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

GRADUATION THESIS
FOOD TECHNOLOGY

IN VIVO EVALUATION OF THE ABILITY TO PREVENT
OVERWEIGHT, OBESITY, AND DIABETES OF 96%
ETHANOL EXTRACTION FROM OLAX
IMBRICATA LEAVES

SUPERVISOR: TRINH KHANH SON
VO THI NGA
HUYNH NGUYEN LINH CHI
STUDENT: TRAN HOANG VU

SKL 0 0 9 1 6 3

Ho Chi Minh City, August, 2022


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
PREVENT OVERWEIGHT, OBESITY, AND DIABETES
OF 96% ETHANOL EXTRACTION FROM OLAX

IMBRICATA LEAVES
Name:

TRAN HOANG VU

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


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
PREVENT OVERWEIGHT, OBESITY, AND DIABETES
OF 96% ETHANOL EXTRACTION FROM OLAX
IMBRICATA LEAVES
Name:

TRAN HOANG VU

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


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ACKNOWLEDGEMENTS
First of all, I would like to express my appreciation and sincere thanks to Assoc. Prof. Trinh
Khanh Son, PhD. Vo Thi Nga, and Eng. Huynh Nguyen Linh Chi, the professors who have helped
me with this thesis. Beside instructing and helping me to complete my graduation thesis, the
advisors have also provided me with lot of helpful advisements that myself can apply in my future
and professional development.
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 31st,2022

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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 31st ,2022

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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
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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

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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

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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 4.1. Body weight of mouse...............................................................................................35
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

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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 4.1. Result from ether extraction ........................................................................................33
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

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LIST OF ABBREVIATION
High fat ethanol-200
(mg/kg.w/day)
Half maximal Inhibitory
concentration

AGIs

Alpha-glucosidase inhibitors

HFE-200

AUC

Area under the curve

IC50

AE

After eating

JNK

c-Jun N-terminal Kinase

ATP

Adenosin triphosphat

LDL

Low-density lipoprotein

BE

Before eating

LFD

Low-fat diet

BGL

Blood glucose level

MAE

Microwave assisted extraction

CETP

Cholesteryl ester transfer protein

MAG

Monoacylglycerol

DAG

Diacylglycerol

ND

Normal diet

DNA

Deoxyribonucleic acid

NRC

National Research Council

EAE

Enzyme assisted extraction

PLE

Pressurized liquid extraction

ER

Endoplasmic reticulum

OGTT

Oral glucose tolerant test

FA

Fatty acid

SFE

Supercritical fluid extraction

FFA

Free fatty acid

SF

Supercritical fluid

GLUT2

Glucose transporter 2

SWE

Supercritical-water extraction

GLUT4

Glucose transporter 4

T1D

Type 1 diabetes

HDL

High-density lipoprotein

T2D

Type 2 diabetes

HF

High fat

TAG

Triacylglycerol

HFA

High fat Acarbose-100
(mg/kg.w/day)

TG

Triglyceride

HFD

High-fat diet

UAE

Ultrasonically assisted extraction

HFE

High fat ethanol

VLDL

Very low-density lipoprotein

VHFD

Very high-fat diet

WHO

World Health Organization

HFE-50
HFE-100

High fat ethanol-40
(mg/kg.w/day)
High fat ethanol-100
(mg/kg.w/day)

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SAMPLE CODE
Experimental group
ND

Code
135

HFD

362

HFA

946

HFE-50

785

HFE-100

275

HFE-200

571

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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 α-glucosidase inhibitory activity and to be approximately 1.5 times more resistant to αglucosidase 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.

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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
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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

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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 710 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
3