Nghiên cứu độc tính, tác dụng điều trị nhồi máu não giai đoạn cấp trên thực nghiệm và lâm sàng của chế phẩm trúng phong hoàn tt tiếng anh

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INTRODUCTION
Of the disorders of the nervous system, stroke is the third leading
causes of death, followed heart disease and cancer death. About 20% of
people is affected by stroke. Approximately 80% of strokes are
infarction. The percentage of patients with infarction was between 80%
and 85% of stroke in and infarction is a leading cause of disability.
In addition to methods of modern medicine, there are many kinds
of drugs from traditional medicine which is very effective in treating
acute stroke, for example, Angongniuhuang, Huatuo Zaizao Pills.
However, these imported drugs are very expensive while the domestic
demand of them have been increased rapidly. Thus, there exists a need
for safe folk drugs which are more effective and easily available at low
cost. “Trung phong hoan” is a traditional medicine derived from the
ancient Vietnamese remedy in the name of “Thần dược cứu mệnh”, in
which the dried mandarin peel is added to. “Trung phong hoan” was
processed to granules so as to treat wind strike which is characterized
by numbness in the mouth and eyes, drooling, limb spasms, aphasia,
hemiplegia. This drug is composed of four ingredients, namely,
earthworm, black bean, sweet leaf and dried mandarin peel, which
completely meets the above criteria. In addition, although it is
commonly used in folk, there has not been any complete and systematic
study on yet.
Therefore, this study was conducted aiming to:
1. Determine the acute toxicity and semi-chronic toxicity and
evaluate the effectiveness of treatment for “Trung phong hoan” on
experimental animals
2. Evaluate the supporting effectiveness of the “Trung phong hoan”
composition on patients with acute ischemic stroke.
PRACTICAL SIGNIFICANCE AND NEW DISTRIBUTION OF
THE STUDY

In the search for new ways to treat stroke, pre-clinical studies are
required. Thus, it is necessary to conduct pre-clinical researches on
animals. In this study, the modeling infarction in mice was successfully
carried out by photochemical method which combines between Krypton
laser 568nm, a laser power of 6mW and Rose Bengal. The laser dose
was 20mg/kg body weight. Although modeling infarction using
photochemical method have been used for a long time and applied in


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rats, it maybe the first successful rat model in Vietnam. Moreover, we
changed some steps during modeling, in particular, the way of introduce
Rose Bengal into the circulatory system. Especially, Krypton laser
system was set for this study only to simplify the procedure, shorten
the time of manipulation and increase the success rate of stroke
modeling.
Research on therapeutic effects of traditional medicines for acute
ischemic stroke is a new direction. According to current statistics on
study in Vietnam, this is the second research on acute ischemic stroke
treatment followed by the research namely, “Angongniuhuang”. It is always
needed to continue to seek effective and safe herbal medicines, and as such
it is attractive to scientists from all over the world.
Clinical trials and experiments of this research were performed in a
rigorously systematic way. The remedy was processed to pills composed
of available four ingredients used to treat acute ischemic stroke.
Studying a remedy in order to apply it in ischemic stroke treatment will
contribute to illuminate the effects of treatment of traditional medicine,
which has practical and scientific significance.
TABLE OF CONTENTS
Introduction

CHAPTER 1: Literature Review
37 pages
CHAPTER 2: Methodology
18 pages
CHAPTER 3: Results
46 pages
CHAPTER 4: Discussion
31 pages
Conclusion
This study includes 49 tables, 16 charts, 14 figure, 01 drawing, 14 appendix
pages and 141 references (Vietnamese 70, English 36 and Chinese 35).
CHAPTER 1
LITERATURE REVIEW
1.1. Ischemic stroke
* Cerebral infarction
Cerebral infarction is caused by the sudden loss of blood circulation
because of a sudden lack of blood flow to whole or a part of cerebral
arteries or carotid arteries or a vein of the brain in rare. Clinically Clinically
cerebral infarction suddenly or acutely exhibits focal neurologic signs, in
which hemiplegia is common.


3
Supratentorial ischemic stroke includes cerebral hemisphere injury
caused by lesions in anterior brain arteries, middle brain arteries, anterior
choroidal arteries, posterior communicating arteries or branches of these
arteries and a part of posterior cerebral artery or branches of posterior
cerebral artery.
* Causes and pathogenic mechanisms of cerebral infarction: Cerebral
infarction includes coagulation, embolus, and lacunar infarct.

* Drug treatment of acute ischemic stroke
Stroke is a medical emergency and urgent treatment is essential. The
sooner a patient receives treatment for a stroke, the less percentage of
death and disability is likely happen.
Ideally, patients with stroke should be treated in in the intensive and
comprehensive care units. According to the current trend in Vietnam,
patients with stroke are often treated at Resuscitation & Emergency
Departments or stroke center under the following principles:
1. The ABC’s of first aid: Airway, Breathing, and Circulation
2. Managing cerebral edema; maintaining water and electrolyte
balance.
3. Specific treatment depending on stroke types.
4.Symptomatic treatment, treatment complication, anticonvulsants;
Controlling the blood pressure; not reducing the blood pressure
too fast; keeping blood pressure at a high limitation level: from
160/90 mmHg to < 185/100 mmHg (recommended by the
American Stroke Association).
5. Nursing care for stroke (to prevent superinfection of the lungs
and the urinary tract), ensuring adequate daily nutrient
requirements of patients, early rehabilitation (prevention of ulcer,
muscular atrophy, joint stiffness, and so on).
6. Surgical treatment, methods for assessing intervention
mechanical tool, remove blood clots from the circulation by
recombinant plasminogen.
7. Early relapse prevention therapy
1.2. The concept of stroke in traditional medicine
* “Wind Strike”
The term ”stroke” has not been mentioned in traditional medicine.
However, based on the clinical manifestations of stroke, it is labeled a
wind disease. It is characterized by sudden collapse, insensibility,

