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Nghiên cứu độc tính và hiệu quả điều trị vết thương phần mềm của cao lỏng LT tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING

MINISTRY OF DEFENSE

MILITARY INSTITUTE OF TRADITIONAL MEDICINE

TRUONG MINH TUAN

STUDY THE TOXICITY AND EFFICIENCY
OF LT EXTRACT ON WOUND
Speciality: Traditional medicine
Code: 62720201

DOCTOR THESIS ABSTRACT

HANOI – 2019


THIS STUDY WAS COMPLETED AT
THE MILITARY INSTITUTE OF TRADITIONAL MEDICINE


Science instructors:
Professor, Medicine Doctor NGUYEN MINH HA
Associate Professor, Medicine Doctor DINH VAN HAN

Reviewer 1: Assoc Prof, PhD Le Luong Dong
Reviewer 2: Assoc Prof, PhD Pham Dang Ninh
Reviewer 3: Assoc Prof, PhD Nguyen Tran Thi Giang Huong

The thesis is protected by The National Council at The
Military Institute of Traditional Medicine
At (time)

date

month

2019

YOU CAN READ THIS THESIS IN:
-

National Library
Military Institute of Traditional Medicine Library


3
INTRODUCTION
1. Reason for choosing this subject
In daily life, wound always account for a significant
proportion due to traffic accidents, labor accidents and domestic
accidents. The good management of wounds will be the basis for
treating other wounds such as bone, nerve and blood vessel wounds.
In addition, along with the development of the socio-economy, the
average life expectancy of people has improved markedly, thereby
increasing the chronic diseases, leading to more and more chronic
wounds.
In addition, the situation of antibiotic resistance is becoming
more and more serious and concerned by many scientists, the study
of a new antibacterial drug is very necessary, in which antibacterial
drugs of herbal origin are be concerned.
LT extract is produced from melaleuca trees for the purpose of
treating acute and chronic wounds, with a simple production process,
a rich supply of medicinal herbs can be an ideal solution to meet the
above requirements.
2. Objectives of thesis
1- Research on acute toxicity and subchronic toxicity of LT
extract on experimental animals.
2- Evaluate the effectiveness of LT extract in the treatment of
wounds on experimental and clinical.
3. Scientific and practical significances
Demonstrate the safety of LT extract.
Given the hypothesis of the mechanism of action of LT extract
according to Traditional Medicine and Modern Medicine in the
treatment of wounds. Provide a simple, easy to produce form of
medicine, can be widely applied in isolated conditions due to war or
natural disasters.
The topic has successfully applied experimental wound
research model on experimental animals as a basis for similar
studies.


4

4. The structure of the thesis
Our thesis consists of 133 pages: Introduction (2 pages);
Background (33 pages); Method (24 pages); Results (39 pages);
Discussion (32 pages); Conclusion (2 page); Request (1 page). There
are 158 references (Vietnamese: 40, English: 99, Chinese: 18,
spanish: 1); 51 tables, 4 diagram, 2 chart, 59 figures, 15 appendixes
in the thesis.
Chapter 1: BACKGROUND
1.1. Modern medical concept about wounds
1.1.1. Wound
Wounds are defined as the disruption of the continuity of bodycovered tissues, often related to the loss of organization. Wounds are
classified into 3 main categories: Mechanical wounds, thermal and
chemical wounds, ulcers.
1.1.2. Physiological of wound healing
The normal course of the wound healing process takes place in a
sequence of 3 successive separate stages, but there are interwoven
phases between stages: hemostasis / inflammation, proliferation and
remodeling.

Diagram 1.1: The process of wound healing
1.1.3. Methods for treating wounds
In the treatment of wounds, we must pay attention to two
problems, systemic treatment and topical treatment. In which the
systemic treatment is to ensure the conditions that promote the


5
healing process, eliminate the risk factors of slow wound healing.
Topical treatment goal is to clean the wound, remove dead tissue and
necrosis, excessive secretions, which can cause infection as well as
hinder the growth of granulation tissue and epithelium. The main
methods of wound treatment include: Surgical, Hyperbaric Oxygen
Therapy, VAC, growth factors, laser.
1.2. Traditional medicine treating wounds
Traditional medicine considers that factors affecting the healing
process mainly include “qi zhi xue yu”(blood stasis), “ying qi bu
cong”(stagnation of ying-qi), “jing luo zu zhi”( blockage of channel),
function of viscera are not completed, these factors cause “du re”(
toxic heat), necrotic tissues, stasis, deficiency, they interact with each
other interfering with the healing process. Thereby the treatment can
be reduced to 4 main methods: clearing heat, resolve stasis, removing
necrotic tissues, growth of new skin. These methods mainly use
external drugs, so clinically often add strengthen spleen of drugs to
increase the effectiveness of treatment.
1.3. Some experimental wound models
There are many models proposed to study wounds healing on
experiment, such as the model of causing the whole skin loss, the
model causing burn injury, the model causing chronic wound, model
on regenerated skin from tissue culture.
1.4. Application of melaleuca in treating wounds
Melaleuca cajuputi as well as other melaleuca species, contain
many ingredients that have been shown to be effective in treating
wounds. The process of extracting Melaleuca leaves will yield 2
main products:
* Essential oil: The main ingredient is 1,8-Cineol and 4Terpinenol, has antibacterial and anti-inflammatory effects.
* Melaleuca extract: There are Flavonoid, Ellagic acid,
Anthraquinone, all have antibacterial and anti-inflammatory effect.


