In vivo evaluation of the ability to prevent overweight, obesity, and diabetes of 96% ethanol extraction from olax imbricata leaves

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:

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: TRINH KHANH SON

VO THI NGA HUYNH NGUYEN LINH CHI STUDENT: TRAN HOANG VU

IN VIVO EVALUATION OF THE ABILITY TO PREVENT

OVERWEIGHT, OBESITY, AND DIABETES OF 96%

ETHANOL EXTRACTION FROM OLAX

IMBRICATA LEAVES

GRADUATION THESIS FOOD TECHNOLOGY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

Thesis code: 2022-17116044

Student ID: 17116044 Major: FOOD TECHNOLOGY Advisor: TRINH KHANH SON, Assoc Prof

VO THI NGA, PhD

HUYNH NGUYEN LINH CHI, Eng

IN VIVO EVALUATION OF THE ABILITY TO

PREVENT OVERWEIGHT, OBESITY, AND DIABETES

OF 96% ETHANOL EXTRACTION FROM OLAX

IMBRICATA LEAVES

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

Thesis code: 2022-17116044

IN VIVO EVALUATION OF THE ABILITY TO

Student ID: 17116044 Major: FOOD TECHNOLOGY Advisor: TRINH KHANH SON, Assoc Prof

VO THI NGA, PhD

HUYNH NGUYEN LINH CHI, Eng

Ho Chi Minh City, August 2022

PREVENT OVERWEIGHT, OBESITY, AND DIABETES

OF 96% ETHANOL EXTRACTION FROM OLAX

IMBRICATA LEAVES

In addition, I want to thank the departments and administration at Ho Chi Minh City University of Technology and Education for making it possible for me to study and complete this thesis in the best possible circumstances I also want to thank the professors at the departments of food technology and chemistry for providing me with the resources I needed to finish the thesis and for letting me use their labs

Finally, I want to express my sincere gratitude to my family and friends for their unwavering support and encouragement throughout the writing of this thesis and throughout my life

I have done my best, but with the limitation time of the research process and my limited knowledge, mistakens and errors are hard to advoid I appreciate your sincere suggestions to make this thesis better

My sincere gratitude!

Ho Chi Minh City, July 31 st ,2022

COMMITMENT

I hereby declare that all content presented in this graduation thesis has been done by ourselves, including instructors and students The research content is carried out based on the requirements, design, and guidelines and is validated by the instructor The entire content of the graduation thesis has been checked against plagiarism using Turnitin software and ensures no more than 30% duplication I hereby certify that the content in the writing process is referenced in the graduation thesis from clearly sourced documents that have been correctly and fully cited in accordance with regulations

Ho Chi Minh City, July 31 st ,2022

CONTENT GRADUATION PROJECT ASSIGNMENT Error! Bookmark not defined

ACKNOWLEDGEMENTS ii

COMMITMENT iii

LISTS OF FIGURES xv

LIST OF TABLES xvi

LIST OF ABBREVIATION xvii

SAMPLE CODE xviii

ABSTRACT xix

CHAPTER 1: INTRODUCTION 1

1.1 Reasoning of the research 1

1.2 Purposes of the research 1

1.3 Limits and scope of the research 1

1.4 Scientific and practical significance 1

1.5 Subjects, scope and limitations of the research 2

1.6 Structure of report 2

CHAPTER 2: OVERVIEW 3

2.1 Overview about Olax imbricata 3

2.2 Overview about extration methods 3

2.2.1 Maceration 3

2.2.2 Soxhlet extraction 4

2.2.3 Microwave-assisted extraction (MAE) 4

2.2.4 Ultrasonically assited extraction (UAE) 5

2.2.5 Others extraction methods 5

2.3 Metabolism processes of nutritional compounds in humans 6

2.3.1 Carbohydrate metabolism 6

2.3.2 Lipid metabolism 7

2.3.3 Protein metabolism 9

2.4 Combination model of high-fat diet and low-dose streptozotocin (HFD-STZ-T2D) 10

2.4.1 High fat diet 10

2.4.2 Using streptozotocin 10

2.4.3 HFD-STZ-T2D paradigm 10

2.4.4 Advantages and disadvantages of the HFD-STZ-T2D paradigm 11

2.5 In vivo testing on laboratory animals 11

2.5.1 Origin and classification of mice 11

2.5.2 The advantages and disadvantages of utilizing laboratory animals 12

2.5.3 Conditions for raising laboratory mice 12

2.5.4 Animal Testing Ethics and the 3Rs rule 14

2.5.5 Experimental environment 15

2.6 Previous relevant research 15

2.6.1 Domestic studies 15

2.6.2 Foreign Studies 15

CHAPTER 3: MATERIALS AND METHODS 16

3.1 Materials 16

3.1.1 Olax imbricata leaves 16

3.1.2 Streptozotocin 16

3.1.3 Food ration 16

3.1.4 Experimental animal 17

3.2 In vivo experimental design 18

3.3 Determine sample size 18

3.4 Extraction receiving method 19

3.6 Method for determining the chemical components in Olax imbricata leaves 21

3.6.1 Overview of determination method 21

3.6.2 Equipments and chemicals 21

3.6.3 Procedure 22

3.7 Animal experimentation methods 27

3.7.1 Measuring mouse weight 27

3.7.2 Mouse injection method 28

3.7.3 Oral infusing solution method 28

3.7.4 Glucose tolerance testing 29

3.7.5 Anatomy and cardiac blood collection 30

3.7.6 Measuring organ mass and making visceral tissue templates 31

3.7.7 Locomotor activity method 31

3.7.8 Statistical 32

CHAPTER 4: RESULTS AND DISCUSSIONS 33

4.1 Chemical compounds in Olax imbricata leaves 33

4.2 In vitro α–glucosidase inhibitory activity of extractions from Olax imbricata leaves 34 4.3 Body mass and energy comsumption 35

4.4 Glucose tolerant testing 36

4.5 Blood lipid index 40

4.6 Mass of tissue 42

4.7 Microsurgery of histological structure of organs 43

4.8 Locomotor activity and behavior 47

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 51

REFERENCES 52

APPENDICES 64

LISTS OF FIGURES

CHAPTER 3

Figure 3.1 Olax imbricata leaves 16

Figure 3.2 White mice (Mus Musculus var albino) 17

Figure 3.3 In vivo experimental design 17

Figure 3.4 Extraction receiving method 20

Figure 3.5 Soxhlet refluxing equipment 21

Figure 3.6 Procedure for preparation of extractions 22

Figure 3.7 Qualivative analysis chemical compounds in ether extraction 23

Figure 3.8 Qualivative analysis chemical compounds in ethanol extraction 25

Figure 3.9 Qualivative analysis chemical compounds in hydrolyzed ethanol extraction 26

Figure 3.10 Qualivative analysis chemical compounds in water extraction 26

Figure 3.11 Qualivative analysis chemical compounds in hydrolyzed water extraction 27

Figure 3.12 Injection site 28

Figure 3.14 Oral gavage needle 29

Figure 3.13 Oral infusing solution method 29

Figure 3.15 Mouse anatomy model 30

CHAPTER 4

Figure 4.2 Energy comsumption of mouse 35

Figure 4.3 Fasting blood glucose of experimental mouse 37

Figure 4.4 Glucose tolerant of experimental mouse in week 0, 2nd, 4th, 7th, 10th and 12th 38