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hemiplegia, numbness in the mouth and eyes, limb spasms and aphasia.
A stroke often occurs suddenly and quickly with complicate conditions.
* Clinical type according to traditional medicine
According to traditional medicine books and the modern medical
literature, wind strike is divided into two types: wind on acupuncture
spots and wind on each of the five viscera.
+ Wind on acupuncture spots: The symptoms is mild, patients are not in
a coma, damp wind comes to meridian passage, collateral passage
causing numbness in the mouth, hemiplegia, a tongue with a thin white
coating.
+ Wind on each of the five viscera: The symptoms is severe due to
damp wind comes to zang organs, fu organs, representing in two levels
as belows:
° Blocking syndrome: Stupor, hemiplegia, incomprehensible sounds,
wheeze, numbness in the mouth and eyes, warm limb, floating pulse.
° Escaping syndrome: coma carus, wheeze, sweating, cold limbs,
fecal incontinence, pulled back tongue, moderate pulse.
* The study drug
“Trung phong hoan” is a remedy derivered from the ancient
Vietnamese remedy namely ”Thần dược cứu mệnh”, in which the dried
mandarin peel is added to. “Trung phong hoan” is mentioned in the
book in the name of “Những cây thuốc và vị thuốc Việt Nam”, written
by Prof. Do Tat Loi. At present, it is used to treat ”wind strike”
characterized by numbness in the mouth and eyes, drooling, limb
spasms, aphasia, hemiplegia. 20 patients with stroke in Military
Institute of Traditional Medicine were also administered by “Trung
phong hoan” in the fluid extract form. Initial results showed that the

remedy had positive effects in motor functional recovery using the NIHSS
and MRC score.
CHAPTER 2. SUBJECTS AND METHODS
2.1. Research materials
- Study drug: “Trung phong hoan” of 8.5g/ pill was prepared at the
Center for Eastern medicine research and production - Military Institute
of Traditional Medicine, which reached the standard basis.
- Drugs for the foundation regimen: Cerebrolysin 10 ml; Gliatilin 1g.
Nootropyl 1g). Blood pressure medications are used by the
recommendation of patients with cerebral infarction stroke’ attitude and


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there was no indication of thrombolytic agent (AHA/ASA 2005 Guideline
Update). Aspirin 100mg and Cebrex 40mg were used as experimental
controls.
2.2. Research subjects
2.2.1. Animal testing
Healthy Swiss mice (male an female, 25 ± 2g weight) were provided by
National Institute Of Hygiene And Epidemiology. Healthy Wistar mice
(male an female, 150 – 180g weight) were provided by Vietnam
Military Medical University.
2.2.2.Patient group
120 inpatient regardless of gender with acute ischemic stroke, age 18 to 90,
were treated at Military Institute of Traditional Medicine, 103 Military
Medical Hospital, Military Medical University in the period from 6 - 2016 to
6-2017.
2.3. Research methods
2.3.1. Evaluate acute toxicity and semichronic toxicity
Acute toxicity: The acute toxicity was studied base on guidance of the

Ministry 27 of Health and OECD. The study drug was administered
single ordered dose progression via orally route of 10.4g/kg to 52g/kg by
10 mice per lot. The health status as well as the number of dead mice was
recorded at 72 hours interval. The LD 50 was calculated by using the
method of Litchfield- Wilcoxon.
The semichronic toxicity of “Trung phong hoan” in mice was
studied base on guidance of the Ministry of Health and OECD, WHO.
Thirty mice were divided randomly into three groups of 10. The mice in
group control had access to distilled water in oral dose of 2 ml/kg/day.
The mice in group 1 had access to “Trung phong hoan” in oral dose of
7.14g/kg (equivalent to human dose). The mice in group 2 had access to
granulated medicine in the name of “Trung phong hoan” in oral dose of
21.42g/kg/day (equivalent to 3 times the human dose). All mice were
administered distilled water or study drugs in 8 weeks continuously, one
time per day at 8 AM. Inclusion criteria for the study population before,
after 4 weeks and after 8 weeks of administering medication were:
general health status, weight, hematopoietic function, liver function,
kidney function, liver histology and kidney histology.
2.3.2. Evaluate the treatment effectiveness of “Trung phong hoan” for
aute ischemic stroke in experimental animals
a) Mice model of ischemic stroke