6
LT extract consists of two main parts, essential oils and extracts,
from which we assess that LT extract also has antibacterial and antiinflammatory effects.
Chapter 2: MATERIALS, SUBJECTS AND METHODS
2.1. Material and research equipment
- LT extract: LT extracted from Melaleuca cajuputi leaves
collected in Phu Quoc, bottled 240ml, the ratio of 1: 1 (1g
medicinal / 1ml LT extract). Standard facility.
- 1% Sulphadiazine silver cream (Silver sulfadiazine)
produced by Hue Central Pharmaceutical Company for use as a
control drug.
2.2. Research subjects
Study on acute, subchronic dermal toxicity and experimental
wound models in rabbits: 138 white rabbits of Newzealand strain
both healthy breeds, weighing 1.8-2.2kg, by the Center Breeding
Testing Institute provides.
Study on antimicrobial susceptibility testing: Using strains of
bacteria Pseudomonas aeruginosa, Staphylococus aureus and
Escherichia coli.
Clinical study: 83 inpatients with acute and chronic wounds
treated at Military Institute of Traditional Medicine and the National
Burns Hospital.
2.3. Research Methods
2.3.1. Study acute and subchronic dermal toxicity of LT extract
Study acute dermal toxicity according to OECD guidelines
and Ministry of Health. Acute experimental wounds toxicity under
the guidance of the Chinese Ministry of Health. Study subchronic
dermal toxicity according to OECD guidelines and Lori O. Lim.
2.3.2. Evaluate the effectiveness of LT extract on experimental
and clinical
2.3.2.1. The effectiveness of LT extract in experimental wound
treatment
* Antimicrobial susceptibility testing


7
According to the method of Vanden Bergher and Vliet link
* Effect of treating experimental wounds:
Causing experimental wounds: Refer to the guidance of
methods to model experimental wounds of Duong Thuy (2003) and
of the Department of Labo to study and apply the treatment of burn
wounds - National Burn Hospital. treatment for 14 days.
Each rabbit has 2 experimental wounds, one wound is treated
with LT extract (zone A1 and A2: research wound), the other wound
will be used as a control group (control wounds).
Control wounds (30 rabbits) divide the group as follows:
Group 1 (Zone B1, n = 10): treated with NaCl 0.9%.
Group 2 (Zone B2, n = 20): Apply 1% Sulphadiazine silver.
Evaluate the effectiveness of treatment: Monitor general
condition, weight, serum biochemistry test and hematology tests.
Evaluate the histopathological and microbiology of wound.
2.3.2.2. Evaluate the effect of wound treatment of LT extract in
clinical
clinical trial, longitudinal and cross-sectional study. The data
were analyzed at pre-treatment times, after 7 days and after 14 days.
* Treatment according to grouping
17 inpatients with acute wounds randomly divided into 2
groups, research group 9 patients, control group 8 patients. Similarly,
66 inpatients with chronic wounds were also randomized to 2 groups,
research group 33 inpatients, control group 33 inpatients.
- Research group: treatment with LT extract ratio 1: 4.
- Control group: treatment with silver sulphadiazine 1%
Wounds are dressing once a day for 14 days.
* Evaluate treatment results
- Monitor systemic conditions, serum biochemistry test and
hematology tests.
- Compare treatment results between 2 groups based on the
criteria of changing wound area, wound healing speed, RESVECH
2.0 scale. Microbiological testing, histopathological and
ultrastructure testing.
2.3.3. Data processing


8
Research results are synthesized and processed by Microsoft
Excel 2010 and SPSS 20.0. Compare parameter variables by t-test,
non-parameter variables by Fisher test, χ2 test.
2.3.4. Medical ethics in research
Providing sufficient information related to the study, patients are
free of drugs and tests. To be considered by the Scientific Council of
the Military Institute of Traditional Medicine and the National Burns
Hospital Le Huu Trac in terms of ethics and acceptance.
Chapter 3: RESULTS
3.1. acute and subchronic dermal toxicity in experimental rabbits
- Acute dermal toxicity and skin irritation: Both in
dermatological and experimental wound studies, rabbits developed
normally. Because there are no dead rabbits, LD 50 of LT extract has
not been determined with the highest dose of 1500mg / kg of weight
on the skin pathway.
- Subchronic dermal toxicity: After 4 weeks and 8 weeks, the
rabbit developed normally, there was no special expression. The
function of hematopoietic function and liver and kidney function
were not abnormal. The resulting anapathology of the liver, kidneys
and skin does not detect damage.
3.2. The effect of LT extract in wound treatment on experimental
and clinical
3.2.1. The effect of LT extract in wound treatment on
experimental
3.2.1.1. Antimicrobial susceptibility testing
At 1/4 ratio, LT extract has bactericidal effect on S. aureus and
P. aeruginosa, has bacteriostatic effect on E. coli.
Table 3.10. Antibacterial effect of LT extract
S. aureus
P. aeruginosa

