Nghiên cứu điều chế cao từ cỏ sữa lá lớn (euphobia hirta l ) và ứng dụng chế biến thực phẩm dinh dưỡng kiểm soát glucose máu tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING

MINISTRY OF HEALTH

NATIONAL INSTITUTE OF NUTRITION

NGUYEN MANH THANG

A RESEARCH ON MAKING EUPHORBIA HIRTA L.
EXTRACT IN APPLICATION FOR PRODUCING
NUTRITIOUS FOOD TO CONTROL
THE GLUCOSE IN BLOOD

DOCTORAL THESIS ON NUTRITION
Code: 9720401

SUMMARY OF DOCTORAL THESIS

Hanoi – 2020


THE WORK WAS COMPLETED AT
NATIONAL INSTITUTE OF NUTRITION

Science instructors:
1. Prof. Dr. Nguyen Cong Khan
2. Assoc. Prof. Dr. Truong Tuyet Mai

Reviewer 1:
Reviewer 2:
Reviewer 3:

The thesis is presented and approved from Thesis Council at
Institute level at the National Institute of Nutrition
Time: ……….o’clock, day……… month…… year……..

The thesis can be found at:
- The National library
- The Library of the National Institute of Nutrition


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INTRODUCTION
Relevance of the study
In recent decades, studies of plants that can help preventing and treating diabetes
and diabetes complications are increasingly drawing the attention of scientists around the
world. The asthma plant with the scientific name Euphorbia hirta L., belonging to
Euphorbiaceae family, a wild plant that grows in humid lands in tropical countries,
including the southern provinces of Vietnam. The field survey shows that Binh Duong is
a province with satisfactory weather and soil conditions for the development of plants in
the castor family. Many studies around the world such as India, Malaysia, and Japan
have shown the effects of Euphorbia hirta L. plants on diabetes through the mechanism
of controlling blood glucose, inhibiting enzymes α-amylase and α-glucosidase by
conjugation. Flavonoids are found in this herb. Up to now, in our country, there are
limited systematic studies on Euphorbia hirta L. applying this medicinal source to the
production of nutritious food to meet the needs of public health care. With this regard,
the thesis titled "A research on making Euphorbia hirta L. extract in application for
producing nutritious food to control the glucose in blood” was carried out.
The objectives of the study
1. To describe the botanical characteristics and identify some of the chemical
components of the Euphorbia hirta L.

2. To make and evaluate the safety and effectiveness of this plant’s extract in
controlling blood glucose
3. To design a technological process to produce nutritious beverages from
Euphorbia hirta L.used in blood glucose control
Research content
1. Studying the botanical characteristics and analyzing some chemical components
of the Euphorbia hirta L.
2. Working on the process of extracting Euphorbia hirta L., evaluating the safety
and efficiency of the extract in controlling blood glucose.
3. Building a technological process to produce nutritious beverages from
Euphorbia hirta L. extract used in blood glucose control.
Research hypothesis
1. At present, there is a limitation of well-ground and systematic scientific studies
on the botanical characteristics and chemical components of Euphorbia hirta L. in
different regions of Vietnam.
2. The extract from Euphorbia hirta L. that has been studied to undergo optimal
conditions to achieve appropriate flavonoid content will be able to inhibit the enzymes
α-amylase and α-glucosidase in vitro, control blood glucose in diabetic rats.
3. Euphorbia hirta L. extract products can be used as raw materials to create
various products, including nutritious drinks with high-quality and food safety, allowing
the community to access and use them with ease.
New contribution of the study
- Provides scientific evidence on the botanical characteristics and some chemical
components of naturally grown Euphorbia hirta L. in Binh Duong, proving the blood
sugar-lowering mechanism of Euphorbia hirta L. is due to its ability to inhibit


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carbohydrate hydrolytic enzymes such as α-amylase and α-glucosidase. This is the
scientific basis to demonstrate the effect of Euphorbia hirta L. (Euphorbia hirta L.) in

reducing the glycemic index in mice with diabetes on an experimental scale.
- The research has built a technological process for extracting Euphorbia hirta L.
on the basis of determining the appropriate technological conditions. Many diabete
products (capsules, powder or liquid) can be produced from this extract with ease.
products in diabetes prevention (in capsule, powder or liquid). The author has
successfully tested and trial-produced a flavonoid-rich nutritional beverage from
naturally sourced milk grass. This is an area in Vietnam that has not yet been developed
much, and should receive more attention in the coming time to contribute to improve
community health.
Structure of the thesis
The thesis consists of 148 pages (excluding references and appendixes), in which
there are 2 pages of the introduction; 1 page of research objectives and hypothesis; 48
pages of literature review; 26 pages of the subjects and method of the study; 41 pages of
the results; 25 pages of the discussion; 2 pages of the conclusion and 1 page of the
recommendations.
The thesis consists of 26 tables, 33 figures and 167 references, which include 58
references in Vietnamese, 107 references in English and 02 websites.
Chapter 1: REVIEW
1.1. Overview of disease pattern: Current situation and intervention solutions
to facilitate the prevention and treatment of diabetes and blood glucose disorders
1.1.1. Current state of diabetes
Currently, the disease pattern in Vietnam has many changes compared to this a
decade ago. It is a rapid rise in untransmittable chronic diseases in terms of prevalence
and mortality rates. This is the result of a shift in demographic patterns, with the
proportion of the elderly approaching 11%, which is considered to reflect an aging
population and result from socio-economic, diet and life style changes. Thus, we are
facing the enormous challenge of the double burden of this disease. One of the most
common untransmittable chronic diseases is diabetes.
In 2019, the American Diabetes Association introduced diagnostic criteria for
detecting diabetes based on blood glucose (capillary or venous) and HbA1c levels.

Result
Dignostic criteria
FPG (no caloric intake for at least 8 h) �7.0 (mmol/L).
2h-PG (a glucose load containing the equivalent of 75-g anhydrous
Diabetes
glucose dissolved in water) �11.1 (mmol/L).
Or HbA1c �6.5%
FPG (no caloric intake for at least 8 h) from 5.6 to 6.9 (mmol/L).
Pre2h-PG (a glucose load containing the equivalent of 75-g anhydrous
diabetes
glucose dissolved in water) from 7,8 to 11,0 (mmol/L).
Or HbA1c from 5.7% to 6.4%


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Accoding to the latest IDF report in 2017, about 425 million people aged 20-79
have diabetes, equivalent to 1 in 11 people with diabetes. This number will be 629
million people (up 48%), equivalent to 1 in 10 people with diabetes by 2045. In Vietnam,
the Ministry of Health reported that in 2017 there were 3.5 million adults with diabetes,
equivalent to 6% of the population, and it is expected that by 2040 there will be 6.1
million adults who can have diabetes. This seriously affects the public health and socioeconomic development of the country. Therefore, it is very important to find solutions to
help prevent and treat diabetes through blood glucose control in the community,
including a combination of many solutions, from nutritional solutions, enhancing
physical activity, reasonable diet, lifestyle changes to the use of plant-based products,
especially polyphenols-rich plants are trending in current research application.
1.1.2. Intervention solutions to support the prevention and treatment of
diabetes mellitus and blood glucose disorders
The main measures in the prevention and treatment of diabetes and blood glucose
disorders include changing lifestyles, having a reasonable diet and increasing physical
activities, in addition to using effective products and drugs for controlling blood glucose.