Figure 4.5 Area under the curve of glucose tolerant of experimental mouse 39

Figure 4.6 Fat tissue of experimental mouse 44

Figure 4.7 Liver tissue of experimental mouse 45

Figure 4.8 Kidney tissue of experimental mouse 46

Figure 4.9 Renal corpuscles of kidney of experimental mouse 47

Figure 4.10 The distance traveled by experimental mouse before and after consuming food during weeks 0, 2nd, 4th, 7th, 10th, and 12th 49

Figure 4.11 Movement of experimental mouse at week 0, 2th, 4th, 7th, 10th and 12th 50

LIST OF TABLES

CHAPTER 3

Table 3.1 Nutrient compositons in ND and HFD 16

Table 3.2 Equipments in determining the chemical components method 21

Table 3.3 List of chemicals 22

Table 3.4 List of reagents 22

CHAPTER 4

Table 4.2 Result from ethanol extraction 33

Table 4.3 Result from hydrolyzed ethanol extraction 33

Table 4.4 Result from water extraction 33

Table 4.5 Result from hydrolyzed water extraction 34

Table 4.6 In vitro result of extraction from Olax imbricata leaves 34

Table 4.7 Blood lipid indicators of experimental mouse 40

Table 4.8 Mass of liver, kidney and fat of experimental mouse 42

LIST OF ABBREVIATION

AGIs Alpha-glucosidase inhibitors HFE-200 High fat ethanol-200

(mg/kg.w/day)

Half maximal Inhibitory concentration

CETP Cholesteryl ester transfer protein MAG Monoacylglycerol

EAE Enzyme assisted extraction PLE Pressurized liquid extraction

HFA High fat Acarbose-100

HFE-50 High fat ethanol-40

Very high-fat diet

HFE-100 High fat ethanol-100

World Health Organization

ABSTRACT

In this study, Olax imbricata leaves were extracted with 96% ethanol, then the bioactivity of the extract was evaluated by in vitro and in vivo tests For in vitro testing, extracts was found that

have glucosidase inhibitory activity and to be approximately 1.5 times more resistant to

α-glucosidase than acarbose (type 2 diabetes drug) From there, in vivo testing began with groups of white mice (Mus Musculus var albino) used to evaluate the effects of different doses of extracts

(50, 100 and 200 mg/kg/day)comparisons with acarbose The results after 3 months of testing showed that the average body weight of the groups of rats decreased gradually with the extract doses from 50mg/kg to 200mg/kg Besides, the results after testing show that the extract has the ability to inhibit α-glucosidase activity, positively impact, significantly reduce overweight, obesity, type 2 diabetes through results on glucose tolerance testing, blood lipid index, body weight, histological and locomotor behaviors compared with acarbose

CHAPTER 1: INTRODUCTION

1.1 Reasoning of the research

Obesity seems to be an extremely intricate multi-factorial disease Since 1980, the prevalence of overweight and obesity in the world has expanded many times It rises regardless of age, gender, race, or geographical region According to research [21], however, the bulk of those

at risk for obesity are senior citizens and women [21] The World Health Organization (WHO) also recognizes obesity as a global pandemic; the numbers of people suffering from this disease is rapidly increasing and is predicted to hit 300 million by 2025 [119] Obesity is a medical disorder

in which excess body fat accumulates to an extent that might negatively impact health, reduce life expectancy, or aggravate health conditions [105] Over time, excess fat is deposited, with lipid buildup predominantly in the form of triglycerides in adipose tissue and a volume increase in skeletal muscle, liver, and other organs and tissues [52] Social life, mobility, and quality of life can be negatively impacted by obesity More seriously, the increase in the prevalence of obesity has led to an increase in the incidence of other diseases including diabetes, stroke, cardiovascular disease, hyperlipidemia, more severe cases such as cancer, infectious liver disease [116] Obesity, polycystic ovary syndrome, and osteoarthritis Consequently, obesity must be effectively prevented and treated The most prevalent treatment is weight loss through good diet and physical activity In addition, obesity can be alleviated through the use of weight loss medications However, these treatments can induce several adverse effects, including diarrhea, vomiting, even neurological abnormalities [110] Therefore, there is a need for an alternative strategy with minimal or no adverse effects for treating obesity and its associated consequences Due to their minimum side effects and maximum usefulness, the development of natural anti-obesity medications is gaining importance nowadays [81] In light of this trend, we sought to evaluate the

capacity of Olax imbricata leaves extraction to prevent overweight and obesity in in vivo testing

1.2 Purposes of the research

This research was conducted with the aim of: (1) Assessing the effect of the extraction on the ability of experimental animals to control weight gaining and energy consumption; (2) Evaluating the effect of the extraction on the glycemic control of experimental animals; (3) Evaluating the effect of the extraction on the control of blood lipid indexes of experimental animals; and (4) Evaluating the effect of the extraction on the ability of experimental animals to control motor behavior

1.3 Limits and scope of the research

This study evaluated the efficacy of the extracts on prevent and reduce disease status in experimental animal models (mouse) of overweight, obesity, and diabetes when HFD-STZ-T2D paradigm In addition, the focus of this study was only on evaluating the outcomes based on body weight, energy intake, blood fat, fasting blood sugar, blood glucose tolerance, tissue microsurgery (liver, kidney, fat), and locomotor activity of experimental animals

1.4 Scientific and practical significance

Throughout history, medicinal plants have played a vital role in maintaining and restoring human health Vietnam has an abundance of herbal resources Therefore, medicinal plants have been used since antiquity to cure numerous severe ailments with positive effects Our study was

undertaken to control and prevent diseases associated with Olax imbricata leaves extraction on

experimental animals suffering from overweight, obesity, and loss of glycemic control and high blood fat levels This research will initially serve as the scientific foundation for the development

of an extraction of natural origin in the form of a functional meal to aid in the treatment of diseases

caused by nutritional problems In the future, the Olax imbricata leaves extraction may serve as

an effective alternative for certain commercially accessible medications From there, contribute to the diversification of items that promote illness treatment and prevention

1.5 Subjects, scope and limitations of the research

- Subjects of the research: Olax imbricata leaves and the male white mice (Mus Musculus

var albino) Olax imbricata leaves of the poplar tree were collected in February 2022 at Research

and Production of Medicinal Materials Center in Phu Yen Province, Vietnam Male white mice (5-week-old, average weight 13g±1g) were purchased in early April 2022 at the Pasteur Institute

in Ho Chi Minh City, Vietnam

- Scope of the research: This study focused on evaluating the ability to control and prevent

disease of Olax imbricata leaves extraction on the physiological indicators of experimental

animals (mouse) suffering from irritation causing overweight disease, obesity by high-fat diet combined with STZ injection

- Limitations of the research: to evaluate the ability of the Olax ỉmbricata leaves extraction

to control and prevent diseases on body weight, blood lipids, blood sugar, tissue structure microsurgery, and to evaluate the behavior and activities of experimental animals

1.6 Structure of report

Chapter 1: Introduction

Chapter 2: Overview

Chapter 3: Materials and Methods

Chapter 4: Experimental results and Discussion

Chapter 5: Conclusions and Recommendations

CHAPTER 2: OVERVIEW

2.1 Overview about Olax imbricata

Olax is the biggest genus in the Olacaceae family (including 40 species) People all around

the world use plants of the Olax genus, Olacaceae family, for the aim of curing and protecting health [85] The genus Olax grows extensively throughout Africa, South Asia, and Australia, and

a wide range of biological functions have been attributed to it [118] Some species of the Olax

genus, which are native to the tropical woods of Asia and Africa, are also involved in numerous

biological processes [85] Olax subscorpioidea is a shrub or tree that grows to a at least 10 meters

high in Nigeria Their body includes numerous therapeutic characteristics

Olax imbricata, member of the Olacaceae family, native to the tropics, and well-suited to

sandy soils, sunny and dry conditions, such as those found in the central of Vietnam and Phu Yen province [118] This is a 5-meter-tall shrub with slanting branches; the leaves are oblong and 7-

