Evaluate the supporting effectiveness of the “Trung phong hoan” composition on patients with acute ischemic stroke.. Research materials - Study drug: “Trung phong hoan” of 8.5g/ pill was
Trang 1Of the disorders of the nervous system, stroke is the third leadingcauses of death, followed heart disease and cancer death About 20% ofpeople is affected by stroke Approximately 80% of strokes areinfarction 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 treatingacute stroke, for example, Angongniuhuang, Huatuo Zaizao Pills.However, these imported drugs are very expensive while the domesticdemand of them have been increased rapidly Thus, there exists a needfor safe folk drugs which are more effective and easily available at lowcost “Trung phong hoan” is a traditional medicine derived from theancient Vietnamese remedy in the name of “Thần dược cứu mệnh”, inwhich the dried mandarin peel is added to “Trung phong hoan” wasprocessed 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, whichcompletely meets the above criteria In addition, although it iscommonly used in folk, there has not been any complete and systematicstudy 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 arerequired Thus, it is necessary to conduct pre-clinical researches onanimals In this study, the modeling infarction in mice was successfullycarried out by photochemical method which combines between Kryptonlaser 568nm, a laser power of 6mW and Rose Bengal The laser dosewas 20mg/kg body weight Although modeling infarction usingphotochemical method have been used for a long time and applied in
Trang 2rats, it maybe the first successful rat model in Vietnam Moreover, wechanged some steps during modeling, in particular, the way of introduceRose Bengal into the circulatory system Especially, Krypton lasersystem was set for this study only to simplify the procedure, shortenthe time of manipulation and increase the success rate of strokemodeling.
Research on therapeutic effects of traditional medicines for acuteischemic stroke is a new direction According to current statistics onstudy in Vietnam, this is the second research on acute ischemic stroketreatment followed by the research namely, “Angongniuhuang” It is alwaysneeded to continue to seek effective and safe herbal medicines, and assuch it is attractive to scientists from all over the world
Clinical trials and experiments of this research were performed in arigorously 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 willcontribute to illuminate the effects of treatment of traditional medicine,which has practical and scientific significance
TABLE OF CONTENTS
Introduction
CHAPTER 3: Results 46 pages
CHAPTER 4: Discussion 31 pages
Conclusion
This study includes 49 tables, 16 charts, 14 figure, 01 drawing, 14 appendixpages 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 circulationbecause of a sudden lack of blood flow to whole or a part of cerebralarteries or carotid arteries or a vein of the brain in rare Clinically Clinicallycerebral infarction suddenly or acutely exhibits focal neurologic signs, inwhich hemiplegia is common
Trang 3Supratentorial ischemic stroke includes cerebral hemisphere injurycaused by lesions in anterior brain arteries, middle brain arteries, anteriorchoroidal arteries, posterior communicating arteries or branches of thesearteries and a part of posterior cerebral artery or branches of posteriorcerebral 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 Thesooner a patient receives treatment for a stroke, the less percentage ofdeath and disability is likely happen
Ideally, patients with stroke should be treated in in the intensive andcomprehensive care units According to the current trend in Vietnam,patients with stroke are often treated at Resuscitation & EmergencyDepartments 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 electrolytebalance
3 Specific treatment depending on stroke types
4.Symptomatic treatment, treatment complication, anticonvulsants;Controlling the blood pressure; not reducing the blood pressuretoo fast; keeping blood pressure at a high limitation level: from160/90 mmHg to < 185/100 mmHg (recommended by theAmerican Stroke Association)
5 Nursing care for stroke (to prevent superinfection of the lungsand the urinary tract), ensuring adequate daily nutrientrequirements of patients, early rehabilitation (prevention of ulcer,muscular atrophy, joint stiffness, and so on)
6 Surgical treatment, methods for assessing interventionmechanical tool, remove blood clots from the circulation byrecombinant 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 awind disease It is characterized by sudden collapse, insensibility,
Trang 4hemiplegia, 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 medicalliterature, wind strike is divided into two types: wind on acupuncturespots 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 passagecausing numbness in the mouth, hemiplegia, a tongue with a thin whitecoating
+ Wind on each of the five viscera: The symptoms is severe due todamp wind comes to zang organs, fu organs, representing in two levels
* The study drug
“Trung phong hoan” is a remedy derivered from the ancientVietnamese remedy namely ”Thần dược cứu mệnh”, in which the driedmandarin peel is added to “Trung phong hoan” is mentioned in thebook 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, limbspasms, aphasia, hemiplegia 20 patients with stroke in MilitaryInstitute of Traditional Medicine were also administered by “Trungphong hoan” in the fluid extract form Initial results showed that theremedy had positive effects in motor functional recovery using the NIHSSand 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 therecommendation of patients with cerebral infarction stroke’ attitude and
Trang 5there 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 VietnamMilitary 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 MilitaryMedical Hospital, Military Medical University in the period from 6 - 2016 to6-2017
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 administeredsingle 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 wasrecorded at 72 hours interval The LD50 was calculated by using themethod of Litchfield- Wilcoxon
The semichronic toxicity of “Trung phong hoan” in mice wasstudied base on guidance of the Ministry of Health and OECD, WHO.Thirty mice were divided randomly into three groups of 10 The mice ingroup 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 of7.14g/kg (equivalent to human dose) The mice in group 2 had access togranulated medicine in the name of “Trung phong hoan” in oral dose of21.42g/kg/day (equivalent to 3 times the human dose) All mice wereadministered distilled water or study drugs in 8 weeks continuously, onetime 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
Trang 6- 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 asuitable 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 radicalsformed caused oxidation and injury vascular endothelial cells located atthe position of irradiation and then activated platelet aggregationmechanism, 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 formblood clots, which leads to stroke Motor measurement results andmotor decline points of this group were used to compare with the results