LT1
1/8
1/4

MBC
LT2
1/16
1/4

LT3
1/8
1/4

LT1
1/16
1/4

MIC50
LT2
1/32
1/4

LT3
1/16
1/8

E. coli

1/2

1/2

1/2

1/4

1/4

1/4

Bacterial strain


9

3.2.1.2. Effect of experimental wound treatment
* Experimental wound development
Table 3.15. Speed of wound healing in rabbits
Speed of wound healing (cm2/day)
Day

zone A1
(LT extract)
(n=10)

D0-D7

2,31±0,14

2,17±0,13

2,25±0,34

2,31±0,32

D7-D14

0,57±0,045

0,52±0,052

0,55±0,11

0,57±0,10

D0-D14

1,44±0,078

1,34±0,075

1,4±0,17

1,44±0,16

p

zone B1
zone A2
zone B2
(NaCl 0,9%) (LT extract) (Sulphadiazin
(n=10)
(n=20)
silver) (n=20)
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,01
pA2-B2>0,05
pA1-A2>0,05

The Speed of wound healing in zone A1 is faster than B1 (p
<0.05), zone A2 is equivalent to B2 (p> 0.05).
* Experimental wound bacteria
Table 3.17. Rate of Microbiological testing positive of wounds
zone A1
(LT extract)
(n=10)

zone B1
(NaCl 0,9%)
(n=10)

zone A2
(LT extract)
(n=20)

zone B2
(Sulphadiazin
silver) (n=20)

amount

%

amoun
t

%

amount

%

amount

7

70

6

60,
0

17

85

18

D7

2

20

7

70,
0

6

30

5

D14

1

10

6

60,
0

2

10

1

p0-14

<0,00
1

Day

D0

>0,05

<0,00
1

p

%
pA1-B1>0,05

90,0 pA2-B2>0,05

pA1-A2>0,05
pA1-B1<0,05
25,0 pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
5,0 pA2-B2>0,05
pA1-A2>0,05

<0,00
1

The rate of Microbiological testing positive of wounds
decreased in 3 zones A1, A2 and B2 (p <0.001). At D7 and D14, the


10
rate of Microbiological testing positive of wounds of zone A1 was
smaller than B1 (p <0.01), zone A2 was equivalent to B2 (p> 0.05).
Table 3.18. Density of bacteria on wound
Density of bacteria on wound (bacteria /cm2)
zone A1
(LT extract)
(n=10)

zone B1
(NaCl 0,9%)
(n=10)

zone A2
(LT extract)
(n=20)

D0

722,86±293,2
8

705,0±303,65

pA1-B1>0,05
745,59±298,1
711,94±247,23 pA2-B2>0,05
9
pA1-A2>0,05

D7

402,5±233,04

541,25±181,77

435,0±272,79

D14

90,0±54,08

381,79±128,36

92,5±53,46

Day

p

zone B2
(Sulphadiazin
silver) (n=20)

p

pA1-B1<0,005

455,0±282,31 pA2-B2>0,05
102,5±60,48

pA1-A2>0,05
pA1-B1<0,001
pA2-B2>0,05
pA1-A2>0,05

p0-7<0,001; p7-14<0,001; p0-14 <0,001 p0-7<0,001; p7-14<0,001; p0-14 <0,001

The local bacterial density in zones was significantly reduced
compared to before treatment (p <0.001). At D7 and D14, the zone
A1 bacteria density is lower than the B1 (p <0.005), zone A2 is
equivalent to B2 (p> 0.05).
Table 3.19. Distribution of bacteria in the wound on rabbits
Day

D0

Bacterial
strain
S.aureus
E.coli
K. pneumoniae

D7

S.aureus
E.coli
K. pneumoniae

D14

S.aureus
E.coli
K. pneumoniae

zone A1
(LT extract)
(n=10)
amount
%
2
20
2
20
3
30
0
0
1
10
1
10
0
0
1
10
0
0

zone B1
(NaCl 0,9%)
(n=10)
amount
%
3
30
1
10
2
20
5
50
4
40
3
30
5
50
4
40
4
40

zone A2
(LT extract)
(n=20)
amount
%
7
35
2
10
8
40
1
5
2
10
3
15
0
0
1
5
1
5

zone B2
(Sulphadiazin
silver) (n=20)
amount
%
10
50
3
15
5
25
2
10
3
15
1
5
0
0
1
5
1
5