10 centimeters long (hairless); the peduncle is 6-8 millimeters long; the inflorescences are in the leaf axils and have a small, white calyx; the plant flowers from December to January and bears

fruit in August [118] Olax imbricata, is known as a herb with antioxidant, anti-inflammatory,

antibacterial, anti-infective, and preventative properties against a variety of gynecological disorders and has been used in Vietnam as a traditional treatment for diabetes [85] In the study [77], the presence of polyphenol chemicals, flavonoids, glycosides, saponins, tannins, alkaloids, and antibacterial and antioxidant activities were demonstrated in the poplar tree [77] According

to [85], in 2018, Vo Thi Nga et.al, effectively isolated 2 phenolic compounds, 3 phenol glycosidic

compounds, and leonuriside A from the chemical composition of Olax imbricata [85] Using triterpenoid glycoside isolation and in vitro α-glucosidase inhibition tests to continue the work in

2019, findings were obtained by separating triterpenoid glycosides [118]

2.2 Overview about extration methods

Various extraction techniques are now employed to create plant extractions containing physiologically active compounds Some old methods are still being enhanced, while others are still being created In the presence of solvents, extraction is the process of extractioning active chemicals from materials of natural origin (plants, animals, etc.) Extraction is the process of eliminating undesired components from a substance in order to recover the desired soluble component in the presence of solvents [10] During extraction, the solvent diffuses into the substance and dissolves polar chemicals [95] The goal of the extraction procedure is to separate the plant's soluble metabolites from its insoluble constituents In addition, the extraction method plays a role in determining the quality and quantity of the ingredients [10]

2.2.1 Maceration

Maceration, a technique employed in winemaking that has been used extensively to the study

of plants with a wide variety of biological activity In order to soak beams, plant material (in the form of flakes or powder) is soaked in a suitable solvent The mixture of extraction material and extraction solvent was stored for a minimum of three days (3-7 days) at room temperature with intermittent stirring Note that plant matter must be completely submerged in the solvent [1] The extraction mixture is recovered and the residue is filtered upon completion of the extraction step The solvent was then removed from the extraction using drying or water heating The same basic

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 [1] The optimal ratio of solvent to coarse residue is 10:1 (v/w) [95] During extraction, the mixture was heated Reflux heating employs the same idea as soaking the beams; both are submerged in

an appropriate solvent Due to temperature catalysis, the extraction period with reflux heating is less than that with beam soaking Reflux is appropriate for the extraction of heat-stable chemicals

or hard substances [87]

2.2.2 Soxhlet extraction

Soxhlet extraction is the standard extraction technique with greater extraction efficiency compared to other conventional procedures [123] This is a method of continuous solid/liquid extraction The finely powdered sample is wrapped in a filter paper bag and inserted in the sample tube of the Soxhlet device in this approach At the bottom of the equipment is a flask in which the extraction solvent is heated The solvent evaporates into the sample tube, exposing the component

to be extractioned, then condenses in the condenser and drips back into the sample tube When the liquid reaches the siphon tube, it will flow into the flask located at the bottom [97] As a result, the solvent will transport the extraction downward, facilitating the process of product recovery This cycle of evaporation and condensation is repeated until the complete amount of compound to be extractioned from the sample has been extractioned [1]

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 [87] The solvent recovery is satisfactory, the system is affordable, and it is simple to operate [97] There is no need to filter the residue after extraction [1] This approach is not ideal for the extraction of heat-sensitive components such as enzymes, alkaloids, esters, etc

2.2.3 Microwave-assisted extraction (MAE)

Microwave-assisted extraction (MAE), is a technique that utilizes microwave energy to isolate the chemicals of interest from the sample of raw material The microwave radiation has a frequency range of 300MHz to 300GHz and a wavelength of 1cm to 1m [1] These waves are electromagnetic radiation that is non-ionizing [97] Microwave radiation interacts with the dipole bonds of polar materials (consisting of samples and solvents) to heat the surface of the substance, which is then transported via conduction These dipole linkages traveling around molecules will break the hydrogen bonds, releasing polar components from the sample and allowing the solvent

to easily enter [87] Even if plant materials have been dried to remove water, they still contain a small quantity of moisture This water content is heated by the collision of microwaves, resulting

in an increase in internal pressure The pressure exerted on the cell wall of a plant causes the cell

to enlarge The increase in pressure over time causes cell disintegration, hence increasing the exposure of cellular components to solvent [97]

The variables that influence MAE are solvent selection, the ratio of solvent to raw material, irradiation temperature, irradiation time, and microwave power [130] The MAE extraction method reduces extraction time and solvent usage in comparison to standard extraction methods [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

Additionally, the solvent's dielectric characteristics influence the extraction procedure Consequently, the optimization of these parameters is crucial [97] The MAE extraction approach

is restricted to small-molecule phenolic chemicals such as phenolic acids (gallic acid and ellagic acid) and quacertin, as these molecules are stable at heating conditions up to 100oC 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]

2.2.4 Ultrasonically assited extraction (UAE)

In the food and pharmaceutical industries, this method of extraction has been regularly employed for decades [130] This technique employs ultrasonic pulses between 20kHz and 2000kHz [87] The method's mechanism of action is based on the phenomena of cavitation [130] Due to the presence of ultrasonic vibrations in the solution, the extraction of UAE will generate small air bubbles inside the solvent [100] Over time, the size of the bubbles will increase until a critical size is reached The bubble subsequently deflates, releasing a great deal of energy This procedure can generate high temperatures (5000K) and pressures (1000atm) [130] The surface contact between the solvent and the sample is increased due to the mechanical action of the sound waves produced by ultrasonic waves Under the influence of ultrasonic waves, the physical and chemical properties of the material are altered and the plant cell wall is broken down, enabling the release of chemicals and boosting the mobility of solvents into the cells plant cells [87] UAE heat transfer in the direction from the exterior to the interior of the plant cell The MAE approach, on the other hand, transmits heat in the reverse way [130] In this technique, the plant material must

be dried, pounded into a fine powder, and sieved appropriately The prepared material sample and solvent are then introduced into the ultrasonic extraction apparatus Without the use of heat, energy from high-frequency sound facilitates extraction [1]

The frequency, intensity, temperature, and duration of ultrasound can have a direct impact

on the extraction process's effectiveness In addition to sample properties (moisture, size, etc.), solvent type, solvent volume, and solvent concentration are significant factors for effective extraction [130] The advantage of the UAE approach is that it is easy to implement and very inexpensive, thus it may be used on a small or large scale This technique helps to reduce extraction time and solvent consumption [135] Nevertheless, the use of ultrasonic energy at frequencies above 20 kHz can result in the production of free radicals [87]

2.2.5 Others extraction methods

In addition to the aforesaid extraction process, there are several current extraction methods

in use today, such as: pressurized liquid extraction (PLE), supercritical fluid extraction (SFE), enzyme assisted extraction (EAE), supercritical-water extraction (SWE)…