In recent decades, researches on plant species capable of helping to prevent diabetes are
of greater concerns. Many plants have been recommended for use as a supplement or as
an alternative to diabetic medicines, mainly based on the polyphenol and flavonoid
compounds available in plants
1.2. Overview of Plant Polyphenol Compound Group
There exist about 1200 plants worldwide that have been acknowledged to have
positive effects on diabetic patients. Each plant may contain one or many different active
ingredients which can limit hyperglycemia such as polyphenols, flavonoids, alkanoids,
terpenoids, anthocyanins and other active ingredients isolated from plants. Polyphenol is
a compound found in natural plants with color and smell. Polyphenols are composed of
benzene rings with different OH radicals in different groups: non-flavonoids and
flavonoids.
In plants, flavonoids exist in two forms: the free form (aglycon) and the sugarlinked form (glycoside). The research results have shown that flavonoids act as insulin
stimulants or mimic the function of insulin, affecting the activity of enzymes in sugar
metabolism, which helps slow the breakdown and absorption of carbohydrates in the diet
by limiting the hydrolysis of straight or branched oligosaccharides such as dextrin,
maltose and maltotriose to glucose production, thereby blocking glucose absorption.
Currently, Vietnamese scientists continue to study plants that have been used
according to traditional experience in preventing diabetes such as bitter melon,
cyanosis…. In the southern provinces, Euphorbia hirta L. has been widely used in the
treatment of some diseases such as dysentery, thirst, diabetes, and high polyphenol
content. Therefore, the research to promote the potential of using naturally grown
Euphorbia hirta L plants in diabetes prevention and treatment is essential. This is the
reason why the author chose Euphorbia hirta L. as the main raw material to conduct the


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research and takes into account the industrial production of nutrient-rich drinking water
from Euphorbia hirta L plants as a health supplement in the future, contributing to the
diversification of beverage products as well as supplementary products for the

prevention and treatment of diabetes.
1.3. Euphorbia hirta L.
Euphorbia hirta L. belongs to the castor family (Euphorbiaceae). Herbaceous plant,
growing all year around, is 30-60 cm high with tap-root of 3-5 mm diameter. The stem is
usually less branched, branching from the center or above, light red, with white latex and
with many long, yellow-brown protective hairs. Leaves are simple, in opposite pairs with
two small triangular leaves or hard hairs, size 0.8-1.7 mm, and deciduous. The petiole is
13.5 mm long, the leaf blade is oblong, elongated, or lanceolate, 10-50 x 3-6 mm in size,
sometimes with purplish spots along the midrib. Both sides are hairy thick. The leaf
blades are round, slightly deviated. Leaf margin on the lower part is fully or partially
serrated, the upper part is half serrated, pointed leaf tip, or conical. The inflorescence
grows in leaf axils, with stalks 25 mm long, all parts hairy, and single flowers. A flower
cluster consists of 4-5 male flowers, female flowers have short stalks, high gourds,
protruding from the total bell-shaped, 3-compartment pots, sparse hairs, 2-lobed stigma.
In the Medicinal Plants and Animals document, Euphorbia hirta L is mentioned to
help control blood glucose in the animals. In fact, this plant has been used by the
Southern people according to the Oriental medicine method to treat diabetic patients.
This plant can be considered as a potential source of natural raw materials to support
diabetes prevention and food production in general and nutrient-rich beverages in
particular.

Euphorbia hirta L.
1.4. Overview of extraction and preparation of plant extracts
Extraction is a technological process that involves separating the pharmacologically
active parts from inactive ingredients in the plant tissue using solvents. The finished
products are usually impure, in fluid or semi-solid (thickened), or powdered (highly
dried) forms. This is the first step to separating the desired product from natural
ingredients. Extraction methods include: solvent extraction, distillation, pressing and
sublimation according to the respective principles. In which, solvent extraction is a
widely used method. In industry, commonly used methods include: extraction, soak

extraction or reflux extraction. The rest of the methods, such as microwave assisted,


5
ultrasonic, supercritical enzymes or fluids, apply for specific purposes or groups of
active ingredients that are susceptible to thermal degradation.
Flavonoids are considered to be quite resistant to heat and have a good solubility
in water. Its applicable extraction methods include immersion, percolation, soxhlet or
reflux extraction. In Euphorbia hirta L., flavonoid compounds, polyphenolic and tannin
are quite polar and easily soluble in water. Therefore, it is possible to use reflux
extraction with water or a liquid alcohol solvent system. However, reflux extraction with
water is more economical and produces higher extraction efficiency, with the collected
extract can be easily used in beverage preparation.
1.5. The production technology of herbal beverage for blood glucose
prevention and control.
The trend of using beverages with natural ingredients, especially those extracted
from herbs and fruits, is growing. This type of beverage is a refreshing drink, providing
the body with nutrients and has become popular in the market, will gradually replace
carbonated drinks. The production line for beverages prepared from natural or synthetic
sources is shown in the figure below.
Chapter 2: SUBJECTS AND METHODS OF THE STUDY
2.1. Research materials
2.1.1. Main material
Euphorbia hirta L. plants,collected in Binh Duong province, dried to the humidity
≤ 11%, cut into segments 4-6 cm long and stored in a plastic bag, kept in a dry and
ventilated place. The minimum time from harvesting to putting into experiments is 30
days. From this raw material, the author has conducted preparing Euphorbia hirta L.
extract. In the study, the author uses a number of other materials such as: Honey,
Ginseng, sweeteners, Cinnamon, Ginger, Water and aluminum package cans.
2.1.2. Chemicals and tools

- In this study, the main chemicals used include: absolute ethanol, methanol,
quercetin, L-aspartate and L-alanin substrate, Blocked p-nitrophenyl maltoheptaoside
(BPNPG7) substrate, p-nitrophenyl substrate - α-D-glucopyranoside (PNPG), quercitrin;
reagents used in qualitative reactions and chromatography reagents (Mayer, Dragendorff,
Bouchardat, dd FeCl3 5%, dd gelatin 1%, dd lead acetate 10%, Lugol, Fehling A,
Fehling B, vanillin- sulfuric acid).
- Tools: Glucose dosing kit, HbA1c (Wako Pure Chemicals, Japan), syringes,
needles and basic laboratory tools
2.1.3. Experimental animals
In the study, the experimental animals were the white mice of Swiss albino used in
the assessment of acute toxicity and blood glucose decrease and Wistar white rats used
in semi-chronic toxicity.
2.1.4. Device
Basic devices used in the laboratory such as analytical balance, optical meter, oven,
moisture meter, microscope, automatic blood analyzer, ... In test production, the devices