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- Establishment of an rat model of focal cerebral infarction using a
photochemical method: To induce intravenous coagulation, a colored
photosensitive was injected to the circulation. Then, a light with a
suitable wavelength was used to radiate the vein that is needed to block
from the outside. This leaded to photosensitive activation at the position
of irradiation, which caused free radical formation. The free radicals

formed caused oxidation and injury vascular endothelial cells located at
the position of irradiation and then activated platelet aggregation
mechanism, which formed blood clots at this position.
b) Evaluate the treatment effectiveness of “Trung phong hoan” in
mouse model
36 mice were divided randomly into four groups as follows:
- Normal group (n = 6): The mice in this group were not treated to form
blood clots, which leads to stroke. Motor measurement results and
motor decline points of this group were used to compare with the results
of mice with stroke.
- Non-treatment stroke-induced group (n = 10): Movement of strokeinduced mice were evaluated at day 1, 3, 7, 14 after stroke inducing and
they were examined the brain specimens at day 3, 7, 14 after stroke
inducing.
- Stroke-induced Cebrex group (n = 10): Mice were administered orally
Cebrex in dose of 38.4mg/kg weight/day after stroke inducing inducing.
Movement of stroke-induced mice were evaluated at day 1, 3, 7, 14
after stroke inducing and they were examined the brain specimens at
day 3, 7, 14 after stroke inducing.
- Stroke-induced group treated with TPH (n = 10): Mice were
administered orally TPH after stroke inducing inducing. Movement of
stroke-induced mice were evaluated at day 1, 3, 7, 14 after stroke
inducing and they were examined the brain specimens at day 3, 7, 14
after stroke inducing.
* Evaluation criteria
- Evaluate the level of decline in motor: Mice of post-stroke inducing
groups were evaluated the level of decline in motor at day 1, 3, 7 and 14
after surgery. The group that was not induced stroke were evaluated
once only.
- The total length of movement distance and the average velocity for 5
minutes of groups were measured at day 1, 3, 7 and 14 after stroke

inducing.


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* TTC staining: In order to measure the infarct size and since then, the
foci of infarction could be calculated, we used TTC staining method.
* Lesion volume measuring: Brain slices has different colors after TTC
staining. Red or pink staining indicates normal tissue, white indicates
infarction.
* Study place and study time: Experimental testing and effectiveness
evaluation of the remedy in the name of “Trung phong hoan” in mice
model was performed at the Department of Pathophysiology – Military
Medical University in the period from 25 November, 2015 to 30
December, 2015.
2.3.2. Evaluate effects of the drug “Trung phong hoan” on patients
with acute ischemic stroke
a) Treatment method
- Control group (Group 2): was treated at the A12 Department/ Military
Institute of Traditional Medicine and Stroke Department A14, 103
Military Hospital using recommended regimen of WHO as follows:
- Study group (Group 1): was treated as the same as the group 2 with
“Trung phong hoan” combination by oral administration, 6 pills/ 3 times
daily.
Maintenance of patients’ life using the ABC rule:
Ensure breathing and circulation are adequate to deliver oxygen to
the body (heart rate adjustment, using blood pressure medications to
reduce the blood pressure if it is higher than 180/100 mmHg and to
increase if it is lower than 100/60 mmHg).
Using Manitol to reduce cerebral edema; antiplatelet drug like Aspirin
100mg orally, once per day after the breakfast; neuroprotective and

nutritional support drug of Cerebrolysin, 10ml per day (02 ampoules),
that is injected into veins slowly in the morning/afternoon. Gliatilin 1g in dose
of 02 ampoules/day, that is injected into muscles in the morning/afternoon.
Nootropyl 1g in dose of 02 ampoules/day, that is injected into veins in the
morning/afternoon.
* Adjusting water and electrolyte balance
b) Follow up the patients’ conditions
- Patients’ conditions were followed up and evaluated. All study criteria
were recored in the medical file at day 1 and 15.
- Side effects at patient clinic were monitored daily and continuously
during the study time.


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c) Monitoring evaluation criteria
Clinical monitoring and evaluation criteria
Clinical criteria according to modern medicine: Participants were
neurological examined, monitored and evaluated at day 1 and day 15.
Clinical criteria were assessed by Glasgow score, MRC scale, and
NIHSS. (Appendix 5, 6, 7).
b. Approaches to Criteria in traditional medicine: Participants were
examined and evaluated by quadrangular. Syndrome Differentiation is
based on the eight diagnostic principles. Diseases are differentiated via
fu-organs. Diseases are classified according to traditional medicine:
catching meridian and collateral.
c. Paraclinical monitoring criteria
At day 01 and 15: Total blood tests were performed, which included
erythrocyte, leukocyte, white blood cell, platelets counting, hemoglobin
test; biochemical blood test (Glucose, total protein, AST, ALT, GGT,
total bilirubin, Urea, Creatinine); a cranial CT scan

Side effects monitoring at patients:
- Clinic: fatigue, headache, dizziness, itching, and other manifestations
on gastrointestinal, cardiovascular, respiratory, kidney, urinary organ
were monitered.
- Paraclinic testal: Changes in hematopoiesis function, liver function and
kidney function were monitered
d. Standards for evaluating treatment effects
Results were assessed by Glasgow score, MRC scale and NIHSS score.
e. Evaluating the results by characteristics of traditional medicine
Participants were examined and evaluated for changes in symtoms
(before and After stroke treatment of study groups) via quadrangular
(inspection, listening and smelling examination, inquiry, and palpation),
including tongue textile, tongue coating, and pulse rate.
2.4. Data collection and analysis: After collection, data was analyzed
using statistical methods medical methods with SPSS statistical
software v.20.0 for Window.
2.5. Ethical considerations in clinical research: After studying the the
toxicity of the remedy and its effects on experimental animals, proofing
its safety and positive effects, this study was approved to human
volunteer testing by the Military Institute of Traditional Medicine
Council for Science and Medical Ethics Committee of 103 Military
Medical Hospital.