Pressurized liquid extraction (PLE) is a technique used to extraction solid or semi-solid samples using high temperature (up to 200oC) and pressure (about 1500psi) Under conditions of high temperature and high pressure, the liquid state of the solvent persists [66] The PLE extraction approach is substantially faster (about 30 minutes) [54] and requires significantly less solvent than previous methods [97] This extraction condition increases the mass transfer rate, solubility of the analyte chemicals, and diffusion of the solvent into the extractioned material PLE can also be automated, which facilitates the process of quality control [54] The extraction solvent for the

supercritical fluid extraction (SFE) technique is supercritical fluid (SF) SF has equal solubility and diffusivity to liquids and gases, respectively This characteristic allows SF to dissolve a wide variety of natural substances Their solvation properties alter drastically at their critical points for modest pressure and temperature variations [135] Carbon dioxide (S-CO2) is often employed in solvent-free extraction (SFE) due to its advantages, which include a low critical temperature (31oC), selectivity, inertness, low cost, nontoxicity, and suitability for compound extraction [87] Thermal instability S-CO2 is suitable for the extraction of non-polar natural substances, such as lipids and volatile oils, due to its low polarity [135] Although S-CO2 has poor solubility for polar compounds, the addition of tiny volumes of ethanol and methanol improves polar molecule extraction [87]

Enzyme assisted extraction (EAE): Cell membranes and cell walls are composed of macromolecules including polysaccharides and proteins The extraction of natural substances is influenced by the coagulation and denaturation of proteins at elevated temperatures [135] Due to the presence of enzymes, the EAE approach facilitates the breakdown of the cell wall, cell membrane, and intracellular macromolecules Consequently, biological chemicals contained inside natural materials are readily discharged [19] In EAE, cellulose, α-amylase, and pectinase are frequently employed [135] These enzymes can be extractioned from a variety of sources, including bacteria, fungus, plants, and animal tissues It has been demonstrated that EAE procedures boost the extraction efficiency of antioxidants, such as phenolics, flavonoids, anthocyanins, and carotenoids [130]

2.3 Metabolism processes of nutritional compounds in humans

2.3.1 Carbohydrate metabolism

Three essential dietary components-carbohydrates, lipids, and proteins-must be present for the organism to live and develop normally Specifically, carbohydrates make for a considerable share of the body's total nutritional requirements and are regarded its primary source of energy [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]

The majority of carbohydrate metabolism takes place in the mouth and small intestine, specifically the duodenum When carbohydrates come into touch with salivary enzymes in the oral cavity, the metabolic process begins [64] The principal salivary gland enzyme is α-amylase : carbohydrate that is quickly exposed to α-amylase following oral administration The enzyme α-amylase hydrolyzes carbohydrates by cleaving the α-1,4 glycoside linkages without affecting the α-1,6 glycoside bonds, producing maltose, maltotriose, and short-chain dextrins The contents are then moved to the stomach Because the presence of hydrochloric acid provides low pH environment in the stomach [14], any remaining α-amylase in the food is promptly rendered inactive The food is subsequently transported to the small intestine When food that has been digested enters the duodenum, the pancreas secretes α-amylase and bicarbonate The pancreatic enzyme α-amylase is responsible for hydrolyzing carbohydrates Bicarbonate neutralizes acidic gastric fluids in order to enable enzyme action [42] Similar to the oral cavity, only α-1,4 glycosidic crosslinks are severed when pancreatic α-amylase is present This indicates that amylopectin is partially digested, whereas sucrose and lactose are not hydrolyzed Oligosaccharides and

disaccharides are indigestible On account of their presence on the cilia of intestinal mucosal cells, disaccharides can be degraded into monosaccharides α-glucosidase is a key enzyme in disaccharides [53] The rate of starch digestion is governed by the activity of α-glucosidase [111] This enzyme hydrolyzes α-1,4 glycoside linkages of dextrins or oligosaccharides into glucose for intestinal epithelial and blood absorption [109], [111] Almost entirely absorbed via the intestinal mucosa, monosaccharides enter the bloodstream and go to various bodily tissues [64] The pancreas secretes the hormone insulin to assist reduce blood sugar and transport glucose into cells and liver when blood sugar levels rise Glucose then undergoes glycolysis to generate adenosine triphosphate (ATP) for energy production [103] Insulin activates the liver's GLUT-2 transporter

to absorb glucose Through glycogenesis, glucose is stored in the liver in the form of glycogen Through glycolysis and gluconeogenesis, glycogen is converted into glucose when the organism

is hungry or active This helps the body generate energy [88], [103]

After carbohydrates are entirely digested, glucose is produced in the blood At this moment, blood glucose levels are elevated The pancreas plays a crucial function in lowering blood sugar levels by secreting insulin [103] If the body has difficulty managing the metabolism of carbohydrates and lipids, pancreatic insulin production and insulin sensitivity decline gradually This is the cause of human type 2 diabetes [24] Diabetes is characterized by hyperglycemia, which

is associated with the development of chronic complications [73] According to [31], individuals with diabetes had 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) Normal fasting blood glucose levels in laboratory animals are 199mg/dL, whereas the blood glucose levels indicative of type 2 diabetes in laboratory animals are: pre-diabetes 200-249mg/dL and diabetes >250 mg/dL [31]

2.3.2 Lipid metabolism

Dietary lipids are an important source of energy, accounting for approximately 30% of daily energy intake [61] There are approximately 95% long-chain triglycerides (TG) and 5% off phospholipids, cholesterol, sterols, and fat-soluble vitamins The majority of fat is metabolized as triglycerides About 95% of all fat is efficiently absorbed in the intestine, while the remaining 5%

is excreted in the stool [53], [61] Because fats are insoluble in water, emulsification is required for fat digestion in the digestive tract [11] Only when TG is hydrolyzed into fatty acids (FA) and monoglycerides (MG) can the body absorb it [61]

Most lipid digestion occurs in the small intestine The oral cavity is the initial site of food intake Here, lipids are only mechanically digested; as a result of chewing, food is broken into small pieces and transported to the stomach [42], [61] In the stomach, the primary factor contributing to the emulsification of lipid particles is intestinal motility [59] Triacylglycerol (TAG) digestion is initiated by the action of gastric lipase on the surface of emulsified granules [50] Lipase is a water-soluble enzyme that can only act on the surface of fat particles and can operate in the stomach's acidic environment [53], [61] At pH 3.0-6.0, lipase hydrolyzes TAGs into FAs and glycerides Continuously secreted from the serous glands, lipase accumulates in the stomach between meals when gastric pH is between 3.0 and 3.0 [36] Only 20 to 30% of fat is digested in the stomach, and there is no hydrolysis of phospholipids and cholesteryl esters [50] Next, the food enters the small intestine The majority of fats from the stomach to the small intestine are TAGs and diacylglycerol (DAG) The pancreas secretes enzymes (including lipase

and esterase) and bicarbonate into the duodenum to create a neutral environment that maximizes lipase activity The gallbladder simultaneously releases bile salts into the duodenum [42], [53] These lipid granules must be emulsified because large lipid particles prevent lipase from performing hydrolysis Bile salts have an amphoteric structure that aids in emulsification by breaking up large hydrophobic lipid droplets into smaller lipid droplets [61] Small lipid droplets have a large surface area and provide pancreatic enzymes with easy access Compared to that in the stomach, the duodenal emulsion is more stable Phospholipids are applied to the fat particles