At the D7, the rate of Microbiological testing positive of S.
aureus in A1 area was significantly reduced compared to B1 (p
<0.05), area A2 was equivalent to B2 (p> 0.05). At D14, the rate of
Microbiological testing positive of S. aureus and K. pneumoniae in


11
A1 area was significantly reduced compared to B1 (p <0.05), area A2
is equivalent to B2 (p > 0.05).
* Change the morphological test of the wound
Table 3.20. Change the number of neovascular at the wound
number of neovascular / Unit area
p
zone
A1
zone
B1
zone
A2
zone
B2
Day
(LT extract)
(n=10)

(NaCl 0,9%)
(n=10)

(LT extract)
(n=20)

(Sulphadiazin
silver) (n=20)

D0

1,7±0,67

1,8±0,42

1,8±0,89

1,9±1,02

D7

4,7±1,57

3,3±1,16

4,75±1,33

4,65±1,23

D14

5,1±1,66

4,9±1,73

5,0±1,21

5,15±1,18

p

p0-7 <0,001;p7-14 < 0,05;p0-14 <0,001

pA1-B1>0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1>0,05
pA2-B2>0,05
pA1-A2>0,05

p0-7 <0,001;p7-14 < 0,05;p0-14 <0,001

All regions increase the number of neovascular. At D7, the
number of neovascular in A1 is higher than that of B1 (p <0.05), area
A2 is equivalent to B2 (p> 0.05). At D14, the number of neovascular
in A1 region compared with B1 and A2 compared with B2 is similar
(p> 0.05).
Table 3.21. Change the number of inflammatory cells at the
wound in the rabbit
number of inflammatory cells / Unit area
zone
A1
zone B1
zone A2
zone B2
Day
p
(LT extract)
(n=10)

(NaCl 0,9%)
(n=10)

(LT extract)
(n=20)

(Sulphadiazin
silver) (n=20)

D0

48,7±6,5

49,2±6,78

49,75±7,69

50,35±8,31

D7

28,9±4,72

38,0±7,8

27,45±4,25

26,85±3,66

D14

14,6±6,19

27,2±8,36

13,75±3,86

14,15±3,13

p

p0-7<0,05;p7-14<0,05;p0-14<0,05

pA1-B1>0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05

p0-7<0,05;p7-14<0,05;p0-14<0,05

Regions reduce the number of inflammatory cells. At D7
and D14, the number of cells in zone A1 was less than in B1 (p
<0.05), zone A2 was equivalent to B2 (p> 0.05).


12
Table 3.22. Change the number of fibroblasts at the wound
Change the number of fibroblasts / Unit area
zone
A1
zone B1
zone A2
zone B2
Day
(LT extract)
(n=10)

(NaCl 0,9%)
(n=10)

(LT extract)
(n=20)

(Sulphadiazin
silver) (n=20)

D0

10,5±4,79

10,2±3,82

10,9±4,6

10,3±3,63

D7

48,4±6,17

41,9±6,33

45,5±8,4

46,95±8,9
3

D14

52,8±6,63

44,3±9,62

51,9±7,72

55,2±9,37

p

p0-7<0,05;p7-14<0,05;p0-14<0,05

p

pA1-B1>0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05
pA1-B1<0,05
pA2-B2>0,05
pA1-A2>0,05

p0-7<0,05;p7-14<0,05;p0-14<0,05

The regions increase the number of fibroblasts. At D7 and D14,
the number of region A1 fibroblasts was greater than B1 (p <0.01),
area A2 was equivalent to B2 (p> 0.05).
3.2.2. Effect of wound treatment of LT extract in clinical
3.2.2.1. Effects of LT extract in treating some patients with
acute wounds
* Local development of acute wound
Table 3.28. Modified acute wound size
Group

n

Research
group

9

Control group 8
p

D0
31,07±13,7
5
38,79±18,3
7
p>0,05

wound size (cm2)
D7

D14

24,62±11,66

18,32±9,64

30,6±13,68
p>0,05

22,94±10,2
1
p>0,05

p
p0-7,p7-14,p0-14 < 0,001
p0-7,p7-14,p0-14 < 0,001

The size of the wound in both groups gradually decreased at the
time of the study (p <0.001). Comparison between the 2 groups at
the same time was not statistically significant (p> 0.05).
Table 3.30. RESVECH 2.0 score in the acute wound group
Criteria
Size

Research group (n=9)
Control group (n=8)
D0
D7
D14
D0
D7
D14
3,22±0,79 2,89±0,87 2,44±0,68 3,5±0,87 3,13±0,6 3,0±0,5

p
p0-7<0,01


13
Depth
Edges
Exudate
Bed
Infection
Total
p

3,11±0,74 2,44±0,68
2,56±0,5 2,11±0,57
1,44±0,83 0,89±0,87
3,11±0,87 2,33±0,67
4,11±1,37 2,22±1,31
17,56±3,86 12,89±3,78