of mice with stroke
- Non-treatment induced group (n = 10): Movement of induced mice were evaluated at day 1, 3, 7, 14 after stroke inducing andthey were examined the brain specimens at day 3, 7, 14 after strokeinducing
stroke Strokestroke induced Cebrex group (n = 10): Mice were administered orallyCebrex in dose of 38.4mg/kg weight/day after stroke inducing inducing.Movement of stroke-induced mice were evaluated at day 1, 3, 7, 14after stroke inducing and they were examined the brain specimens atday 3, 7, 14 after stroke inducing
- Stroke-induced group treated with TPH (n = 10): Mice wereadministered orally TPH after stroke inducing inducing Movement ofstroke-induced mice were evaluated at day 1, 3, 7, 14 after strokeinducing and they were examined the brain specimens at day 3, 7, 14after stroke inducing
* Evaluation criteria
- Evaluate the level of decline in motor: Mice of post-stroke inducinggroups were evaluated the level of decline in motor at day 1, 3, 7 and 14after surgery The group that was not induced stroke were evaluatedonce 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 strokeinducing
Trang 7* 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 indicatesinfarction
* Study place and study time: Experimental testing and effectiveness
evaluation of the remedy in the name of “Trung phong hoan” in micemodel was performed at the Department of Pathophysiology – MilitaryMedical University in the period from 25 November, 2015 to 30December, 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, 103Military 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/ 3times daily
Maintenance of patients’ life using the ABC rule:
Ensure breathing and circulation are adequate to deliver oxygen tothe body (heart rate adjustment, using blood pressure medications toreduce the blood pressure if it is higher than 180/100 mmHg and toincrease if it is lower than 100/60 mmHg)
Using Manitol to reduce cerebral edema; antiplatelet drug like Aspirin100mg orally, once per day after the breakfast; neuroprotective andnutritional 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 themorning/afternoon
* Adjusting water and electrolyte balance
b) Follow up the patients’ conditions
- Patients’ conditions were followed up and evaluated All study criteriawere recored in the medical file at day 1 and 15
- Side effects at patient clinic were monitored daily and continuouslyduring the study time
Trang 8c) Monitoring evaluation criteria
Clinical monitoring and evaluation criteria
Clinical criteria according to modern medicine: Participants wereneurological examined, monitored and evaluated at day 1 and day 15.Clinical criteria were assessed by Glasgow score, MRC scale, andNIHSS (Appendix 5, 6, 7)
b Approaches to Criteria in traditional medicine: Participants were
examined and evaluated by quadrangular Syndrome Differentiation isbased on the eight diagnostic principles Diseases are differentiated viafu-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, hemoglobintest; 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 organwere monitered
- Paraclinic testal: Changes in hematopoiesis function, liver function andkidney 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, proofingits safety and positive effects, this study was approved to humanvolunteer testing by the Military Institute of Traditional MedicineCouncil for Science and Medical Ethics Committee of 103 MilitaryMedical Hospital
Trang 9CHAPTER 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 wasdetected as well as no abnormal symtoms within 72 hours afteradministering 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 innormal activity and agile They also had nice eyes, silky fur, and drystool No abnormal characteristics in all 3 lots were found during thestudy 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 incomparison with before the test There was no statisticallysignificant difference with respect to the degree of change in micebody 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 afteradministering 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 andALT activity in mice blood of both study group 1 and study group 2 incomparison with the control group and comparison between before, afteradministering 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 creatinineconcentration in mice blood of both study group 1 and study group 2 incomparison 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 (boththe control group and two study groups)
Trang 103.1.2.7 Microscopic morphology of liver and kidney
Microscopic morphology of livers:
+ Control group: Normal liver image characterized by hepatic cellsarranges radically in normal The central veins of liver and liversinusoid were mildly congestive Portal tracts were thin, some areaswere 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 Portaltracts were thin and fibers were found scattered through them
+ Study group 2: Normal liver image- characterized by hepatic cellsarranges radically The central veins of liver and liver sinusoid weremildly congestive Portal tracts were thin, lymphocytes and fibers werefound scattered through them
Microscopic morphology of kidneys:
+ Control group: Normal kidney image with enlargement of theglomerulus, blood vessels were congestive which were consisted oferythrocytes inside, the Bowman capsules were enlarged and contained clearliquid Blood vessels of the renal interstitium were mildly congestive.+ Study group 1: Normal kidney image with enlargement of theglomerulus, blood vessels were congestive which were consisted oferythrocytes inside, the Bowman capsules were enlarged and containedclear liquid Blood vessels of the renal interstitium were mildly congestive.+ Study group 2: Normal kidney image with enlargement of theglomerulus, the Bowman capsules were enlarged and contained clearliquid Tubule epithelial cells had normal morphology Blood vessels ofthe renal interstitium were mildly congestive
3.1.3 Evaluation results of positive effects of “Trung phong hoan”
in both periods of 4 minutes and 6 minutes
Trang 11Fig 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 3days after laser irradiation Results show that in groups in which mice wereradiated 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 6minutes, injuries were found in all mice
Fig 2: Mice brain imaging at day 3 after inducing ischemic stroke
Trang 12a 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 laserirradiation groups for 4 minutes and 6 minutes Results show that there was
no statistically significant difference with respect to the volume of bloodclots between groups
Table 1: The percentage of success of the method of inducing stroke
by photochemical method
The volume of blood clots was also was measured by method described inthe study method part Results of the volume of blood clots (mm3; 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 therewas no statistically significant difference at the time study
Fig 1: Ischemic stroke volume was measured at day 3, 7 and 14
after stroke inducing