to stabilize the emulsion The duodenal emulsion is composed of TGs, cholesteryl esters, partially ionized FAs, monoglycerides, and bile salts Although active pancreatic lipase is secreted, pancreatic co-lipase is required for digestion [42], [50] Cofactor co-lipase attacks lipid particles effectively It binds to the surface of emulsified lipid droplets and stabilizes the pancreatic lipase-lipid interaction [59], [61] Pancreatic lipase hydrolyzes TAGs at their sn-1 and sn-3 sites, releasing free fatty acids (FFA) and 2-monoacylglycerol (MAG) After that, pancreatic lipase hydrolyzes MAG further to produce glycerol and FAs Phospholipids are the second largest source of fat in the digestive tract These phospholipids, primarily lecithin (phosphatidylcholine), are hydrolyzed

at the sn-2 site by pancreatic phospholipase-A2 to produce FFAs and lysolecithin (lysophosphatidylcholine) Cholesterol esterase from the pancreas hydrolyzes cholesteryl esters into free cholesterol and FFA [124] Together with fat-soluble vitamins, products of lipid digestion form complexes with bile acids [75] The result is the formation of 2-nm-diameter micelles [61] The jejunum absorbs the hydrolysis products of TG and cholesteryl ester, FA, 2-monoglyceride, and free cholesterol [29], [50] Fat absorption is primarily facilitated by diffusion and protein transport as an intermediary transporter [58] Micelles of mixed composition migrate

to the cell surface and diffuse passively into intestinal mucosal cells Nonetheless, bile acids are not absorbed by cells [75] When the lumen of the intestine exceeds the intracellular concentration (concentration gradient), diffusion occurs across epithelial cells [58] Fatty acids with medium chain length (C4-C12) are amphoteric, water-soluble, and thus readily absorbed They are bound

to serum albumin before entering the bloodstream and being transported to the liver Short-chain FFAs are lipid-soluble and water-soluble, but they are not absorbed in the small intestine Short-chain FFAs are almost exclusively absorbed in the large intestine [42] After penetrating mucosal cells, lipid recombination occurs at the endoplasmic reticulum (ER) to maintain the concentration gradient required for diffusion and preparation for transport to tissues At the ER, monoacylglycerol acyltransferase esterifies MAG with FFAs to form DAG, which is then converted to TAG by diacyglycerol acyltransferase Choline transferase and ethanolamine transferase catalyze the combination of DAG with choline and ethanolamine for the synthesis of phospholipids Choletol is absorbed by enterocytes and esterified by acyl-coA: cholesterol acyltransferase in the ER [58], [75] It is difficult to transport lipids from mucosal cells into the blood because lipids do not dissolve in water Thus, TAGs combine with cholesteryl esters and approximately 30% of free nonpolar and hydrophobic cholesterol to form the core of mucosal cells Amphoteric compounds, including phospholipids, apoproteins, free cholesterol, and some saturated triglycerides, surround the outer membrane This combination produces the compound chylomicron Chylomicrons are lipoprotein-related compounds [43] The bipolar outer membrane

of lipoproteins facilitates their mobility in the blood Therefore, lipoproteins transport dietary

lipids to tissues and the liver [39]

Lipoproteins are classified as chylomicrons, very density lipoproteins (VLDL), density lipoproteins (LDL), and high-density lipoproteins (HDL) [41] Through the lymphatic vessels, chylomicrons transport digested fats from the intestinal lining cells into the bloodstream The lipoprotein lipase enzyme located on the blood vessel wall will then hydrolyze the TG contained in chylomicron into FAs that will be transported to muscle tissue, heart tissue - where it

low-is used for energy - or adipose tlow-issue - where it low-is stored More than 90% of TAGs in chylomicrons are absorbed by tissues during their brief time in the systemic circulation The remaining cholesterol will be absorbed by the liver, bound to lipids synthesized in the liver to form VLDL, and then secreted into the blood [75] VLDL is abundant in triglyceride granules and is the primary transporter of endogenous fat from the liver to the tissues [39], [61] After nearly all TAGs are removed, VLDL transforms into LDL [41] LDL lipids are predominantly composed of cholesterol esters and cholesterol [75] LDL's primary function is therefore to transport endogenous cholesterol to tissues [41] LDL contains approximately 70% of plasma cholesterol [29] and is regarded as the cause of atherosclerosis Particularly, atherosclerotic lesions in the aorta and coronary arteries are caused by elevated LDL levels [88] HDL plays a crucial role in the reversal

of cholesterol transport [75] The transport of cholesterol and phospholipids from peripheral tissues to the liver, where they are degraded and eliminated, can be utilized for bile acid synthesis [29], [39] Numerous studies have demonstrated that plasma with a high concentration of HDL can reduce the risk of atherosclerosis and cardiovascular disease HDL possesses antioxidant, anti-inflammatory, antithrombotic, and vasodilator qualities Consequently, it has the potential to promote arterial health [34]

2.3.3 Protein metabolism

Protein is an indispensable nutrient for nutritional homeostasis in humans After entering the body, proteins must normally undergo a series of complex breakdown processes to form simple molecules that are easily absorbed [30] The digestion of proteins begins in the stomach, where the acidic pH denatures the proteins The main cells of the stomach secrete an abundance of the inactive proteases pepsinogen They are transformed into active pesins by the stomach's low pH Pepsin degrades polypeptides and proteins into amino acids and oligopeptides [61] When the stomach's acidic mixture enters the small intestine, the low pH triggers the release of a hormone into the bloodstream To neutralize HCl in the stomach, secrentin stimulates the pancreas to secrete bicarbonate into the small intestine Consequently, the pH of the small intestine increases abruptly

to approximately 7.0 [84] The gastric phase of protein digestion may not play a significant role, but the pancreatic phase is essential [30] When food reaches the upper portion of the small intestine (duodenum) [84], endocrine cells secrete cholecystokinin and secretin, which trigger the release of hydrolytic enzyme precursors (inactive form) by pancreatic cells [36] These enzymes are activated in the duodenum, where an increase to pH 7.0 deactivates pepsin in the stomach This proenzymes' activation is initiated by enteropeptidase [30] It transforms inactive trypsinogen into active trypsin [36] From there, a series of proteolytic reactions leads to the formation of fully activated trypsin, chymotryopsin, elastase, and carboxylpeptidases A and B [30] These enzymes convert peptides into free amino acids and oligopeptides (about 2 to 6 amino acid residues) Oligopeptides then diffuse to the epithelium of the villi Here, the peptidase enzyme in the brush

border membrane cleaves to generate dipeptides, tripeptides, and free amino acids [53], [61] The small intestine will absorb these compounds Na+ co-transport is associated with amino acid absorption Co-transport of protons is associated with peptide absorption [125] This mixture is transported into the epithelial cells of the small intestine, where it then enters the villous capillaries and travels to the liver Thanks to aminotransferase, amino acids are converted into keto acids and amino groups in the liver In it, keto acid will undergo oxidation to CO2 and H2O, or it can be converted to glucose for energy production The amino groups will be recycled to produce new amino acids or other nitrogenous compounds In contrast, if it is not utilized, the body excretes it via the urea cycle [84]

2.4 Combination model of high-fat diet and low-dose streptozotocin (HFD-STZ-T2D) 2.4.1 High fat diet

There are numerous high-fat diets for rodents, however the majority of them vary in both fat content and fat source When 40 to 60% of calories are derived from fat, metabolic problems, hypertension, obesity, and the generation of inflammatory cytokines can result Although there is

no clear definition, a diet with 10% of calories from fat is called a low-fat diet (LFD), whereas 30–50% would be termed a high-fat diet (HFD), and more than 50% would be deemed an HFD This diet is highly heavy in fat (VHFD) Diabetes can be induced using both HFD and VHFD levels [37]