1,89±0,57 3,38±0,86 2,75±0,66
1,67±0,47 2,5±0,5 2,38±0,7
0,56±0,68 1,38±0,7 1,13±0,6
1,78±0,63 2,5±0,71 2,25±0,66
1,0±0,82 4,13±1,05 2,86±1,17
9,33±2,4 17,38±2,96 14,5±2,96
Presearch-control >0,05

2,38±0,7
1,75±0,43
0,63±0,48 p7-14<0,01
1,75±0,43 p0-14<0,01
1,5±1,22
11,0±2,55

Compare before and after in the same group all of p<0.01
Compare indicators at the same time between 2 groups p> 0.05
* Microbiological testing in acute wound
Table 3.33. Average bacterial density in acute wound group
Average bacterial density (bacterial /cm2)
Research group (n=9)
Control group (n=8)

Day
D0
D14
p

601,67±444,72
222,78±210,35
<0,05

680,63±504,04
204,38±263,72
<0,05

p
>0,05
>0,05

The local bacterial density of wound between 2 groups at the
same time was similar (p> 0.05). Compare before and after in the
same group was statistically significant (p <0.05).
3.2.2.2. Effects on chronic wounds
* Occurrence of chronic wound
Table 3.40. Resize in chronic wound groups
Size of wound (cm2)
p
Group

n

D0
D7
D14
Research group 33 36,21±26,68 30,22±19,9 24,41±16,41
Control group 33 38,9±21,86 33,28±18,6 28,13±16,29
p
>0,05
>0,05
>0,05

p0-7; p7-14; p0-14< 0,001
p0-7; p7-14; p0-14< 0,001

Comparing wound size in the same group at the study time was
statistically significant (p <0.001). Comparing at the same time
between the two groups was not statistically significant (p> 0.05).
Table 3.41. RESVECH 2.0 scale of chronic wound group
Criteri
a

Size

Research group (n=33)

Control group (n=33)

D0
D7
D14
D0
D7
D14
3,33±0,88 3,12±0,88 2,85±0,86 3,48±0,96 3,27±0,93 3,03±0,97

p


14
Depth

3,12±0,54 2,85±0,56 2,36±0,59 3,18±0,63 2,67±0,8 2,18±0,83

Edges

2,64±0,59 2,39±0,6

Exudate 1,45±0,56
Bed

1±0,6

1,94±0,6 2,55±0,61 2,3±0,58 1,85±0,66
0,36±0,48 1,42±0,7 0,91±0,62 0,48±0,56 Presearch-control
>0,05

2,88±0,59 2,39±0,6 1,97±0,46 2,94±0,69 2,67±0,72 1,94±0,78

Infection 3,15±1,28 2,12±1,15 1,18±1,14 3,48±1,23 2,48±1,16 1,33±1,01
Total
p

16,58±3,43 13,88±3,05 10,67±2,67 17,06±3,75 14,3±3,59 10,82±3,46
p0-7<0,01, p7-14<0,01, p0-14<0,01
p0-7<0,01, p7-14<0,01, p0-14<0,01

The previous comparison after the indicators in the same group
are statistically significant (p <0.01). Comparing the indicators at the
same time between two groups, the difference was not statistically
significant (p> 0.05).
* Microbiology of chronic wound
Table 3.43. Distribution according to the strain of infection in
chronic wound group
Day

D0

D14

Bacterial strain
S.aureus 1
E.coli 2
P.aeruginosa 3
Enter. Faecalis 4
S.aureus a
E.coli b
P.aeruginosa c
Enter.faecalis d

Research group
(n=33)

p

amount

%

amount

%

17
7
10
1
7
5
4
1

51,52
21,21
30,30
3,03
21,21
15,15
12,12
3,03

16
7
8
2
7
6
2
1

48,49
21,21
24,24
6,06
21,21
18,18
6,06
3,03

p1-a<0,05, p2-b>0,05
p3-c<0,05, p4-d>0,05

p

Control group (n=33)

>0,05
>0,05
>0,05
>0,05
>0,05

p1-a<0,05, p2-b>0,05
p3-c<0,05, p4-d>0,05

The distribution of bacteria at the same time between two
groups is similar (p> 0.05). Comparison before and after density of
S.aureus and P.aeruginosa in both groups decreased (p <0.05).
Table 3.44. Positive infection rate in chronic wound group
Day
D0

Research group (n=33)

Control group (n=33)

amount

%

amount

%

31

93,94

30

90,91

Presearch-control

>0,05


15
D14
p0-14

17

51,52
<0,001

16

48,49

>0,05

<0,001

The comparison between the 2 groups at the same time was not
statistically significant (p>0.05). The previous and after comparison
in the same group was statistically significant (p <0.001).
Table 3.45. Local bacterial density in chronic wound groups
Local bacterial density (bacterial /cm2)
Day

Research group (n=33)

Control group (n=33)

P

D0
D14
p

889,24±499,45
317,58±388,98
p0-14 <0,001

960,91±508,85
294,09±387,89
p0-14 <0,001

>0,05
>0,05

At the same time, the bacterial density between the two groups
is equivalent (p> 0.05). Compared before and after in the same
group, the bacterial density in both groups decreased significantly (p
<0.001).
* Change the morphology of chronic wounds
Change on histopathology
Before treatment: The lesions in both groups were similar,
including thick epidermis, degenerate epidermal cells, basal ulcers,
fibrous connective tissue, mononucleosis infiltrates, intact Sparse,
dark, fibrous cells. Rare blood vessels.