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includes: ordinary oven; Herbal cutting machine; Pharmaceutical grinder; Ultrasound
with heating; Rotary distillation machine; Pumping equipment; Automatic mixer;
Canned machine system; Pasteurization system.
2.1.5. Research location
After directly field surveying the naturally grown Euphorbia hirta L. in Binh Duong,
the researcher has collected the fresh (original) Euphorbia hirta L., washed it and
transported it to Hanoi to conduct experiments. The author has carried out the following
studies: Plant characteristics and chemical components of Euphorbia hirta L. in the
Department of Medicinal Materials, Hanoi University of Pharmacy; Euphorbia hirta L.
preparation at the Department of Plant Chemistry under the Institute of Medicinal
Materials; Evaluation of acute and semi-chronic toxicity in the Department of
Pharmacology and Biochemistry of the Institute of Medicinal Materials; Determination of

inhibitory activity of α-amylase and α-glucosidase enzymes at the National Institute of
Food Safety and Hygiene under the Ministry of Health; Efficiency evaluation was
conducted at the Center for standard laboratory animal research and production under the
National Institute of Hygiene and Epidemiology; Formulating the formula and process of
nutritional beverage products from Euphorbia hirta L.extract at the Aroma Alcohol and
Beverage Joint Stock Company; Test production and canning at the Institute for Fruit and
Vegetable Research and Community Acceptance Assessment in Hanoi and Hung Yen.
2.2. Research Methods
2.2.1. Surveying the botanical characteristics and chemical components of the
Euphorbia hirta L.
2.2.1.1. Plant characteristics study
Morphological characterization and scientific name determination: Euphorbia hirta
Lsamples were described according to the analytical description method, compared with
the genus Euphorbia key, the description of Euphorbia hirta L. species in Plants Chinese
Journal and the Course of Identifying and the Vietnamese Oil Contractor surname of
Prof. Dr. Nguyen Nghia Thin to identify scientific names.
Description of stem and leaf micro-anatomy: The stems and leaves are cut microsurgery by hand cutting machine, bleached with Cloramin B, and dyed by double dyeing
method. Look under a microscope, take pictures and describe microscopic features.
Description of stem powder, leaves, flowers, fruit: Dried medicinal herbs (stem,
leaf, flower, fruit) in an oven at 60oC, then crushed by boat and mortar. Sieve the fine
powder, use a lance needle to take the medicinal powder onto the glass slide, already
drip a drop of distilled water, and put the lamina on. Observe under a microscope, take
pictures and describe powder characteristics.
Take photos of micro-anatomical features and powder with Canon camera. Image
processing by software PHOTOSHOP CS6.
2.2.1.2. Chemical component research
Qualitative groups of compounds by chemical reaction: Conducted according to the
method stated in the Document Research Methodology of Medicinal Materials,

7
Department of Medicinal Materials - University of Medicine and Pharmacy, Ho Chi
Minh City, Ho Chi Minh City. .
Qualitative n-hexane, chloroform, and ethyl acetate extracts by thin layer
chromatography
- Extraction of the large foliar above ground milky fraction:
- Medicinal herbs (1.0320 kg, H = 9.4%) boiled with distilled water for 1 hour,
sometimes adding distilled water. Then hot filter by vacuum pump. Add more distilled
water and perform the second and third extraction as above. Combine the extracts and
concentrate the extracts to the ratio of 1 kg of medicinal herbs: 1 L of extract, allow to
cool. The obtained extract is extracted with n-hexane, chloroform, and ethyl acetate
solvents, resulting in n-hexane fraction, chloroform fraction, ethyl acetate fraction,
respectively. Store and recover the extract fractions under reduced pressure and
evaporate the corresponding fractional bites, n-hexane bites (0,1001 g), chloroform bites
(0.5912 g), ethyl acetate bites (14.4499 g)
- Prepare solutions for chromatograph:
Test solution: Place 1 mg of bite each fraction into 3 test tubes and then add 1 ml of
methanol to each tube; Standard solution: Quercitrin solution of 0.1 mg / ml in methanol
and rutin solution of 0.1 mg / ml in methanol.
- Reagent: vanilin-sulfuric acid
- Implementation process:
The F254 silica gel thin sheet was activated at 110oC for 1 hour. Spot the extracts
on the plate and develop with the corresponding solvent system. Observe the stain in
normal light, then look under ultraviolet light (UV254 and UV366). Show traces with
vanillin-sulfuric acid reagent. The ethyl acetate fraction was compared with quercitrin
and standard rutin.
Quantification of flavonoids present in Euphorbia hirta L. dairy plants by
photometric method
- Prepare stock standard solutions
Accurately weigh 0.98 mg of standard quercitrin, place into a 10 ml volumetric

flask, add 6 ml of methanol, and shake to dissolve completely. Make up to the mark with
methanol to obtain a stock standard solution S of concentration 98 μg / ml.
- Survey the maximum absorption
Accurately take 1 ml of the stock standard solution to a 10 ml volumetric flask, add
0.3 ml of 10% aluminum chloride solution R, 0.3 ml of 1M sodium acetate solution R,
make up to volume with distilled water, Shake well, then let stand for 30 minutes.
Conduct spectral scan from 200-600 nm. Spectral scan results show that the solution has
3 absorption peaks at the wavelengths of 207.5 nm, 270.5 nm and 412.0 nm. Therefore,
choose the wavelength of 412.0 nm as the photometric wavelength.
- Construction of standard roads
 Prepare standard series: Get exactly 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1,2; 1,3 ml of
stock standard solution S, in turn, into 8 10-ml volumetric flasks, evaporate the solvent,


8
allow to cool, add 1 ml of methanol shake to dissolve, add 0.3 ml. 10% aluminum
chloride solution R, 0,3 ml sodium acetate solution 1 M R, add distilled water to the
mark, shake well, obtain a series of standard symbols respectively S1, S2, S3, S4, S5. ,
S6, S7, S8 have respective concentrations of 5.88; 6.86; 7.84; 8,82; 9.80; 10.78; 11.76;
12.74 μg / ml.
 Building a calibration curve: Measure the absorbance of the S1 - S8 standard
solution series at a wavelength of 412.0 nm with a blank sample prepared by taking
exactly 1 ml of methanol, 0.3 ml of aluminum chloride solution 10%. Make up to the
mark with 0.3 ml of 1M sodium acetate solution R into a 10 ml volumetric flask.
The results of the absorbance measurement of the standard series and the graph
showing the linear correlation between the absorbance and quercitrin concentration are
shown in the following table and figure.
The dependence of the absorbance on the standard quercitrin concentration
Độ hấp thụ
Nồng độ quercitrin (μg/ml)

