed in a ceramic crucible and evaporated until dry A small amount of high-proof ethanol is then added, followed by further evaporation until dry The presence of essential oil is indicated by a distinct, mild scent.
Carotenoids were analyzed by evaporating 5mL of the extraction in a porcelain crucible until dry, followed by the removal of the solvent and any essential oil aroma A few drops of concentrated H2SO4 were then added, and the appearance of a dark blue or greenish-blue to light blue solution indicates the presence of carotenoids.
Triterpenoids were identified by evaporating 5mL of extraction in a porcelain crucible until dry, followed by dissolving 0.5mL of acetic anhydride and adding 0.5mL of chloroform The mixture was placed in a test tube, and 1-2mL of concentrated H2SO4 was carefully added along the test tube's wall using a Pasteur pipette At the interface of the two solution layers, a color change to red-brown or red to purple was observed, while the top layer gradually turned green or purple, indicating the presence of triterpenoids such as phytosterols or triterpenes.
Mayer reagent: white precipitate - light yellow
Bouchardat reagent: brown red precipitate
Dragendorff reagent: orange red precipitate
Hager reagent: orange yellow precipitate
Alkaloids were identified by evaporating 10mL of the extraction in a porcelain crucible until dry Subsequently, 2-4mL of a 1% HCl solution was added, and the mixture was distributed evenly into six test tubes Five different reagents (Mayer, Bertrand, Bouchardat, Dragendorff, and Hager) were introduced into five of the test tubes, while one standard control tube was left without reagents The presence of alkaloids was indicated by increased turbidity or precipitation in the test tubes compared to the control.
To test for the presence of coumarin, apply a few drops of the extraction onto filter paper and allow it to evaporate until dry Next, add a few drops of a 10% KOH solution and gently dry the sample Cover half of the dot with a coin and expose it to 365nm UV light After a few minutes, remove the coin; if the covered area exhibits weaker luminescence than the exposed area but eventually matches in intensity, this indicates the presence of coumarin.
The presence of free anthraquinone was confirmed using the Borntrager reaction, where the addition of 1 mL of a 10% NaOH solution to 5 mL of the extract resulted in a pink to red color in the alkaline layer.
Flavonoids were identified by evaporating 10mL of extraction in a ceramic crucible The Cyanidin reaction was utilized by dissolving 2mL of ethanol in the solution and transferring it to a test tube A small amount of metallic magnesium powder and 0.5mL of concentrated HCl were added The presence of flavonoids is indicated by a pink to red hue in the solution.
3.6.3.3 Determination of compounds in ethanol extraction
The ether extraction used to determine the following groups of compounds:
Describe the qualivative analysis of ethanol extraction:
To assess the presence of anthocyanosides, 2-3 drops of a 10% HCl solution were mixed with 1 mL of the extract A pink to red coloration indicates the presence of anthocyanosides, while the solution changes to blue upon alkalinization with a 10% NaOH solution.
Proanthocyanidins were quantified by mixing 5 mL of the extract with 2 mL of a 10% HCl solution and heating the mixture for 10 minutes A color change to pink or crimson indicates the presence of proanthocyanidins.
Add 5 drops of a Gelatin solution containing 2 milliliters of salt to 2 milliliters of extraction and vigorously shake Compared to the original solution, a white precipitate indicates the presence of tannin [98]
Animal experimentation methods
Mice were weighed daily during the adaptive feeding phase until the second STZ injection Before the extraction and acarbose administration, their weights were recorded 3 to 4 times per week using an MF-CDT electronic balance The weighing procedure involved placing a specialized weighing box on the scale, calibrating it to zero, adding the mouse, and noting the displayed weight Daily and weekly weight records were maintained to calculate the consumption of extraction and acarbose by the mice.
Figure 3.11 Qualivative analysis chemical compounds in hydrolyzed water extraction
28 addition, the weight of mice recorded throughout the year revealed a substantial difference between experimental groups
To prepare STZ at a dose of 40 mg/kg, mice should be fasted for 12 hours prior to injection Use a 1cc needle to draw the appropriate amount of STZ based on the mouse's weight Employ the "double hand" technique to physically restrain the mouse: place it on the cage lid, gently pull back its tail with the dominant hand, and secure the neck with the other hand to prevent bites Once the mouse is held firmly, invert it to expose the belly and check for signs of life by observing thoracic movement To minimize infection risk, clean the lower abdomen with a cotton swab soaked in 70% alcohol Administer the injection into the lower right or left corner of the abdominal cavity to avoid damaging internal organs Inject STZ quickly and decisively, then monitor the mice for 24 hours.
48 hours after injection to determine their behavior
The extraction and acarbose were precisely prepared and dissolved in distilled water Mice received daily dosages of 50, 100, and 200 mg/kg body weight of the extraction, while acarbose was administered at a dosage of 100 mg/kg body weight per day After reconstitution, both substances were sealed and stored in a refrigerator at 10 °C to inhibit bacterial growth and maintain the biological activity of the extraction, ensuring they were used within one day.
Note: A and B are the two injection sites on the mouse, placed between abdominal cavity and rear thigh
Before dosing, ensure to shake the extraction and acarbose thoroughly To protect the rat's oral cavity, administer the solution using an "oral gavage" needle directly into the mouth Dilute the extraction so that each rat receives only 0.05 mL per dose This method of orally administering a small volume of extraction, combined with oral gavage, allows for precise control over the infusion amount.
The oral solution infusing method involves immobilizing the mouse heads in an upright position The "oral gavage" needle is then carefully inserted from the mouth, through the pharynx, and into the stomach, allowing for the direct administration of substances such as extraction or acarbose into the stomach of the mice.