Research group, LT02-1, HEx400 Control group, LT15-1, HEx400
Figure 3.19. The bed of the ulcer is the degenerate connective
tissue (1), mononucleosis infiltration (2), the cells degenerate, the
dark cytoplasm (3).
After treatment: the bed of the ulcer has sparse connective
tissue, increasing the number of fibroblasts, proliferating
neovascular.


16

Research group, LT07-2, HE x400

Control group, LT16-2, HE x400

Figure 3.22. Bed of ulcer after treatment. Increased fibroblast
penetration (1), neovascular (2)
Compared to the pre-treatment samples, the after of were
cleaner, without or less fibrin, without detecting necrotic tissue. The
majority of the ulcers have thin connective tissue, do not form strips.
Most of the samples showed an increase in fibroblast count, increase
neovascular.
Change on ultrastructure testing
Before treatment: The major injuries include:
+ On TEM: The main characteristics include sparse extracellular
matrix, fragmented collagen fibers, swelling, disintegration,
penetration of inflammatory cells, macrophages, degenerative
fibroblasts inactivation.

Research group, LT01-1, TEM x 1500

Control group, LT09-1, TEMx1500

Figure 3.24. Collagen fragments dissolve on the sparse of
extracellular matrix (1). Penetration of neutrophil (2).
+ On SEM: The surface is not flat, there is no epidermis on the
surface. There are many different cells, including alternating red
blood cells and leukocytes, mainly white blood cells of lymphocytes.


17

Research group, LT06-1, SEMx1500

Control group, LT10-1, SEMx1500

Figure 3.26. On the surface of wound, there are many red blood
cells (1), white blood cells (2).
After treatment: The major injuries include:
+ On TEM: Most samples show decreased inflammation.
Immobilized fibroblasts, increased activity, increased collagen
synthesis.

Research group, LT03-2, TEM x 1000

Control group, LT15-2, TEMx1000

Figure 3.30. Bed of ulcer. Increased activity of fibroblasts (2),
neovascular (3), monocytes (1).
+ On SEM:
The surface is uneven, there is no epidermis on the surface, no
feathers and hairs. Appears more collagen on the surface and many
neovascular.


18

Research group, LT06-2, SEMx1500

Control group, LT09-2, SEMx1500

Figure 3.32. Image of collagen proliferation on the bed of
ulcers after treatment.
TEM and SEM ultrastructure tests in both groups showed
positive changes after 14 days of treatment, reduced inflammation,
proliferative process occurred, many fibroblasts appeared, collagen,
neovascular.
Chapter 4: DISCUSSION
4.1. Acute dermal toxicity, skin irritation and subchronic toxicity
of LT extract
4.1.1. Acute dermal toxicity and skin irritation of LT extract
We conducted acute toxicity study both on healing skin and on
experimental skin loss wounds.
Regarding the selection of the initial dose, it should be selected
based on the information related to dermal toxicity of similar
substances, if there is no information as a basis for determining the
dose, a dose of 200mg / kg to start dose. LT extract is a completely
new product of melaleuca leaves, no similar preparations yet, so we
have no information as a basis for determining the dose. The basis
for us to choose the main dose due to the maximum rate of drug can
reach 1: 1, 10% of the area of rabbit body needs 1.5ml / kg,
equivalent to the dose of 1500mg / kg weight, we conducted a
secondary dilution from 1: 1 to 1:32, equivalent to a dose of
1500mg / kg - 46.9mg / kg weight. Other way, according to the
guidelines on OECD's superior skin irritation and corrosion test
(2002), it is only required to apply 0.5ml of reagent on rabbits with