Lần 1
Lần 2
Lần 3
Trung bình
5,88
0,233
0,233
0,233
0,233
6,86
0,280
0,280
0,280
0,280
7,84
0,321
0,321
0,321
0,321
8,82
0,366
0,365
0,366
0,366
9,80
0,394
0,392
0,397
0,394
10,78

0,432
0,433
0,434
0,433
11,76
0,480
0,480
0,480
0,480
12,74
0,516
0,516
0,516
0,516

The graph showing the linear correlation between the absorbance and quercitrin
concentration shows that in the survey concentration range, the absorbance and
quercitrin concentration have a very close linear correlation with the correlation
coefficient R = 0.9989 (coefficient of determination R2 = 0.9978).
Linear regression equation: D = 0.0406.C - 0.0004 (*)
In which: D: Absorption of the solution.


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C: Concentration of solution (μg / ml).
- Prepare the test solution
Accurately weigh about 5 g of medicinal powder into a 250 ml conical flask, add
75 ml of distilled water, and boil for 30 minutes. Filter hot through a filter paper into a
250 ml beaker. Add distilled water to the conical flask for the second and third extraction
as above. Combine the extracts, evaporate the water to bite. Then add 20 ml of methanol

to biting beaker and ultrasonic for 10 minutes, to settle, filter through filter paper into
100 ml beaker. Repeat the above dissolution procedure until the resulting extract reacts
negatively with iron (III) chloride (about 4-5 times). Place the methanol extract in a 100
ml volumetric flask, add sufficient methanol to the mark where the test solution T is
obtained.
- Measure the absorbance of the test piece
Accurately take 0.4 ml of test solution T into a 10 ml volumetric flask, evaporate
the solvent by evaporating it in a water bath, allow to cool, add exactly 1 ml of
methanol, 0.3 ml of 10% aluminum chloride solution ( R), 0,3 ml of 1M sodium acetate
solution R, make up to volume with distilled water, and shake to obtain symbol solution
T1. Allow T1 solution to stand for 30 minutes and then photometric at 412.0 nm with a
blank prepared by taking exactly 1 ml of methanol, 0.3 ml of 10% aluminum chloride
solution R. 1M sodium acetate R into a 10 ml volumetric flask, make up to volume with
distilled water, and mix.
Quantitative determination of total flavonoids in the upper part of the ground with
milk leaves 6 times larger under the same conditions.
From the absorbance of the test sample, calculate the total flavonoid concentration
in the corresponding test solution using Equation (*).
The total flavonoid content in Euphorbia hirta L. is calculated by the formula
(**):
c×k
Total flavonoid content (%)
=
× 100 (**)
m × 102 (100 - H)
In which:

C : fluid level (μg/ml).
m: Khối lượng dược liệu (g).
k : Hệ số pha loãng (k=25).

H : Độ ẩm (%).
- Experimental procedure: Preparing Euphorbia hirta L.extract and removing
impurities; React with 10% aluminum chloride solution in water; Investigate the maximum
absorption; Investigate the linear range; Use the calibration curve technique to determine the
concentration of analyte present in the quantitative sample. Total flavonoid content was
calculated according to quercitrin standard in absolute dry medicinal herbs.
- Processing experimental results
Experimental results are stored and statistically processed using Microsoft excel
software.
2.2.2. Euphorbia hirta L. preparation, evaluating the safety and effectiveness of
the product in blood glucose control
2.2.2.1. Investigation of factors influencing on the preparation of large-leaved


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milkgrass
Apply the quantitative method of total flavonoid compounds according to the
established method. Using the one-factor change method, the other factors remain the same,
after choosing the appropriate value of that factor, this value is used for subsequent studies,
the author chooses the extraction process. Export together with 10g pharmaceutical / batch
scale to explore the main factors affecting the preparation process.
Experiment 1: Investigating the effect of temperature on the extraction process
Experiment 2: Surveying the effect of the ratio of solvents and medicinal herbs
Experiment 3: Investigate the effect of extraction times
Experiment 4: Investigate the effect of extraction time
Experiment 5: Surveying the effect of the size of medicinal herbs on the extraction
process
2.2.2.2. The method is highly obtained from extracts of Euphorbia hirta L
Dried Euphorbia hirta L. (moisture <10%), chopped 1-3 cm, extracted 3 times with
water at 95oC, the volume of solvents / dry Euphorbia hirta L is 10/8/8 times in turn.

Extraction in the corresponding time is 2.5 hours / 2 hours / 1.5 hours, respectively. The 3time extracts were filtered, combined extract, condensed at reduced pressure (evaporation
temperature 60oC), dried in a vacuum oven at 50oC to high dry (humidity <5%).
2.2.2.3. Evaluate the safety of extracts of Euphorbia hirta L.
After investigating the factors affecting the preparation process, Euphorbia hirta L.
extract was obtained under the right conditions, the author used the above product in
subsequent studies:
Assessment of acute toxicity
- Rats were raised for 3 days before the experiment to adapt to the experimental
conditions. Before the experiment, the rats fasted for 12 hours, let the water drink
according to the needs of the mouse, the rats were divided into experimental groups (10
mice each), each group of mice were given different doses of the sample based on into
calculation and exploration. Give the hamster a drink by using a syringe with a needle
tip to gently insert the test sample into the rat's stomach.
- Test sample: Extract is crushed in water with different concentrations. Rats were
given high oral doses at different doses per gram. Rats were given a test dose of different
doses of 0.2ml / 10g rat body weight. After the test samples were taken, the rats were fed
and drank sufficiently, and kept in a laboratory with a climate that ensures all rats'
activities were normal. Monitoring and observing the rat behavior, activity, eating,
excretion and the number of rats alive and dead in 3 days (72 hours).
- Find the maximum dose without any dead mice (LD0) and minimum dose to
100% of mice die (LD100). Try adding two to four intermediate doses between the two
above doses to determine LD50
LD50 is calculated according to the method Behrens-Karber.
- Formula for calculation : LD50 = LD100 -  (d x z)
n