Diets high in fat inhibit the PI3K/AKT pathway and modify insulin action in target tissues, hence inducing insulin resistance and promoting the onset of type 2 diabetes In addition, insulin-stimulated tyrosine phosphorylation was lowered in both insulin receptors and IRS in mice fed a high-fat diet By activating c-Jun N-terminal Kinase (JNK) in the insulin signaling pathway, a high-fat diet suppresses the IRS and induces inflammatory responses that further inhibit this pathway [37] Animals fed a high-fat diet have decreased levels of GLUT4 at the cell surface, which plays a crucial role in insulin resistance

2.4.2 Using streptozotocin

Streptomycetes achromogenes, is capable of synthesizing streptocotozin (methyl-3-nitrosoureido)-Dglucopyranose] STZ is a free radical that can also cause damage to DNA and cells STZ is often administered in a single large dosage or repeated modest doses [37] Streptozotocin contains nitro radicals that harm β-cells, whereas the deoxyglucose moiety is responsible for the transfer of native molecules across β-cells Since STZ transports into pancreatic β-cells via glucose transporter 2 (GLUT2), other organs that also express this transporter, such as the kidneys, liver, and intestines, are also affected There is an increase in poly-ADP-ribose synthase activity when DNA is alkylated or broken, leading to cell damage STZ promotes rapid and massive β-cell necrosis leading to the Type 1 diabetes (T1D) pathway at low dosages [37] Multiple modest doses of STZ, on the other hand, led to partial destruction of β-cells, inflammation, and progressive loss of activation β-cells, which resembles the pathophysiology of type 2 diabetes (T2D) STZ use is related with metabolic alterations, which are often noticed during the first 2–8 weeks following exposure [37]

[2-deoxy-2-(3-2.4.3 HFD-STZ-T2D paradigm

For the onset of diabetes, animals must ingest an HFD for a predetermined amount of time

to induce insulin resistance This time duration varied among the conversion studies, but in the

majority of trials, a two-week diet followed by a low dosage of STZ elevated blood glucose levels within 3-7 days after STZ injection The amount of streptozotocin and the condition of fasting or not fasting can impact blood glucose levels Blood glucose will be less varied when fasting than when not fasting In other trials, intraperitoneal STZ injections of 30–40 mg/kg and a diet containing 40–60% calories from fat were utilized to induce HFD-STZ-T2D [37]

Blood glucose levels and the severity of diabetes are correlated with STZ dosage Based on prior research, the blood insulin levels in the HFD-STZ-T2D model may be higher, lower, or unchanged compared to those in the control group In the majority of studies, however, insulin levels increased following an HFD and reduced after STZ administration The amount of STZ injection is a significant element in reducing blood insulin levels; high doses result in the death of many cells, resulting in a fast decline Although blood insulin levels are a reliable indicator of the T2D stage, additional research has refined 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

Due of their omnivorous nature, small size, quiet habit, ease of handling, short generation interval, easy availability, and cost-effectiveness, rodents (such as mice) are the species of choice for inducing HFD-STZ-T2D Although genetic models of diabetes provide a better understanding

of the disease's development, they are costly, difficult to maintain, and unavailable for screening experiments normally In addition, in other diabetes models, such as STZ-nicotinamide and newborn STZ diabetic mouse, animals require rather high doses of STZ (>50 mg/kg), and the development of hyperglycemia is primarily attributable to the direct loss of pancreatic-cells The HFD-STZ-T2D model shares similar metabolic characteristics and natural history progression with the human T2D model This approach also generates sustained and long-lasting hyperglycemia, as reported by [99], where mice fed HFD for 4 weeks after two injections of 30 mg/kg STZ exhibited a steady rise in blood glucose four to 8 weeks after STZ administration [37] The greatest downside of this paradigm is the lengthy test duration, which increases test costs In addition, mice can be killed if the injection dose of STZ is not managed [46]

2.5 In vivo testing on laboratory animals

Important in the drug development process, the safety clinical trial of a new medicine is

dependent on in silico (virtual computer simulation) and in vitro (test-tube experiment) and in vivo (animal testing) [40] Currently, in vivo testing is a crucial link between in vitro testing success and human safety Long time ago, in vivo testing was mandated before a medicine could enter

human clinical trials [40]

Utilizing animals in research has been and continues to be a prevalent practice Numerous research have utilized animals such as mice, rabbits, fish (such as zebrafish, salmon), guinea pigs, amphibians (frogs), primates, dogs, cats, etc assist The primary objective of these studies is to test potential treatments for infectious and noncommunicable diseases for toxicity and medication efficacy Animals operate as experimental instruments In addition, they are employed in research

to develop pharmaceutical items like vaccines, antibiotics, etc The advancement of medical technology has increased the number of animals employed in research [27]

2.5.1 Origin and classification of mice

Mice are among the most often utilized animals in scientific research [32] They are prized

for characteristics such as their small size, short breeding period, ease of reproduction, and superior genetics Specifically, they share morphological, physiological, and genetic similarities with humans [16] Consequently enhancing the dependability of all research results [114] Mice are ideally suited for sepsis, obesity, diabetes, cancer, intestinal issues, organ transplants, etc.-related research [32] Future improvements in mouse models will include the creation of "humanized" mice containing human genes, cells, tissues, and organs in order to create therapies for human ailments [16]

2.5.2 The advantages and disadvantages of utilizing laboratory animals

2.5.2.1 Advantages

Due to their omnivorous habit, small size, calm disposition, ease of handling, and long lifespan, mouse are the preferred species for scientific research, particularly medical and epidemiological studies Quick production, simple accessibility, and cost-effectiveness Additionally, due to the similar metabolic characteristics and natural history of mice and people, mice were selected for the experiment rather than a healthy human subject [37]

2.5.2.2 Disadvantages

A figure in the United States indicates that more than 100 million dogs, mice, cats, and rabbits, etc., die annually as a result of laboratory chemical studies They are frequently burned, poisoned, incapacitated for hours, abused, have organs removed, or, worst of all, have holes punched into their bodies [45]

Animal corpses produce several species of bacteria, viruses, and parasites during decomposition The treatment involves a vast scale and the application of specific chemicals and sophisticated equipment In the absence of the aforementioned circumstances, animal carcasses are likely to release a large quantity of harmful compounds into the environment (water, soil and air) Long-term buildup of these chemicals will also have direct effects on humans For decades, attempts have been undertaken to limit the use of chemicals on animals to minimize the damage it causes, as a result of the environmental repercussions of animal testing on chemicals [89]

2.5.3 Conditions for raising laboratory mice

For in vivo testing to be accurate, laboratory animal care must be tightly regulated When

animals illed or under stress, results cannot be relied upon [114] Therefore, it is vital to establish and control the experimental setting in accordance with the Guidelines for the Care and Use of Laboratory Animals, in order to ensure that the animals' physiological condition remains optimal throughout the duration of the research methodology [89] Some aspects, including housing, temperature, humidity, ventilation, lighting, noise, nutrition, etc., must be developed and closely governed