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CHAPTER 3. RESULT
3.1. Experimental research results
3.1.1. Results from acute toxicity tests
- Mice were administered “Trung phong hoan” in highest dose of
52g/kg weight, (50 folds of dose for human) but no lethal mouse was

detected as well as no abnormal symtoms within 72 hours after
administering and during 7 days of monitoring were found.
3.1.2. Semichronic toxicity
3.1.2.1. General health status
During the experiment, rats in all 3 lots still ate as usual. They were in
normal activity and agile. They also had nice eyes, silky fur, and dry
stool. No abnormal characteristics in all 3 lots were found during the
study time.
3.1.2.2. Changes in mice body weight: The body weight of mice in all
3 groups (control group and 2 study groups) was increased in
comparison with before the test. There was no statistically
significant difference with respect to the degree of change in mice
body weight between the control group and study groups (p>0.05).
3.1.2.3. Effects of “Trung phong hoan” on hematopoiesis function
There was no statistically significant difference with respect to the quantity
of erythrocytes, hemoglobin amount, the avarage volume of erythrocytes,
the quantity of leukocytes and the quantity of platelets of both study group
1 and study group 2 in comparison with the control group before and after
administering study drugs (p > 0.05).
3.1.2.4. Effects of TPH on liver function
There was no statistically significant difference with respect to AST and ALT
activity in mice blood of both study group 1 and study group 2 in
comparison with the control group and comparison between before, after
administering study drugs (p > 0.05) at the time of 2 weeks and 4 weeks.
3.1.2.5. Effectiveness of “Trung phong hoan” on kidney function
There was no statistically significant change with respect to urea and creatinine
concentration in mice blood of both study group 1 and study group 2 in
comparison with the control group (p > 0.05).
3.1.2.6. Histological results of 4 weeks after administering
Macroscopy: No abnormal change in macroscopic scale was found in heart,

lungs, liver, spleen, pancreas, kidneys and digestive organ of all mice (both
the control group and two study groups).


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3.1.2.7. Microscopic morphology of liver and kidney
Microscopic morphology of livers:
+ Control group: Normal liver image characterized by hepatic cells
arranges radically in normal. The central veins of liver and liver
sinusoid were mildly congestive. Portal tracts were thin, some areas
were infiltrated mononuclear cells which consists of lymphocytes.
+ Study group 1: Normal liver image with intact hepatitic cells, the
central veins of liver and liver sinusoid were mildly congestive. Portal
tracts were thin and fibers were found scattered through them.
+ Study group 2: Normal liver image- characterized by hepatic cells
arranges radically. The central veins of liver and liver sinusoid were
mildly congestive. Portal tracts were thin, lymphocytes and fibers were
found scattered through them.
Microscopic morphology of kidneys:
+ Control group: Normal kidney image with enlargement of the
glomerulus, blood vessels were congestive which were consisted of
erythrocytes inside, the Bowman capsules were enlarged and contained clear
liquid. Blood vessels of the renal interstitium were mildly congestive.
+ Study group 1: Normal kidney image with enlargement of the
glomerulus, blood vessels were congestive which were consisted of
erythrocytes inside, the Bowman capsules were enlarged and contained
clear liquid. Blood vessels of the renal interstitium were mildly congestive.
+ Study group 2: Normal kidney image with enlargement of the
glomerulus, the Bowman capsules were enlarged and contained clear
liquid. Tubule epithelial cells had normal morphology. Blood vessels of

the renal interstitium were mildly congestive.
3.1.3. Evaluation results of positive effects of “Trung phong hoan”
on experimental model
3.1.3.1. Results of performing experimental a animal model
in stroke research
Results shows that laser irradiation in two minutes did not cause
loss of circulation of the middle brain arteries of all mice of study
groups. In groups wherein mice were radiated by laser in the period
from 4 to 6 minutes, it is realized circulation of the middle brain arteries
was lost was lost after the first laser irradiation. After the second laser
irradiation at the position that is below the position of the first laser
irradiation, the circulation capacity of the middle brain arteries was lost
in both periods of 4 minutes and 6 minutes.


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Fig. 1: The middle brain arteries imaging of mice in coagulation process
caused foci of infarction and the method of induction of ischemic stroke.
a. The middle brain arteries imaging after cranial exposure. b. The
middle brain arteries imaging while Krypton laser irradiation. c. Loss
of circulation of the middle brain arteries imaging when blood clots
appearing. d. The method of induction of ischemic stroke: after the first
laser at the division position of the middle brain arteries, circulation of
the middle brain arteries was lost, the second laser irradiation was
performed at the position that is below the position of the first laser
irradiation in the same period.
To ensure the exist of lesion, TTC staining was performed with mice brain 3
days after laser irradiation. Results show that in groups in which mice were
radiated by laser within 2 minutes, no lesions in brain was found in all slices.

While in both mice groups radiated with laser within 4 minutes and 6
minutes, injuries were found in all mice.

Fig. 2: Mice brain imaging at day 3 after inducing ischemic stroke


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a. Mice brain were irradiated by Krypton laser in 2 minutes. b. Mice
brain were irradiated by Krypton laser in 4 minutes. c. Mice brain
were irradiated by Krypton laser in 6 minutes.
The volume of blood clots was measured and compared between two laser
irradiation groups for 4 minutes and 6 minutes. Results show that there was
no statistically significant difference with respect to the volume of blood
clots between groups.
Table 1: The percentage of success of the method of inducing stroke
by photochemical method

Ischemic stroke volume (mm3)

The volume of blood clots was also was measured by method described in
the study method part. Results of the volume of blood clots (mm 3; Mean ±
SEM) at day 3, day 7 and 14 were 267.7 ± 42.7; 219.7 ± 4.4; and 205.7 ±
35.1, respectively (Fig. 3.6).
Thus, the volume of blood clots tended to reduce over time, but there
was no statistically significant difference at the time study.