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Inside:
d: difference of the next two doses

z: the average number of rats died between successive doses
n: number of rats in 1 lot
Semi-chronic toxicity assessment
Rats were kept stable for 5 days before conducting the study, fed with standard
food, drinking free water. White rats are randomly divided into 3 plots:
- Control group (n = 10): drink water with a volume of 1ml / 100g rat
- Test group dose 1 (n = 10): drink extract of Euphorbia hirta L.extract with dose of
0.30 g / kg rat
- Test group dose 2 (n = 11): drink Euphorbia hirta Lextract of milk grass extract
with a dose of 3.0 g / kg rat
Rats were given high leaf milk extract extract continuously for 30 days. Weigh the
rats weekly for weight monitoring. To monitor liver function, kidney function,
hematopoietic function, mice were drawn blood at 4 different times: before taking the
sample (N0), 15 days after drinking (N15), 30 days after drinking (N30) ) and 15 days
after stopping. Rat blood was collected into 2 types: Type 1 blood was anticoagulated by
EDTA (to determine the hematological parameters), type 2 blood was left automatically,
centrifuged serum to do biochemical test.
Monitor liver function, kidney function, hematopoiesis and histopathology
Data processing
The results are presented as mean value / subtract standard error: M ± SEM (M: mean
value of each batch, SEM: standard error) and statistically compared using SPPSS 16.0
software. Use Mann Whitney and Wilconxon test to test an average of 2 groups at 2
different times. The result is considered statistically significant when p <0.05.
2.2.2.4. Evaluate the blood glucose control effect of extracts
of Euphorbia hirta L
Investigation of inhibitory activity of α-amylase enzyme in vitro
The inhibitory activity of α-amylase was determined based on the method using
Blocked p-nitrophenyl maltoheptaoside (BPNPG7) substrate. The enzyme inhibitory
capacity was determined by the IC50 index. IC50 is defined as the concentration (µg /
mL) of the sample subject to 50% inhibition of enzyme activity.

Investigation of inhibitory activity of the enzyme α-glucosidase in vitro
The α-glucosidase inhibitory activity was determined based on the p-nitrophenylα-D-glucopyranoside (PNPG) substrate method. The enzyme inhibitory capacity was
determined by the IC50 index. IC50 is defined as the concentration (µg / mL) of the
sample subject to 50% inhibition of enzyme activity.
Evaluation of blood glucose control effects on experimental animal models.
Increased blood glucose in mice by peritoneal injection of streptozocin solution (STZ)
mixed in cold 0.9% normal saline at a dose of 100 mg / kg. After 7 days of STZ injection,
mouse blood collected to measure blood glucose. Rats with blood glucose ≥ 10 mmol / L


12
were included in the follow-up study. Rats are divided into 4 (n = 10 / group):
Group 1 (physiological controls): healthy mice, drinking distilled water; Group 2
(pathology): mice with hyperglycemia, drinking distilled water; Group 3 and 4: mice with
hyperglycaemia, drinking Euphorbia hirta L. with doses of 250 and 500 mg / kg,
respectively. Rats received the study sample or water for 8 consecutive weeks. To the last
day of the 8th week, 1 hour after taking the study sample, take mouse venous blood,
centrifuge serum to quantify blood glucose and HbA1c according to the manufacturer's Kit.
How to evaluate the results: Compare the blood glucose values after taking the test
8 weeks with the control group. The experimental data were processed by one way
ANOVA analysis, using Mann-Whitney test.
2.2.3. Large foliar application for the production of nutritional beverages for
blood glucose control
2.2.3.1. Technology method
Selection of recipe
The main ingredient to make canned milk grass nutritional drink is Euphorbia hirta
L. According to the traditional medicine and pharmacy documents, the water of milk
grass has a pale yellow color, a slightly acrid taste. In order for milk grass drink products
to retain the characteristic flavor of milk grass, the author has researched to improve the
color and flavor with medicinal herbs with flavor properties such as honey, sweetener,

cinnamon, and ginger. The choice of blending formula needs to meet two requirements:
At what proportion of the auxiliary ingredients helps to increase the perceived quality of
the color and sweetness of the product and ensure that the product retains its strong
flavor. appearance of milk grass without being overwhelmed by the flavoring by the
auxiliary materials.
Formular Formular Formular Formular Formular
Formular
1 (CT1)
2 (CT2)
3 (CT3)
4 (CT4)
5(CT5)
Euphorbia hirta L.
X
X
X
X
X
extract
Honey bee
X
X
X
X
Sweeteners
X
X
X
X
Cinnamon

X
Ginger
X
Gingseng
X
Packaging method
The mixed product is packaged in 2 types of packaging: aluminum cans and white
glass bottles with a capacity of V = 200 ml. Carry out grafting and sterilization at 900 °
C for 15 minutes and then monitored for changes in the quality of the product when
stored at room temperature. After 6 months, determine the chemical, physical, and
microbiological parameters of the product.
Method of determining the time of pasteurization
Products packed in aluminum cans and glass bottles are pasteurized at 90oC,
holding time a: 5, 10, 15 minutes. Products are monitored to be stored at room


13
temperature. After 6 months, determine the chemical, physical, and microbiological
parameters of the product.
2.2.3.2. Testing methods
Physical and microbiological criteria: use reliable methods to determine.
Preparation efficiency: The extraction efficiency is determined by the ratio of the
extracted high mass to the extracted volume of medicinal powder
- The formula is as follows:

- Inside:
- M: Extraction efficiency(%)
- M2: High volumes are obtained(mg)
- M1: The amount of medicinal powder (Euphorbia hirta L. powder) is put into
extraction(mg)

2.2.3.3. Evaluation of food quality and safety
Assessing food quality and safety according to the QCVN 6-2: 2010 / BYT
National Technical Regulation for non-alcoholic beverages and the Vietnamese standard
TCVN 7041: 2009 on non-alcoholic beverages.
2.2.3.4. Evaluate community acceptance
The Euphorbia hirta L. nutritional drink product, when it was tested, was assessed
for community's acceptance by a taste score test conducted on a sample size of 98
mature consumers in Hanoi and Hung Yen. average 24-65 years old, both men and
women, currently not smoking, not suffering from nose and throat diseases and
voluntarily participating in the study.
Chapter 3: RESULTS
3.1. Botanical characteristics and chemical components of Euphorbia hirta L.
3.1.1. Botanical characteristics
3.1.1.1. Morphological characterization
Herbaceous plant, annual life, 20-50 cm high, has white latex. Stems erect
sometimes folded, cylindrical, 1-4 mm diameter, light green (when young) to pink, redpurple (when mature), divided into several segments, the nodes near the roots are often
short more than 2-3 cm long, the upper segments 3 to 7 cm long, the nodules between
the trunk segments slightly bulging. Leaves are simple, opposite, in a row, with 2 leaves
with thin, hard hair, 1-2 mm long on either side of the petiole. Leaf stalks short, 1-3 mm
long, hairy. Leaf blade oval or long elliptical, young leaves 1-2 cm long, 0.4-0.8 cm
wide; The leaf base is asymmetric, one side is rounded, one side is shield-shaped, the
leaf edge is serrated, the leaf tip is pointed
3.1.1.2. Determine the scientific name of the research sample
Based on the morphological characteristics of the study sample, comparing with the
classification key Euphorbia, the description of species Euphorbia hirta L. of the
Chinese plant lice, and the GS. Dr. Nguyen Nghia Thin with the help of TS. Nguyen