19
an area of 6cm2, much smaller than our area. reagents that we use.
Thus our dosing range has included the recommended starting dose
when there is no relevant information, and is also high enough to be
able to reveal acute dermal toxicity and skin irritation.
The results showed that LT extract did not cause acute dermal
toxicity or local skin irritation at all test doses despite the highest rate
of 1: 1 equivalent to the dose of 1500mg / kg weight. Because there
are no dead rabbits, the lethal dose of 50% of animals (LD50) of LT
extract has not been determined.
4.1.2. Subchronic dermal toxicity of the LT extract
In order to have a basis to choose the dose used for toxicity
studies as well as in clinical studies, we had to study the
antimicrobial susceptibility testing in vitro test before, the results
showed that the LT extract rate 1:4 is the lowest rate that has the
effect of resistance to the bacterial strains used in the study, so we
have chosen this ratio as the expected rate of clinical use as well as
for subchronic toxicity studies. Other way, the LT extract at 1: 4 ratio
is 1.5ml / kg on our rabbits, which is equivalent to 375mg / kg, while
we expect only 3-6ml to be covered depending on the area of injury,
equivalent to the dose of 15-30mg / kg for people weighing about
50kg. So the dose we use for toxicology studies is higher than the
expected dose to be used in clinic, ensure disclosure of unwanted
effects.
After 8 weeks of dressing, no abnormal changes in the general
condition, blood function tests, liver cell damage, liver and kidney
function, and morphological tests of liver, kidney and skin were
detected.
In summary, the LT extract did not cause subchronic dermacl
toxicity in experimental animals despite the similar clinical rates (1:
4) and the highest ratio was 1: 1 equivalent to the dose of 1500mg /
kg weight for 8 weeks.
4.2. Discuss the effectiveness of LT extract on experimental and
clinical


20
4.2.1. Treatment effect on experiment
4.2.1.1. Antimicrobial effects tested in vitro
Through research results, it can be seen that LT extract has good
results with Gram (+) bacteria. This mechanism of action was
explained by Cowan MM (1999) because the membrane of Gram (+)
bacteria does not have lipopolysaccharide molecules to prevent the
penetration of fatty components such as essential oil, the
hydrophobic part of the essential oil has the ability to directly
combine with the double phospholipid layer of the cell membrane to
increase ionic permeability causing bilateral membrane metabolism
and osmosis of components in bacterial cells, or degrading bacterial
enzyme system.
Research results show that LT extract at 1/4 ratio has the effect
of bactericidal with S. aureus, P. aeruginosa and bacteriostatic effect
with E. coli.
4.2.1.2. The therapeutic effect of LT extract on experimental
animal wound models
After 14 days of treatment, in all 30 rabbits, no abnormal
changes in general condition, hematopoietic function tests, liver cell
damage, liver and kidney function were detected. This reaffirms the
results of the previously conducted toxicity study.
In terms of antibacterial effects, microbiological tests showed
that high-grade wound treatments of LT extract had a marked
reduction in both positive bacterial culture rates as well as local
bacterial density, the level of reduction equivalent to group used 1%
silver sulphadiazine and better than NaCl 0.9% group. The most
significant reduction was the subsequent infection with S. aureus and
K. pneumoniae, with E. coli, the reduction was less. This is entirely
consistent with Huynh Kim Dieu's comment (2011) when it is
assumed that Melaleuca trees have many different strains and these
strains have differences in antibacterial properties, but most of them
have strong impacts on S. aureus and its effect is weak on E. Coli.


21
Regarding anti-inflammatory, histopathology test results showed
that at D7 and D14, the group treated with LT extract had
inflammatory cell density lower than the NaCl 0.9% group (28.9 ±
4.72 compared to 38.0 ± 7.8 cell /unit area, 14.6 ± 6.19 compared
with 27.2 ± 8.36 cell/unit area), equivalent to the 1% silver
Sulphadiazine group (27.45 ± 4.25 compared with 26.85 ± 3.66
cell/unit area, 13.75 ± 3.86 compared with 14.15 ± 3.13 cell/unit
area). This effect can be explained by the fact that in LT extract
contains melaleuca oil, the anti-inflammatory properties of melaleuca
essential oil have been proved by many studies. The antiinflammatory mechanism is due in part to inhibiting the production
of mediators of inflammatory processes induced by
lipopolysaccharides such as TNF-α, IL-1β and IL-10 in monocyte
and peripheral blood monocytes. Prostaglandin E2, or it may also be
possible to inhibit the production of superoxyd in monocytes caused
by antigens but not for neutrophils. In addition, there are other active
ingredients that have anti-inflammatory effects such as flavonoids,
ellagic acid, anthraquinone.
Regarding the stimulating effect on the wound, from the results
of the study, the speed of wound healing in the group using LT
extract (1.44 ± 0.078 cm2 / day in A1 area and 1.4 ± 0.17 cm2 / day
in zone A2) and 1% silver sulphadiazine (1.44 ± 0.16 cm2 / day)
were similar and faster than the NaCl 0.9% group (1.34 ± 0.075cm2 /
day). histopathology test also showed that Lt extract group had
fibroblast proliferation, increasing the number of neovascular
equivalent to the 1% silver sulphadiazine group, better than the NaCl
0.9% group. This may be explained by the excessive inflammatory
response that inhibits the development of fibroblasts, when reduced
inflammation results in the development of fibroblasts to increase
division and increase migration to taste wounds. LT extract and silver
sulphadiazin both have anti-inflammatory effects, so the efficiency of
fibroblast proliferation in these groups will be better.