Fig. 1: Ischemic stroke volume was measured at day 3, 7 and 14
after stroke inducing

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3.1.3.2. Evaluation results of positve effects of “Trung phong hoan”
on the model

Fig. 2: Evaluation scores of decline in motor over time after stroke
inducing of each group.
Results show that the scores of the group treated with TPH equivalent to
the Cebrex group. They were always statistically significantly lower in
comparison with the stroke – non treatment group at day 1, 3, 7 after
stroke inducing, and there was no statistically significant difference in
comparison with normal group at day 14. TPH has positive effects on
reducing the level of movement disorders stroke-induced mice, the
positive effects of this drug was equivalent to Cebrex.
b) Motor ability of mice was improved by Trung phong hoan
We also evaluated the positve effects of Trung phong hoan by
calculating distance traveled and speed of mice in groups after stroke
inducing. One day after stroke inducing, and there was no statistically
significant reduction with respect to distance traveled in comparison
with the normal group in all groups with stroke-inducing. There was no
statistically significant difference with respect to the distance traveled
and speed of the TPH group and the Cebrex group in comparison with
the non-treatment group at one day after stroke inducing. It is increased
in statistical difference in comparison with the non-treatment group at 3
days and 7 days after stroke inducing and still tended to be larger than
this of the non-treatment group at 14 days after stroke inducing. In
conclusion, TPH had clearly positive effect on improving the mobility
of stroke-inducing mice in comparison with the non-treatment group,
and the effect was equivalent to the Cebrex group.
We also compared the distance traveled and speed of each group mice over
time so as to evaluate effects of the drug of prolonged use. Results show

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that in the group treated with TPH, the distance traveled and speed of mice
had statistically significant increase at 3, 7 and 14 days in comparison with
one day after stroke inducing. Therefore, in case of prolonged use, TPH
had positive effects on improving the mobility at least until day 14 ngày
after stroke inducing.

Fig. 3: Distance traveled (a) and speed (b) of each group over time
after stroke inducing.
c) “Trung phong hoan” drug had positive effects on reducing the
volume stroke mass

Fig. 4: Ischemic stroke volume over time
From the results of assessing the ischemic stroke volume, the Cebrex
had positive effects fast and reducing the volume of stroke at day 3 after
administering (in comparison with the non-treatment group). However,
these effects did not improve significantly over time. While the group
treated with TPH were also improved as the same as this treated with
Cebrex, characterized by the clearly reduced volume of blood clots at
day 3 after drug administration (in comparison with the non-treatment
group). The volumes of blood clots then increased over time and were
highest at day 7 although the volumes of blood clots were still
statistically smaller in comparison with the non-treatment group. Then


15
the volumes of of blood clots reduced rapidly and reached the smallest
value (in comparison with the non-treatment group and The Cebrex

group) at day 14 after stroke inducing.
3.2. CLINICAL EVALUATION OF RESULTS EFFECTS OF THE
DRUG
3.2.1. Clinical characteristics of patient groups
- Characteristics of age: The average of patients with ischemic stroke
was mainly 50-70. In which the highest percentage of 38/120 (31.67%)
was mainly 61-70. there was no statistically significant difference with
respect to age (p > 0.05) between the two group patients.
- Characteristics of genders: The percentage of men was higher than
this of women with the percentage of male/female was 1.5/1 (in “Trung
phong hoan” group was 1.59/1 and in the control group was 1.38/1),
there was no statistically significant difference with respect to the
percentage of male/female ở hai Study groups (p > 0.05).
- Characteristics of generation time of the two groups: The
generation time of stroke mainly from the midnight to dawn in the TPH
treatment group was 46/70 patients (65.7%), in the control group was
29/50 (58%), in both groups was 75/120 (62.50%) and the difference
was not statistically significant between two groups (p > 0.05).
- From the beginning of stroke till using study drug: Most
hospitalized patients with ischemic stroke in this study were acute, which
was within 1 - 3 days of 70.84%, and < 24h of 29.14%.
- Risk factors of the patient groups: The percentage of patients with
hypertension was highest, of 80.8%. The next was patients with lipid
metabolism disorders of 75.8%. There was a similarity between the two
groups. The third risk fator was other diseases, such as cardiovascular and
asthma with 12.5%. Other risk factors with less frequency often
mentioned were weather, alcoholism and smoking. There was no
statistically significant difference with respect to the percentage of risk
factors in the two patient groups.
- Characteristics of localised nerve damage of patients before stroke

treatment: The main feature of localised nerve damage often seen in
ischemic stroke in both groups were hemiplegia, which was highest
percentage of 100%. The percentage of patients with central facial palsy
was of 85.8%, language disorders was of 74.2%, sensory disorders was
of 77.5%, pathological reflexes chiếm 75.0%, tendon reflexes was of
76.7%. These are most common symtoms in the study groups. The less


16
common symtom was round muscle paralysis of 43.4%. There was no
signigicant difference between the study groups.
- Characteristics of foci of infarction Before stroke treatment in CT
scan film: The data in the table 3.26 shows that the volume of foci of
infarction was between 20-40mm in both groups, of 61 patients
(50.8%). There was no signigicant difference between the study groups.
There was no signigicant difference between the study groups (p >
0.05).
3.2.2. Treatment results of patients acute ischemic stroke after 15
days treatment
3.2.2.1. Treatment results according to morden medicine
Table 2. Changes in blood pressure, pulse rate, and temperature of
patients with acute ischemic stroke after 15 days treatment
Criteria
Systolic
Before
blood
After
pressure
P
(mmHg)