14
Quoc Huy, a sample of Euphorbia hirta L.studied, has been identified as Euphorbia

hirta L., and belongs to the Euphorbiaceae family
3.1.1.3. Microsurgery characteristics
Characteristics of leaf microsurgery
Characteristics of body microsurgery

Note:
Leaf vein
Dear
1.Feather cover
5. Libe
1. Gandular hair
2.Lower epidermis
6. wood
2. Epidermis
3. Soft tissue
8. Hedgehog
3. Soft shell tissue
4. Thick tissue
10. Upper epidermis
4. Libe
7. Soft tissue cells surround the phloem-wood bundle
5. wood
Blade
11. Feather cover
14. Hedgehog
12. Lower epidermis
15. Upper epidermis
13. Soft tissue
3.1.1.4. Characteristics of medicinal powder: Leaf powder is green, fragrant,
tasteless. Stem powder has a golden brown color, aroma, and a slightly bitter taste. The

flower powder is brown, fragrant, has a slightly bitter taste and the fruit powder is
brown, fragrant, tasteless.
3.1.2. Chemical components determination
3.1.2.1. Qualitative groups of compounds by chemical reaction
Conducting a qualitative reaction of the groups of compounds present in the upper
part of the Euphorbia hirta L. milkgrass grass shows that in the upper part of the ground,
the large-leaved milkgrass has groups of compounds: flavonoid, tannin, saponin,
alcaloid, sterol, reducing sugar , polysaccharide.
3.1.2.2. Qualify the extract fractions by thin layer chromatography
Qualitative extracts of n-hexane, chloroform and ethyl acetate by thin layer
chromatography with the corresponding solvent systems are: toluene-ethyl acetate-acid
formic (7: 1,5: 0,1); toluen-chloroform-methanol (5: 4: 0.5) and toluen-ethyl acetatemethanol-acid formic (3: 6: 1: 1). Comparing with the standard, it was found that in the
ethyl acetate fraction with quercitrin had high density, which showed that quercetrin was
a major ingredient in the ethyl acetate fraction
3.1.2.3. Quantification of total flavonoids in Euphorbia hirta L.
Quantification of total flavonoid in the upper part of the Euphorbia hirta L. field
shows that, the total flavonoid content in the upper part of the Euphorbia hirta L.


15
ground, calculated as quercitrin, is 0.375 ± 0.014% in absolute dry medicinal herbs. The
quantitative results have acceptable accuracy with RSD = 3,676%
3.2. Euphorbia hirta L. preparation, evaluating the safety and effectiveness of
the product in blood glucose control
3.2.1. Modulation of Euphorbia hirta L.
3.2.1.1. Investigation of factors influencing on the preparation of Euphorbia
hirta L.
Effect of temperature
Effect of ratio of solvents and medicinal herbs

Euphorbia hirta L.
medicine, cut into 1-3
cm pieces, humidity
<12%

Effect of

Liquid extract at 95oC;
2,5h. VH2O=10mdl, filter
extraction extracted
times fluid

Effect of extraction time

Medicine
waste
Liquid Extract at 95oC; 2h.
VH2O=8mdl, filter extracted
fluid

Extracted
fluid 1

Extracted
The influence of medicinal
size
fluid 2

Medicine
waste

Liquid extract at 95oC; 1h.
VH2O=8mdl, filter extracted
fluid

Medicine
waste

Extracted
fluid 3

3.2.1.2. Develop a process of high-leaf milk grass preparation in the scale of
o
Liquid extract
at /95
C; 2h.
1kg of medicinal
herbs
batch
V =8m , filter extracted
H2O

dl

fluid

Euphorbia hirta L. extract

Vacuum concentrate at 60 oC,
Vacuum dry at 50oC

16

3.2.2. Evaluate the safety of Euphorbia hirta L.
3.2.2.1. Assessment of acute toxicity
The samples were given to 5 groups of rats at the respective doses of 40.35 g; 30.30
g; 25.84 g; 20.67 g and 16.54 g high / kg body weight. Data on acute toxicity test of
fenugreek extract.
Experimental batch Oral dose (g /kg) N Number of rats per lot d
z
dxz
1
16,54
10 0
2
20,67
10 1
4.13 0.5 2.07
3
25,84
10 5
5.17 3.0 15.51
4
32,30
10 6
6.46 5.5 35.53


17

5
40,35
10 10
8.05 8.0 64.40
117,51
 (d x z)
LD50 = 40,35 - (117,51/10) = 28,6 (g extract/kg)
Thus, the dose LD100 is 40.35 g / kg and the dose LD0 is 16.54 g / kg. LD50 dose
of milk grass extract extract was 28.6 g as high as 147.4 g of medicinal herbs / kg. If the
dose with pharmacological effects in rats is 500 mg high / kg, then this LD50 dose is
about 57 times higher.
3.2.2.2. Semi-chronic toxicity assessment
Evaluation of hematological indicators, biochemical indexes, the ratio of organ
mass to body weight and histological tests of experimental mice showed that milk grass
extract at a dose of 0.3 g / kg and 3.0 g / kg orally for 30 days continuously in white rats
did not affect the hematological indicators such as the number of white blood cells,
erythrocytes, hemoglobin content and biochemical parameters of blood. , general
structure, microscopic.
3.2.3. Evaluation of the effectiveness of fenugreek in controlling blood glucose
3.2.3.1. Investigation of inhibitory activity of α-amylase and
α-glucosidase enzymes
Enzyme inhibitory activity of α-amylase and α-glucosidase of fenugreek extract at
different dilutions:

Based on the graph showing the dependence between sample concentration and αamylase enzyme inhibitory activity, the IC50 value was determined to be 967 µg / mL.
Based on the graph showing the dependence between the sample concentration and the
enzyme α-glucosidase inhibitory activity, the IC50 value was determined to be 53.96 µg / mL.
3.2.3.2. Effect of blood glucose control in experimental animal models
According to some researches in the world, the dose of Euphorbia hirta L. extract
in the range of 100-800 mg high / kg body weight of white mice. Therefore, the author

used a dose of 250 mg / kg and a dose of 500 mg / kg body weight of white mice as a
test dose for the effect of hypoglycemia in an experimental animal model. The results of
changes in blood glucose and HbA1c concentrations after 8 weeks of high-leaf