To support successful cleaning, housing must always be kept clean, pest-free, and in good repair [117] Mouse must be housed in cages that provide for their physical, physiological, and behavioral requirements If an animal's environment does not match its demands, it can have a negative impact on brain development, physiological and behavioral dysfunction, animal health, and scientific value The materials used to construct cages must be sturdy and safe, prevent mice from fleeing, be non-toxic to animal health and research, and not impede cleaning and organization [89] Litter must be added to the cage to assist insulate the animals, absorb moisture, and dramatically reduce odors and pollutants (such as ammonia) in the barn Numerous forms of litter

materials are utilized, such as sawdust, wood shavings, maize cobs, and rice husks [114] One of the basic elements for animal productivity is cage cleanliness The cage as well as the livestock area must be cleaned, cleaned, and cleaned on a regular basis, and the litter material must be changed as needed to keep the animals clean, dry, and comfortable However, frequent cleaning

of cages might lead to cannibalism Change the cage 3 to 4 times per week to maintain it clean, and add scented wax around the breeding area to lessen the odor

The ability of a mouse to maintain a normal body temperature is influenced by the temperature and humidity of its environment No exact ambient temperature has been identified to optimize laboratory mouse' comfort, health, and performance [114] However, prior study trials have demonstrated that mice thrive at temperatures between 20 and 26oC and relative humidity between 30 and 70% [89] It is essential that the temperature and humidity levels are maintained within 1°C of one another If this criterion is met, the animal's metabolic rate will not be altered

An automated system should monitor temperature and humidity to ensure that variations remain within permissible parameters [89]

Ventilation's primary goal is to supply the appropriate amount of air and a steady experimental environment [89] This technology supplies sufficient oxygen, hence reducing the amount of air pollution caused by gases such as CO2 and NH3 Simultaneously, the system will dissipate heat to assist in the removal of body heat from animals, humans, and lighting, consequently managing the temperature and humidity of the cage [114] The ventilation system must be designed in accordance with the size of the house and the number of test animals The efficient ventilation rate for the majority of animal rooms is defined by the number of times per hour that fresh air is introduced [117]

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 [114] Since mouse are nocturnal, their eyes are adapted to dim lighting conditions Light-induced retinal damage occurs in mouse even under typical lighting circumstances (over 60 lux), and exposure to light above 100 lux for more than 16 hours per day can cause blindness In addition to promoting aggression and cannibalism in mouse, a high light intensity also boosts their aggressivity [117] At a height of 1 meter, the maximum permissible light intensity in the culture room is 350 lux [114] In animal rooms, fluorescent lighting is frequently employed as the standard light source Light should be dispersed uniformly over the cultivation area [89] The photoperiod

is more influential than light intensity on the physiological processes of many mouse breeds [114] Mouse need a particular amount of time to adjust to light changes in order to avoid alterations in growth, metabolism, reproduction, endocrine and immunological parameters, as well as behavior

vi within them [17] 12:12 (light: dark) and 14:10 (light: dark) are the most prevalent illumination

cycles employed in in vivo testing [114] Consistency in the light-dark cycle is crucial for obtaining

accurate research results [17]

Noise can have negative impacts on humans and laboratory mice It can affect the ear, digestive tract, immunological, reproductive, neurological, and cardiovascular systems, as well as induce metabolic and behavioral abnormalities [117] Noise levels above 85dB are possibly hazardous to humans and animals [114] Even brief variations in noise levels exceeding 100dB or 160dB can induce eardrum damage, convulsions, and other problems in rodents Ultrasonic waves

are extremely sensitive to laboratory mice Therefore, ambient noise (including ultrasonic waves) should be maintained at or below 50dB [117] Mouse are extremely sensitive to ultrasonic vibrations and use them as a means of communication It is vital to decrease the environmental noise created by ultrasonic waves because it can negatively affect the health of mouse [17] Well-feeding is necessary for the health and performance of laboratory mouse Nutrition is highly difficult due to the fact that nutritional needs change with age, species, health status, and research objective [114] Each day, mouse consume 20% of body weight However, the dietary content and nutritional composition must be changed to meet nutritional needs [17] In the diet, three essential macronutrients-protein, carbs, and lipids-must always be met In addition, tiny amounts of other nutrients, such as fiber, vitamins, and minerals, must be supplied to the diet at all times to guarantee full nutrition In numerous contemporary studies, rat meals are frequently created using NRC diets [89] The American Academy of Nutrition has also created AIN-93, a standard serving size This meal satisfied nearly all of the NRC's 1995 nutritional recommendations for mouse Nevertheless, vitamin B12 concentrations in the AIN-93 diet were 50% below NRC standards Therefore, it is required to supplement the AIN diet with twice as much vitamin B12 [55]

2.5.4 Animal Testing Ethics and the 3Rs rule

Utilizing animals in research has resulted in numerous advancements, including the understanding and treatment of infectious diseases, organ transplantation, etc [86] However, the use of animals in scientific research (drug testing, illness modeling, safety testing, etc.) has been a contentious issue for decades [57] Therefore, it is the obligation of all entities responsible for the care and use of animals for scientific purposes to consider and implement non-animal alternatives

If animal testing is the only method to gather the essential information, the studies must be of the highest quality and appropriately structured [86] In their 1959 publication, "Principles of Humane Experimental Engineering", Russell and Burch proposed the 3Rs rule This method has become a generally acknowledged set of ethical, legal, and guiding principles for the conduct of scientific research on animals in many countries [74] The primary objective of this approach is to reduce the number of animal experiments and to minimize the harm caused to animals during tests This will alleviate ethical issues regarding the usage of animals and improve the quality of test results [25] The 3Rs rule has been broadly applied to animal experiments in the past

Replacement: technique that employs the substitution principle, allowing the study's objective to be attained without the use of animals The use of epidemiological data,

physicochemical analysis, computer modeling, mathematics, in vitro systems, non-sentient

species, cell or tissue cultures, cadavers or human tissues or cells (with permission), and clinical situations are alternatives to the use of animals [86], [112] During the design phase of research, it

is vital to identify all viable methods for testing research hypotheses, including the use of inanimate models and animals with limited perception, such as insects [76] If non-sentient physical alternatives are available, sensation animals should not be used [57] Prior to starting an experiment, the validity and applicability of an animal model must be assessed If unsuitable, it must be changed with a different technique [86]

Reduction: the implementation of the principle of reduction permits the study's proposed objective to be attained with the fewest number of animals In addition, a significant number of

animals are still used to assure the accuracy of the results [57], [86] Using an insufficient number

of animals can lead to incorrect results And excessive consumption will result in waste and ethical issues [86]

Refinement: screening principles involved with the employment of approaches that avoid

or limit potential harm to animals in a study process, including generating discomfort and increasing animal welfare [25], [86] Compromised animal health will result in behavioral, physiological, and immunological problems, which may lead to incorrect results or unwanted alterations that influence repeatability [86]

2.5.5 Experimental environment

The experimental setting satisfies all necessary requirements for in vivo testing: always

assure the hygiene of laboratory animals and the operator, as well as conditions such as lighting, noise, and ventilation Weekly inspections are performed to reduce unnecessary hazards

2.6 Previous relevant research

2.6.1 Domestic studies

Currently, the synthesis of medications capable of efficient disease prevention and treatment from natural herbs is pharmacologists' primary priority [28] Numerous investigations on the medicinal effects of plant extractions tested on animal models have been conducted in our country All of these reports generated favorable outcomes

According to [26], their findings on the biological activity of the methanol extraction of