Diastolic
Before
blood
After
pressure
P
(mmHg)
Before
Pulse rate
(times /
After
minute)
P
Before
Temperature
After
(˚C)
P

TPH group
(n=70)
148.5 ± 12.08
118.3 ± 11.95

Control group
(n=50)
149.5 ± 11.88
120.2 ± 12.41

P<0.05

P<0.05

98.3 ± 11.95
76.1 ± 10.24
p<0.05

100.2 ± 12.41
77.8 ± 10.21
p<0.05

> 0.05
> 0.05

83.1 ± 8.53
72.5 ± 8.04
p> 0.05
36.8 ± 0.35
36.5 ± 0.23
p> 0.05

83.4 ± 9.24
71.3 ± 8.86
p> 0.05
36.9 ± 0.38
36.7 ± 0.19
p> 0.05

> 0.05
> 0.05

p
>0.05
>0.05

>0.05
>0.05

Commence:: After 15 days treatment, the “Trung phong hoan”
group and the control group, there was a statistically significant
reduction in systolic and diastolic blood pressure (p< 0.05).
However, there was no statistically significant difference between the
two groups (p>0.05). There was no statistically significant change with
respect to the pulse rate and temperature between the two groups (p >
0.05).
* The level of consciousness recovery using Glasgow day 15 after
treatment
Table 3. The level of consciousness recovery using Glasgow score
TPH group (n=70) A

Control group (n=50) B


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Glasgow
score
6-8
9 - 12
13 - 14

15
Total
pA-B

Before
stroke
treatment
(1)
n
%
0
0
62.8
44
6
21.4
15
3
15.7
11
1
70
100

After
stroke
treatment
(2)
n
%

0
0
3
9
58
70

p2-1

4.28
12.8
6
82.8
6
100

<0.00
1

Before
stroke
treatment
(3)
n
%
0
0

After
stroke

treatment
(4)
n
%
0
0

29

58.00

5

10.00

10

20.00

8

24.00

11

22.00

37

66.00

100

50

100

50
<0.05

p4-3

<0.05

Commence: At the time of hospitalization, the Glasgow Coma scores of
patients were mainly of 9-12 and there was a similarity between the two
groups with p > 0.05. At day 15 after treatment, there was a signigicant
improvement of the study groups. In particular, patients with scores of 15
points was of 82.86%, in comparison with the control group only of 66.00%.
In which, well treated patients were of 47/58, pretty treated group were of 10
patients, who had the Glasgow scores increased from 9-10 points to 12 - 13
points.

Fig. 5. The level of recovery using MRC grade
Commence: At the time of hospitalization, the percentage of patients with
MRC grades I was high (48.57%), grade II (22.86%), grade III (18.57%),
grade IV (10.00%). There was no significant difference between the two
groups (p > 0.05). At day 15 after treatment, muscle strength had decreased
significantly. In particular, patients with MRC grades V were of 57.14%
compared to the control group that was only of 34.00%.

18

Fig. 6. The level recovery of patients of study groups using NIHSS score

Commence: There was a significant improvement in patients of TPH group
at day 15 after administering with NIHSS score ≤ 4 of 38.57%, NHISS score
of 5 – 15 was of 52.86% and NHISS score of 16 – 20 was of 8.57%. In
which well and pretty treated patients were of 75.72% with the average
percentage was of 24.28%. Before stroke treatment, the average NIHSS
score was 12.57 ± 3.39 points, Min - Max: 5 - 20 points; after treatment
was 7.24 ± 3.16 points; Min - Max: 0 - 19 points.
Table 4. Results of general treatment
Results of treatment
Good
Pretty
Moderate
Bad
Severe, dead
Total
p1-2

TPH group
(n=70)
n
%
45
64.29
19

27.14
6
8.57
0
0
0
0
70
100

Control group
(n=50)
n
%
23
46.00
13
26.00
14
28.00
0
0
0
0
50
100
< 0.05

Commence: The results of “Trung phong hoan” group had good and
pretty result were of 91.43% and that of the control group was of

72.00%. The results of general treatment had a statistically significant
difference between the two groups with p < 0.05. The percentage of
completely recovery patients in the study drug, the group that was
not treated with the study drug with respect to 3 criteria including
Glasgow Coma score, the MRC grade, NIHSS score was 91.43% and


19
72%, respectively. There was a statistically significant differene with
p<0.05.
3.2.2.2. Treatment results according to traditional medicine
The color of tongues changed in the congestion status, such as: The
black tongue at the time before and after treatment was of 68.57% and
37.14%, respectively. The rash and congestion point status at the time
before and after treatment was of 35.71% and 17.14%, respectively.
Since then, it is considered that this drug had positive effects on
congestion. There was a statistically significant difference of before and
after stroke treatment (p< 0.05).
The shapes of tongue were not significantly changed after the treatment.
Thus, the change in tongue shape was not statistically significant (p >
0.05).
There tongue movement had the most significant change. In particular,
the percentage of tongue flexibility before and after stroke treatment
was 18.57% and 84.29%, respectively. There was a statistically
significant difference of before and after stroke treatment (p< 0.05).
There was a signigicantly changed in tongue coating, especially in the
tongue coating thickness moisture and viscosity.
Table 5. Characteristics of pulse in patients before and after
administering study drug according to traditional medicine
Characteristics of

pulse rate
according to
traditional
medicine
Floating
Sunken
Slow
Rapid
Racing
String-like
Firm
Weak

Before stroke
treatment

After stroke treatment

Quantity

Percentage
(%)

Quantity

Percentage
(%)

0
19

16
39
51
40
41
29

0
27.14
22.86
55.71
72.86
57.14
58.57
41.43

0
19
22
16
40
36
45
25

0
27.14
31.43
22.86
57.14

51.43
64.29
35.71

Commence:: The above results shows the treatment effects: the
percentage of rapid reduced most significantly before and after treament
with 55.71% and 22.86%, respectively. The next was racing of 72.86%
and 57.14% before and after treament, respectively. There was no
change in others characteristics of pulse rate.