18
Euphorbia hirta L. extract
Targets

10
10

Blood glucose (mmol/L)
HbA1c (%)
At the
After
At the
After
beginning
8 weeks
beginning 8 weeks
6,0 ± 0,8 6,1 ± 1,9
4,2 ± 0,6 4,3 ± 0,5
*
10,9 ± 1,1 14,3 ± 1,5
4,8 ± 0,9 5,3 ± 0,8

10

11,3 ± 1,2 14,1 ± 2,5

4,9 ± 0,8

5,2 ± 0,5

10

11,2 ± 1,1 10,2 ± 1,3*,a

4,8 ± 0,6

4,7 ± 0,6

n
Mouse group
Physiological evidence
Pathology
Extract of Euphorbia hirta L.
(250 mg/kg)
Extract of Euphorbia hirta L.
(500 mg/kg)

*: p < 0,005 compared with the pathological control group
a
: p < 0,01 compared with physiological group
The concentration of blood glucose in the pathological control group was
significantly higher than the physiological control group (p <0.05), proving that the
model of causing hyperglycemia in white mice was successful. After 8 weeks of
continuous treatment with Euphorbia hirta L. extract extract at a dose of 500 mg / kg
body weight, the blood glucose concentration in the intervention group of diabetic mice

was significantly lower than the pathological control group with p. <0.05, 10.2 ± 1.3
mmol / L compared with 14.3 ± 1.5 mmol / L. Thus, extract of Euphorbia hirta L.
extract with a dose of 500 mg / kg body weight of white mice is effective in treating
diabetes in experimental animal models..
3.3. Euphorbia hirta L. foliar application to test the production of nutritional
beverages for blood glucose control
The obtained Euphorbia hirta Lmilkweed can be tested for the production of
nutritional food products. In the framework of the thesis, the author has created a
Euphorbia hirta L. high-leaf milk grass highly nutritious beverage that is easy to use for
many different subjects, including people with diabetes.
3.3.1. Research and select product formula
The author tested 5 different formula for the blending of high-nutrition beverages with
high-leaf milkgrass, the results of analysis of flavonoid and sugar content.


19

Recipe
CT1
CT2
CT3

CT4
CT5

Materials
Extract of Euphorbia hirta L. 25 g/l
Extract of Euphorbia hirta L. 25 g/l, honey
30 g/l; sweetener 0,3 g/l
Extract of Euphorbia hirta L. Extract of

Euphorbia hirta L. 25 g/l, honey 30 g/l;
sweetener 0,3 g/l, cinnamon 5 g/l
Extract of Euphorbia hirta L. 25 g/l, honey
25
g/l;
sweetener
0,3
g/l;
ginger 10 g/l
Extract of Euphorbia hirta L. 25 g/l, honey 25
g/l; sweetener 0,3 g/l; Ginseng 3g/l.

Total
Flavonoid
content
(mg/l)
501,2

Reduced
sugar
content
(g/l)
0,2

Total
sugar
content
(g/l)
0,2

511,5

19,0

21,2

510,4

19,2

21,3

508,9

15,95

17,70

512,0

15,75

17,50

The results of sensory quality assessment of the above milk grass nutrition drink
blending formulas. Formula 4 (Milk grass extract 25 g / l, honey 25 g / l; sweetener 0.3 g
/ l; ginger 10 g / l) had the highest mean sensory score (8.86 points).), slightly sweet
taste, harmonious acrid taste, pleasant smell and easy to drink are selected for further
studies.
3.3.2. Research product packaging and product pasteurization conditions

Large-leaf milk grass drink samples packed in aluminum cans had higher flavonoid
content in glass bottles, which was 506.7 mg / l compared to 480.4 mg / l. When
assessing sensory, the quality of products in aluminum cans reached 8.68 points, higher
than products in glass bottles. Therefore, overall, using aluminum cans to close CSSL
drinks is more appropriate than using light colored glass bottles.
Euphorbia hirta L. drink samples were pasteurized at 90oC for 10 minutes to
produce products with sensory value similar to pasteurized samples for 5 minutes.
Under these conditions, the color of the product is beautiful, not too brown, a
characteristic aroma, and not as concentrated as the pasteurized sample for 15
minutes. However, pasteurized samples for 5 minutes still contain a certain amount of
yeast and mold, which will affect the quality of the product during storage and do not
guarantee the limits of food safety with criteria specified in QCVN 8-3: 2012 / BYT .
3.3.3. Building a production process for Euphorbia hirta L. nutritional drinks
From the identified technological parameters, the production process of Euphorbia
hirta L. nutritional beverage has been built.


20
Extract of
Euphorbia
hirta L.

Materials
phụ

Mixing

Pre-filter
(100 mesh)

Refine
(100 micron)

0

Scale

Tin can
Heated (100oC)
Filling
Clean

Pair the lid of the can
Packaging scrap
Pasteurization
(90oC/10 phút)
Cool

Insulation

Labels

Labeling

Drinks Extract of
euphorbia hirta L..

3.3.4. Evaluation of food quality and safety
The Euphorbia hirta L. nutritional beverage products in the research and trial
production are tested for quality and safety criteria in laboratories meeting the national

standard TCVN ISO / IEC 17025 at the Institute of Nutrition. Country and Eurofins Sac
Ky Hai Dang Company Limited. Euphorbia hirta L. beverage product with high total
flavonoid content (80.3 mg / 100ml). The physical and chemical criteria and food safety
all meet the safety limit as prescribed in QCVN 6-2: 2010 / BYT, QCVN 8-1: 2011 /
BYT, QCVN 8-2: 2011 / BYT and QCVN 8-3: 2012 / BYT.


21
3.3.5. Evaluate acceptability in the community
There are 85.7% of consumers like the color of milk grass drinks, 89.8% of
consumers like the scent and 87.7% of consumers like the taste of the product
Color
Odor
Taste
General
Level
%
n
%
n
%
n
%
n
Like very much
9,2
9
18,4 18 15,3 15 14,3 14
Like moderately
76,5 75 71,4 70 72,4 71 73,5 72