Hedytis diffusa willd in 2020 The results indicated that the extraction contained a significant

amount of total polyphenols and total flavonoids Consequently, the extraction has exhibited potent anti-oxidant and anti-inflammatory activities In addition, experimental mousr exposed to the extraction demonstrated hepatoprotective properties [26]

2.6.2 Foreign Studies

Extensive research has also been conducted worldwide on in vivo testing of the effects of plant-derived substances In 2015, the anti-obesity effect of an ethanol extraction of Terminalia

paniculata bark was investigated in mouse fed a high-fat diet Compared to untreated mouse, oral

treatment of the extraction decreased food intake and greatly avoided weight gain The extraction also improved blood lipid levels, as demonstrated by a drop in TG, TC, LDL, and an increase in HDL In addition, the increase in faeces lipids and the decrease in blood TG levels revealed anti-lipase activity as well as animal modulation of lipid absorption and transport [81]

According to [91], the extraction from the root bark of Olax mannii controlled the blood glucose level and dramatically reduced blood glucose in male mouse with diabetes caused in vivo

by injection of streptozotocin [91]

The author of [79] evaluated the hypoglycemic, anti-dyslipidemic, and oxidative effects of

Vitellaria paradoxa bark extraction on mice with streptozotocin-induced T2D in 2021 Analysis

of extractions revealed the existence of secondary metabolites, including polyphenols and flavonoids After 4 weeks of treatment with the extraction at three doses of 125, 250, and 500 mg/kg body weight, body weight and food consumption reduced significantly, and blood glucose concentration decreased (particularly at doses of 250 and 500mg/kg) Blood indices, including TG and TC, reduced in the extraction group, whereas the HDL-C index increased dramatically [79]

CHAPTER 3: MATERIALS AND METHODS

3.1 Materials

3.1.1 Olax imbricata leaves

Leaves of Olax imbricata were gathered at the Central Center for Research and Production

of Medicinal Materials in Dong Hoa District, Phu Yen Province, Vietnam To ensure therapeutic capabilities in the leaves, they must be fresh, green, and not wilted, yellowed, or infested with insects The 96% EtOH extraction was created by extractioning chemicals from the leaves of the poplar tree using a 96% ethanol solvent

3.1.2 Streptozotocin

Streptozotocin (Macklin) supplied from the BioLab chemical distribution company in Hanoi

in March 2022, storing it at -40OC to preserve its infectious potential when injected into mice

3.1.3 Food ration

Dietary components in this in vivo testing were a normal diet (ND) and a high-fat diet (HFD) Normal diet (ND) is a commercial Fullvit JP70 produced by Jolly Pet Products in Ohio, United States of America The nutritional profile of ND was identical to that of the AIN-93 standard rat diet [101] HFD is a commercial diet including finely ground Fullvit JP70 and liquid beef fat After purchasing beef belly fat, it will be washed with salt water to eliminate impurities and odors, sliced into pieces, and cooked until it transforms from a solid to a liquid state Then, strain the fat liquid using a sieve, place it in a sealed glass jar, and store it in the freezer at 5oC In our experiment, the high-fat diet comprised of finely crushed commercial feed and beef fat blended in a ratio of 1:1,

Table 3.1 Nutrient compositons in ND and HFD

along with a few drops of vanilla essential oil The preparing HFD feed steps: Melt the beef fat (50g), then add the ND finely powdered commercial feed (50g) while continuously stirring to ensure that feed and fat are thoroughly combined Add three drops of vanilla essential oil to the diet to create aroma and stimulate the experimental animals' ability to eat (mouse) HFD rations will be stored at 5oC and consumed within 24 hours Each day, the HFD meal was prepared to prevent mold conditions that could harm laboratory animals

3.1.4 Experimental animal

In this in vivo testing, the experimental animals were 5-week-old male white mice (Mus

Musculus var albino) weighing 13±1g per head These mice were supplied by the Pasteur Institute

in Ho Chi Minh City, Vietnam Prior to the trial, mice were adaptive feeding for 5 days until they weighed 15±1g/head During the experiment, mouse were grown in conventional settings with humidity ranging from 60 to 70% and a temperature of 30±2oC The 12h light/12h dark cycle was also maintained To ensure the optimal growth of the mice in the experiment, standard food and water (Aquafina-purified water) were provided everyday in copious quantities In addition, mice

Figure 3.2 White mice (Mus Musculus var albino)

Figure 3.3 In vivo experimental design

were given a few drops of vitamin Hagebuttentrunk from Beaphar company, Netherlands

3.2 In vivo experimental design

30 mouses are adaptive feeding until they achieve the required weight of 15g will be randomly divided into six groups (n = 5 per group) and put in three separate groups After batching, groups A (Normal Diet) and B (High Fat Diet) received their respective diets for the duration of the study Group B (HFD) and the rest of group C received the HFD diet for the first 2 weeks Then, at week 3rd and week 4th, they will be given with 2 doses of STZ, respectively (the injections are separated by one week to ensure that the mice get type 2 diabetes at a dose of 40 mg/1kg body weight) before to being administered the extraction From week 5th, the HFD diet was administered

to group C, which included 3 groups receiving extractions from Olax imbricata leaves (HFE-50,

HFE-100, and HFE-200 along with extractions corresponding to doses 50, 100, and 200 mg/kg body weight/day), respectively; amount of food divided into 4 times/day) and 1 group receiving Acarbose (HFA with dose 100 milligrams per kilogram of body weight each day) In the experimental groups, this extraction and acarbose were supplied in two sessions (morning and afternoon) and divided into two equal quantities (0.5 mL/time)

After at least two weeks of ingesting a high-fat diet, mouse displayed a significant increase

in body weight and insulin resistance, as determined by [31] We fed mice a high-fat diet for two weeks in order to produce mice with higher body weight and blood glucose levels The extraction and acarbose were then administered to these mouse groups for comparison with the control group

This in vivo testing lasted 12 weeks (excluding 5 days of acclimatized mice after being

retrieved from the Pasteur Institute in Ho Chi Minh City) Throughout the duration of the experiment, the groups of mice were provided with as abundant amount food and water as possible

to ensure the most accurate results Significant variations were seen between the groups of mice Mice was weighed every 24 hours to determine their daily calorie intake [67] and monthly to determine their development rate [82] At weeks 0, 2nd, 4th, 7th, 10, and 12, the experimental groups were evaluated for glucose tolerance (0-120 minutes) as well as for behavior and movement before and after feeding At week 12th, groups of mice were anatomized in order to get histological samples (liver, kidney, fat) as well as blood from the heart in order to analysis blood lipid indicators (Triglycerides, Cholesterol, LDL-Cholesterol, HDL-Cholesterol and Insulin)

Experimental groupings include:

(A) Experimental group with standard normal diet (ND)

(B) Experimental group with high-fat diet and received 2 doses of streptocotozin (HFD) (C) Consist of 2 groups: experimental group with high-fat diet and received 2 doses of

streptocotozin utilizing 3 doses of 96% EtOH extraction from Olax imbricata leaves

(HFE-50, HFE-100, and HFE-200 along with extraction doses corresponding to (HFE-50, 100, and 200 mg/kg body weight/day); and experimental group with high-fat diet and received 2 doses of streptocotozin utilizing Acarbose (HFA with dose 100 milligrams per kilogram of body weight per day)

3.3 Determine sample size

Before beginning any in vivo testing on experimental animals, it is crucial to identify the

number of animals that will be studied If the sample size is insufficient, the experiment cannot identify biologically significant reactions, resulting in erroneous results If the sample size is