20
Almost patients had the following characteristics:
+Tongue texture: gray, having congestive dots, flabby, and teethmarks.
+ Tongue movement: limited or quiver.
+ Tongue coating: yellow, thick, and moisture
+ Pulse rate: racing, string-like, and rapid.
These above characteristics belongs to phlegm dampness, qi deficiency,
and congestion.
3.2.2.3. Side effects of TPH
a. Side effects of TPH in the clinic trial
There were a patient (1.4%) with nausea and 3 patients (4.2%) with
mild rash, 4 patients with abdominal pain and diarrhea and recovered
after two days. In addition, there was no more features in patients of
both groups during the study time.
b. Paraclinic testal evaluation of side-effects of TPH
There was no difference with respect of the quantity of erythrocytes,
leukocytes, platelets, the level of hemoglobin in blood, cholesterol,
triglyceride, glucose, HDL-C, LDL-C, AST, ALT, Urea and Creatinine
level between the two groups.

CHAPTER 4: DISCUSSION
4.1. Acute toxicity and semichronic toxicity của granulated medicine in
the name of “Trung phong hoan”
4.1.1. Acute toxicity: Acute toxicity study of “Trung phong hoan” drug
in mice in maximum dose of 52.0g/kg weight, which is 5-fold dose of
human in common. The LD50 of TPH was not determined. These results
could be explained by the fact that the ingredients of TPH like
earthworm and dried mandarin peel, which is medicine presenting in
the ancient remedy, using for thousand of years.
4.1.2. Semichronic toxicity: At week 4 after administering “Trung
phong hoan” in dose of 7.14g/kg and 21.42g/kg, the development of mice
did not affected. Particularly, there was no change in hematological
indices, blood biochemistry and macroscopy, liver and kidney
histopathology. These results showed the high level of safety of “Trung
phong hoan”.
4.1.3. The effects of “Trung phong hoan” drug on the experiment
* Induced-stroke model: In this study, we successfully performed the
photochemical- induced stroke model in mice combined with Krypton
laser 568nm, a laser power of 6mW and Rose Bengal Rose Bengal in


21
dose of 20 mg/ kg weight. The optimal duration of laser projection to
induce ischemic stroke was 4 minutes and the percentage of stroke
inducing successfully by this method was 100%.
The reference index of volume of blood clots in Swiss mice initially set up
(Mean ± SEM; mm3) at day 3, 7 and 14 days after stroke inducing was: 267.7
± 42.7; 219.7 ± 4.4; 205.7 ± 35.1 (mm3), respectively. Although modeling
infarction stroke using photochemical method have been used for a long
time and applied in rats, it maybe the first successful rat model in

Vietnam.
*Effects of “Trung phong hoan” on experimental model
- The level of decline in motor: Results show that the score of the TPH
group was equivalent to the Cebrex group and always statistically
significant lower compared to the non-treatment group at day 1, 3, 7
after stroke inducing, and there was no statistically significant difference
compared to the normal group at day 14. TPH has positive effects on
reducing the level of motor disorder in stroke-induced mice. The effects
of this drug was equivalent to those of Cebrex.
- Distance traveled and speed after treatment: TPH had positive effects
on improving the mobility of stroke-inducing mice compared to the
non-treatment group, and its effects equivalent to those of the Cebrex
group.
c) Foci of infarction size volume: To evaluate the positive effects of
this drug over time, we compared the ischemic stroke volume at day 3,
7 and 14 in a group of 3 objected group. The results showed that the
volume of blood clots in non-treatment group were tended to reduce
over time. However, the difference among periods had not statistically
significance. The volumes of TPH group were highest at at day 7 after
stroke inducing while reduced rapidly at day 14. TPH has positive
effects on reducing the volume of blood clots and its effects was
increased and prolonged over time, at least till 14 days after stroke
inducing.
4.2. Effective assessment of TPH on clinic trials
4.2.1. Effects on clinic trials according to modern medicine
- Effective assessment of TPH on clinic trials: at day 15 after “Trung
phong hoan” treatment drug and the control group, the systolic and
diastolic blood pressure were all reduced significantly (p<.05).
However, the differene between two groups was not statistically
significant (p>0.05).