Accept
14,3 14 10,2 10 14,3 14 12,2 12
Dislike
0
0
0
0
0
0
0
0
Cumulative from likes to likes
85,7
89,8
87,7
87,8
Thus, in general, the acceptance criteria for dairy products show that there are no
consumers who do not like the product, the other is to accept the product with a
cumulative ratio from like to very like 87.8%.
Chapter 4: DISCUSSION
4.1. Botanical characteristics and chemical components of Euphorbia hirta L.
The author's study is the first study on plant characteristics, describing the
morphology of the Euphorbia hirta L. in the Southern region, Binh Duong province.
According to published documents around the world, Euphorbia hirta L. contains more
flavonoids than small fodder and other plants of the same species. In order to find out and
add more scientific information about the bigleaf, the author has conducted the project
"Research on the plant characteristics and chemical components of the big leaf milk grass
(Euphorbia hirta L.)" In order to learn in detail, the system on the plant characteristics and
chemical components of the big foliar fodder.In Vietnam, there are not many studies on
Euphorbia hirta L. only a few studies reflect each small and incomplete angles of the

plant such as microscopic characteristics, chemical components. This study of ours
systematically and comprehensively describes the large foliar fodder
4.1.1. Botanical characteristics
Nguyen Thu Hang's research on micro-anatomical comparison between large foliar
and small foliar allows a clear distinction between large foliar and small foliar based on
micro-anatomical features and powder medicine. However, this study did not mention
the chemical components, as well as the scientific identification of these two species of
milk grass. In addition to determining micro-anatomy images for the same results as the
research of Nguyen Thu Hang, the author also identified the scientific name of the big
leaflets, the chemical components of the plants growing naturally in Binh. Ocean.
4.1.2. Chemical components
The author performed a fractional extraction of n-hexane, chloroform, ethyl acetate
extracts, and qualitative extraction by thin layer chromatography confirmed that
quercitrin is a major flavonoid in the plant. This is the scientific basis for the selection of
quercitrin as the standard for quantitative determination by thin layer chromatography
and total flavonoid quantification. The research results of the author not only supplement
important and basic scientific information about the plant characteristics and chemical


22
components of the large-leaf dairy plants naturally growing in Vietnam, but also open
up. direction of application in the food industry.
4.2. Euphorbia hirta L. preparation, evaluating the safety and effectiveness of
the product in blood glucose control
4.2.1. Modulation of Euphorbia hirta L.
The author has studied the extraction conditions to find the suitable conditions for
high-yield large-leaf milk grass preparation, cost reduction, convenient operation, safe,
full specifications, process is stable. In order for products to retain bioactive ingredients
during the manufacturing process, optimizing the high preparation process is the key. In
this study, the author selected suitable specifications to optimize the high preparation

process such as extraction temperature, extraction time, solvent / medicinal ratio, and
number of extraction times. This is consistent with studies of many Vietnamese authors
who have produced products for health protection and enhancement, which are widely
consumed in the market. Studies have shown that flavonoids are active active ingredients
in Euphorbia hirta L. and when extracted with hot water, flavonoids can be extracted
with relatively high levels. In this study, the author used aqueous solvents in extracting
the active ingredient from large green fodder. This is also an environmentally friendly,
safe solvent, with simple equipment investment suitable for most production units in
Vietnam, especially with reasonable production costs.
The large-leaf milk grass extraction process is built on a small scale that is repeated
and is stable at the scale of 1 kg / batch, specifically: the extraction efficiency and
flavonoid content obtained at the scale of 1 kg / batch are equivalent. similar to the small
scale 10g / batch. Hence there is great potential for adoption at a larger scale. Thus, after
examining the factors that affect the performance, the content of total flavonoid
compounds, the convenience of manipulation, towards low cost products, stable and ecofriendly process. In fact, the author has developed a process to prepare the stable and highgrade milkgrass with an average efficiency of 21.8% compared with the input materials,
and the content of total flavonoid in quercitrin is stable and average. 22.4 mg / g flask.
4.2.2. Safety evaluation of Euphorbia hirta L.
4.2.2.1. Assessment of acute toxicity
The author has determined the dose LD0 is 16.54 g / kg, LD100 is 40.35 g / kg,
LD50 dose is 28.6 g high / kg. Ali Esmail Al-Snafi studied on experimental rats to
determine the safety of fenugreek by giving the rat extract at doses of 1, 10 and 50 mg /
kg for 50 days, respectively, compared to the control group.
4.2.2.2. Semi-chronic toxicity assessment
The advantage of this study is that it was done on experimental mice at the Institute of
Medicinal Materials, the Ministry of Health, which is a reputable research facility, a team of
scientists experienced in research and toxicity assessment. level, semi-chronic toxicity on
experimental animal models for Vietnamese medicinal herbs. The research was carried out
in accordance with the process and the indicators were closely monitored.
With the semi-chronic toxicity assessment method, the research results showed that

23
the high leaf milkweed did not affect the general condition of the white rats, did not
affect the hematological indicators such as the number of white blood cells, erythrocytes,
hemoglobin content, hematocrit, platelet count, lymphocytes; does not affect
biochemical parameters of blood such as activity of AST, ALT, creatinine, total protein,
urea, bilirubin, glucose; Does not affect the macroscopic structure, micro as well as the
ratio of liver, kidney, spleen, heart mass compared to the rat body mass. From research
on safety and efficiency on experimental animal models is the basis for product
development in the future.
4.2.3.Evaluation of the effectiveness of fenugreek in controlling blood glucose
4.2.3.1. The ability to inhibit the enzymes α-amylase and α-glucosidase of
flavonoids
Inhibition of carbohydrate hydrolytic enzymes such as α-amylase and αglucosidase is known to delay digestion and absorption of carbohydrates from the small
intestine system. Because of inhibition of these enzymes, the absorption of glucose into
the bloodstream is minimal. The ability to inhibit the enzymes α-amylase and αglucosidase of the sample was assessed by IC50 value. IC50 is defined as the
concentration (µg / mL) of a test sample that can inhibit enzyme activity by 50%, the
higher the inhibitory activity the lower the IC50 value. In this study, based on graphs
showing the dependence between the sample concentration and inhibitory activity, the
IC50 values were determined as 967 µg / mL (α-amylase), 53.96 µg / mL, respectively.
(α-glucosidase). Thus, this research result is consistent with the references and
hypotheses. On that basis, conducting follow-up in vivo studies to confirm the
hypoglycemic effect of moth.
4.2.3.2. Effects of blood glucose control in experimental animal models
According to some researches in the world, the dose of extract of Euphorbia hirta
L. alcohol extract ranged from 100-800 mg high / kg body weight of white rats.
Moreover, according to the traditional medicine, the leaf extract is used at a dose of 4-6
g of medicinal herbs / person / day, so the author uses the extract of 250 mg / kg and
extract of 500 mg / kg to Test the effect of lowering blood glucose. In this study, after 8
consecutive weeks of treatment with Euphorbia hirta L. extract extract at a dose of 500

mg / kg body weight, the blood glucose concentration decreased significantly compared
to the pathological control group with p <0.05. Thus, the extract of Euphorbia hirta L. at
a dose of 500 mg / kg body weight of white mice has the effect of treating diabetes on
the experimental animal model through the criteria of lowering blood glucose
concentration.
4.3. Euphorbia hirta L. application for nutritional food production testing in
blood glucose control
For the first time, the author has successfully built a process to extract the stable
extract from the ground-up part of the large-leaved dairy plant from appropriate modern
technologies, appropriate chemicals, and suitable extraction time. Since then, the
formula has been formulated, selected and successfully produced nutritional drink for