Improper placement of the needle into the lung instead of the stomach can lead to procedural failure Symptoms in a mouse, such as coughing, choking, pain, or nasal discharge, require immediate attention Additionally, excessive force during needle insertion into the mouth can result in serious trauma, including pharyngeal rupture or even death of the rat.
Glucose tolerance testing is a crucial method for diagnosing type 2 diabetes through in vivo testing, currently serving as the sole approach for assessing diabetes status The Oral Glucose Tolerance Test (OGTT) is specifically utilized to diagnose both pre-diabetes and diabetes In our study, we measured the fasting blood glucose levels of mice between two doses of STZ to determine their diabetes status, identifying cases of pre-diabetes and type 2 diabetes.
Figure 3.14 Oral infusing solution method
A study was conducted involving mice that had fasted for 14 hours but were allowed to drink water The mice received an oral administration of 0.2 mL of 20% glucose solution It was crucial to ensure that the glucose was delivered quickly and precisely into the stomach Blood glucose levels were measured intravenously prior to the administration.
To measure glucose levels in mice, blood is drawn from the tail vein at various time intervals after glucose injection Preparation for blood collection requires gloves, gauze, a 22-gauge needle tip, 70% alcohol, povidine, and an animal incubator It is essential to ensure the animal's comfort and avoid stress during the procedure Brushing the tail from its corner to tip is discouraged as it can increase white blood cell counts If veins are not visible, dipping the tail in 40°C water can help The tail should be disinfected with 70% alcohol, and blood collection instruments must also be sterilized to prevent infection The sterile needle is inserted into the vein at a 45-degree angle, located two to three centimeters from the tail's tip Blood is aspirated slowly to prevent collapsing the ventricle, allowing enough blood to fill the test strip for the Bene-Check blood glucose meter After blood collection, gauze is applied to stop any bleeding at the site.
To avoid cross-contamination, each dressing should be used just once Then, return the mouse to its cage and start the timer for the subsequent blood sugar measurement
3.7.5 Anatomy and cardiac blood collection
In in vivo testing, it is crucial to collect blood from laboratory animals with minimal discomfort to avoid influencing study results The technique employed involved cervical dislocation of mice to induce paralysis of the autonomic nervous system, immobilizing the rodent and eliminating pain perception Pharmacological anesthesia was not used in this study, as it could have altered the outcomes.
To immobilize the mouse, place it on a flat surface, apply pressure to its neck, and pull its tail until the vertebrae are stretched Exercise extreme caution during cervical vertebrae extraction to avoid damaging the heart or other organs An open thoracotomy is then performed while the heart continues to beat Insert a needle into the ventricle at a 45° angle, then raise it to 60° to draw blood until the heart collapses Transfer at least 1.0 mL of blood into two tubes containing Lithium Heparin and EDTA, ensuring not to exceed the tube's capacity Mix gently and freeze the tubes for transport to the Pham Ngoc Thach Medical University General Clinic for analysis of blood indicators such as Insulin, TG, CT, HDL, and LDL Hematology samples are examined using the Beckman Coulter AU400 biochemical analyzer.
3.7.6 Measuring organ mass and making visceral tissue templates
After 8 weeks of using extraction, the mice were sacrificed to obtain tissues (fat, liver, and kidney) and cardiac blood Before performing surgery, it is necessary to clean the instruments, the anatomical area and prepare a 10% formalin Ensure the anatomical environment is kept sterile at all times [102] Following are the steps of the surgical procedure: The mice's autonomic nervous system becomes paralyzed when the cervical vertebrae are dislocated On a flat surface, place the mouse on its back Using 70% alcohol, disinfect the skin beneath the abdomen From the abdomen to the chin, make an incision [5] The abdominal cavity is opened by clamping and raising the skin of the lower abdominal muscles and cutting along the ribs Remove every rib to reveal the thoracic cavity Blood drawn from the heart The liver is extractioned by severing the ligaments and choroids connecting it to the stomach, diaphragm, and kidneys When handling the liver, care must be taken to avoid crushing and injuring it [107] Then, detach the kidneys and adipose tissue from the abdominal wall Note that male mice should have their testicles and genital fat removed After collection, the liver, fat, and kidney will be weighed and placed immediately in 10% formalin for preservation [5] The tissue will then be sent to the Technical room of the Department of Pathology at Le Van Thinh Hospital in Thu Duc City, Vietnam Tissue samples were fixed in 10% formalin, paraffin block casting, sample cutting, and Hematoxyline & Eosin (H&E) staining were conducted Upon receiving templates, inspect templates on the microscope iScope model IS.1153- PLI and capture images by EUROMEX CMEX 5 DC5000C
Monitoring animal behavior in response to environmental changes or medicinal herbs is crucial for assessing their biological impact This approach allows us to infer underlying physiological mechanisms Key behavioral metrics include movement velocity, direction, geographical distribution, and typical behaviors In this study, we utilized MATLAB 2019 and the Mouse Activity script to efficiently evaluate motion metrics, such as distance traveled and average speed, in a mouse model.
The study utilizes two transparent plexiglass chambers, each measuring 12×12×12 inches and coated with black paper, to analyze mouse behavior A video recording system captures footage in MOV format at a resolution of 1280×720 pixels and 30 frames per second for a duration of 12 minutes The recorded videos are processed using MATLAB software, where adjustable parameters such as pixel size, number of animals, and frame selection are optimized to extract meaningful data The results include a visual representation of the mouse's journey, a figure illustrating distribution with eccentricity, and an Excel table detailing the distance traveled by each mouse.
All in vivo tests were performed in triplicate, and results are presented as the mean with standard deviation Statistical analysis was conducted using SPSS software (version 20.0, USA), employing methods such as ANOVA and Duncan tests, with a significance level set at p