22
- The change in pulse rate and temperature in both groups had not
statistically significant difference (p > 0.05). The positive effects on
lowering the blood pressure of "Trung phong hoan" drug are considered
due to the fact that it contains earthworm which has positive effects on
lowering the blood pressure, proved by the study of Tran Thi Hong
(MD capsule).
Level of recovery from consciousness: "Trung phong hoan" drug had
positive effects on improving the consciousness of patients with stroke.
According to traditional medicine, acute ischemic strokeis mainly
caused by phlegm is blocked at middle jiao, fluctuated ascending and
descending leads liver to loss of excretion, which leading to gathering qi
to produce fire. Lurking qi is blocked that leads to so congestion, bursts
clogged phlegm, excess viscera and bowels and so on. In which, phlegm
syndrome is prominent.
TPH had positive effects on improving motor function: From the
viewpoint of traditional medicine, the muscle strength is closely related
to the spleen and stomach according to the theory considered that the
spleen is responsible for muscle and four limbs. The spleen brings
nutrients of food to feed muscles. If the spleen qi is efficent, muscles
will hard and four limbs will be flexible. Whereas,if the spleen qi is
deficient, muscles will soft and the muscular tonicity reduces that leads
to tiredness.
TPH had clearly positive effects on improving NIHSS scores because
the average NIHSS score of participants was 14.57 ± 4.39, which is as
the same as the patient group who are with moderately conscious,
motor, and sensitive disorders. Lesions observed on X-ray radiographs
in the table 3.26 were mainly small and medium. Morover, participants

were treated early, particularly within < 24h to 3 days leaded to the
good results as shown in the table 3.23. These results are rather similar
to Glasgow Coma score and MRC scale.
4.2.2. The positive effects of "Trung phong hoan" drug on ischemic
stroke according to traditional medicine
Causes, mechanisms of wind strike are very complicated. However,
wind strike in general and cerebral infarction are all come from the
inside and the outside, in which the inside cause is the mainly reason.
The remedy "Trung phong hoan" contains the following ingredients:
earthworm, sweet leaf that spitting up the blood, unblocking the
meridians and collaterals, disperse wind-heat, remove phlegm together


23
with the effects of dried mandarin peels which warm the middle-jiao,
regulate qi and remove phlegm; black beans which tonify the liver and
the kidney. Therefore, it can be considered that this remedy has positive
effects of dispersing wind-heat, counteracting toxicity, promote blood
circulation and removing blood stasis, unblocking the meridians and
collaterals, removing phlegm, tonify the liver and the kidney.
Wind strike is mainly caused by liver and ying kidney deficiency,
heart-heat wind moving, phlegm-heat inside, phlegm-heat. Thus, there
was string, string-like, rapid, and rapid. Tongue coating is mainly
yellow, tongue textile has congestive points. These characteristics are
totally suitable with syndromes liver kidney ying deficiency, phlegmheat and poor circulation. Therefor, in this study, using the remedy
“Trung phong hoan” which had positive effects on promote blood
circulation and remove blood stasis, disperse wind-heat remove phlegm,
tonify the liver and the kidney to treat post-acute ischemic stroke.
CONCLUSION
By performing the evaluation of acute toxicity, semi-chronic toxicity

and positive effects of “Trung phong hoan” drug on experimental
animals and clinical trials, the results are as belows:
1. Acute toxicity, semi-chronic toxicity and positive effects of
“Trung phong hoan” drug on experimental animals
- In highest dose of 52.0g/kg can be administered by mice and no lethal
mouse was detected. The LD50 of TPH was not determined.
- In dose of 7.14g/kg/day and in dose of 21.42g/kg/day in 4 weeks
continuously did not affect on the general health status, hematopoietic
function, function and morphology of liver and kidneys of mice
administered this drug.
- The results of the stroke model in mice showed that TPH has positive
effects on reducing the level of movement disorders immediately after
administering and this effect was prolonged during the study time. TPH
also significantly reduced the volume of the blood clots compared to the
Cebrex group.
2. The supporting effectiveness of the “Trung phong hoan”
composition on patients with acute ischemic stroke
- "Trung phong hoan" drug had positive effects on improving the
consciousness of patients with stroke via improving Glasgow Coma


24
score. The percentage of good and pretty treatment was of 82.85% and
14.28%, respectively.
- TPH group had positive effects on clearly improving muscle strength
of patients using the MRC grade. Good and pretty treatment was of
74.29% and 18.57%, respectively.
- TPH group had positive effects on improving NIHSS score. At day 15
after treatment: NIHSS score ≤ 4 was of 38.57%, NHISS score 5 – 15 was
of 52.86% and NIHSS score 16 – 20 was of 8.57%. In which well and

pretty treated patients were of 75.72%. Before stroke treatment, the average
NIHSS score was 12.57 ± 3.39 points; after treatment was 7.24 ± 3.16
points.
- TPH group had positive effects on reducing the black tongue status
and increasing the pink tongue percentage as well as increasing
motivation of tongue in the study group. It also had positive effects on
reducing yellow level and viscousity of tongue coating, increasing the
bright of tongue coating; reducing the number of rapid, racing, and
string-like pulse type in the study group.
- TPH did not cause side effectiveness in clinical trials and paraclinic
test:
+ Clinic trials: Some patients had symtoms such as arllegy, diarrhea,
nausea. However, it was not needed to treat them due to the
disappearing of these symtoms after 2 days.
+ Paraclinical test: “Trung phong hoan” did not effect on liver and
kidney functions as well as hematopoietic organs of participants.
RECOMMENDATION
By observing in clinical practice, we realized that “Trung phong hoan”
drug has postitive effects on stroke. Thus, this remedy shoud be applied
to patients with acute ischemic stroke. Moreover, the ingredients of
“Trung phong hoan” drug are available and cheap. Therefore, we
recommend as follows:
1. Continously evaluating the effectiveness of “Trung phong hoan”
drug in ischemic stroke in a larger scale and the study should be
performed to post-acute ischemic stroke.
2. Study the mechanism of action of “Trung phong hoan” drug, such as
mechanism of vasodilation and reducing the blood pressure, the
mechanism of the cholesterol‐lowering effect, its effectiveness on
oxidants...