22
Summary, LT extract safe for wounds on rabbits. The drug has
anti-inflammatory and antibacterial effects, promoting wound
healing process. Good effect in wound treatment, treatment effect is
equivalent to 1% silver sulphadiazin.
4.2.2. Effects of wound treatment of LT extract in clinic
4.2.2.1. The therapeutic effect of LT extract for acute wounds
Regarding the percentage of narrowing of wound area, in the
treatment group of LT extract at D7 and D14, respectively 22.3 ±
5.28% and 43.71 ± 7.32%, while with the group treatment with 1%
silver sulfadiazine was 20.49 ± 9.68% and 38.63 ± 18.35%
respectively, the difference between the two groups was not
statistically significant (p> 0.05) ). Evaluation with RESVECH 2.0
scale also shows the similarity between 2 groups. Thus, the wounds
treated with LT extract has a level of healing equivalent to the
wounds treated with 1% silver sulfadiazine.
Microbiological testing also showed that the density of bacteria
in both groups was significantly reduced, the research group from
601.67 ± 444.72 bacteria / cm2 was 222.78 ± 210.35 bacteria / cm2 ,
the control group from 680.63 ± 504.04 bacteria / cm2 was 204.38 ±
263.72 bacteria / cm2, the level of reduction in both groups was
similar. This result demonstrated the antibacterial effect of LT extract
on acute wounds.
4.2.2.2. Effect of LT extract in chronic wounds
In our research, chronic wounds after 14-day treatment of ulcer
size decreased by an average of 34.01 ± 13.55% in the research
group, equivalent to the control group (30.24 ± 10.23 ), the
difference was not statistically significant (p> 0.05), this result was
much lower than the results of Luong Thi Ky Thuy's TG extract
(90.6% of patients reduced the ulcer size > 50%), this may be
because our treatment time (14 days) is much shorter than that of TG
extract (25.67 ± 14.34 days). Evaluation with RESVECH 2.0 also
showed the similarity between 2 groups, in which the LT extract
group decreased from 16.58 ± 3.43 to 10.67 ± 2.67, in the control


23
group decreased from 17, 06 ± 3.75 to 10.82 ± 3.46. In a study using
chronic antioxidants, Castro also used RESVECH 2.0 as a evaluation
tool, result, average RESVECH 2.0 score reduction 10.16 points,
much better than our result. However, in that study, the course of
treatment was 8 weeks, much longer than our treatment period only
14 days. Thus, it can be seen that the reduction level of RESVECH
2.0 in our study is quite good, showing the effect of LT extract.
Mustoe (2004) has shown that the presence of bacteria along
with the white blood cells they attract will interfere with the healing
process, the chronic wound will not healing without reducing the
number of bacteria. In our study, both the positive rate of wound
culture as well as the bacterial density in both groups decreased
markedly after treatment, in the study group decreased from 889.24 ±
499.45 bacteria/cm2 to 317.58 ± 388.98 bacteria/cm2; in the control
group decreased from 960.91 ± 508.85 bacteria/cm 2 to 294.09 ±
387.89 bacteria/cm2, showing the antibacterial effect of LT extract.
Histopathology and ultrastructure test also showed that most
wounds in both groups reduced inflammation, increased fibroblasts,
increased collagen synthesis, increased angiogenesis, these were all
important stages of wound healing, showing that the wound is
healing smoothly.
Through the above analysis, it can be seen that LT extract has
antibacterial and anti-inflammatory effects, according to traditional
medicine, antimicrobial and anti-inflammatory drugs are all in the
"qing re jie du" group of drugs, Since then we have identified that LT
extract also has the effect of "qing re jie du" when treated wound. On
the other hand, after the treatment process, the wounds were all
cleaner, more streaks had necrosis and pseudomembranous
organization, the wound bed was the organization of new red seeds,
by histopathology and ultrastructure test, can be seen LT axtract also
increased the number of fibroblasts and increased angiogenesis, so it
can be seen that from the perspective of traditional medicine, the LT
extract has the effect of "qu fu sheng ji".


24
In summary, LT extract has a good effect on acute and chronic
wounds, treatment wounds with LT extract have a speed of healing
equivalent to those of 1% silver sulphadiazine. Microbiological tests
and wound morphology also showed similarities between two
groups. According to traditional medicine, LT extract has shown the
effect of "qing re jie du", "qu fu sheng ji", helps the wound heal.
CONCLUSION
From the research results, we draw the following conclusions:
1. Acute and subchronic toxicity of LT extract
LT extract does not cause acute dermal toxicity and
experimental wound toxicity despite testing at the highest rate of 1: 1
(equivalent to a dose of 1500mg / kg of body weight). Because no
rabbits died, the LD50 for external use of administration has not been
determined.
LT extract did not cause subchronic dermal toxicity at 1: 4
(375mg / kg / day) and 1: 1 ratio (1500mg / kg / day) for 8 weeks.
Within 8 weeks of applying the drug, no abnormal changes in the
general condition were detected, liver enzyme activity tests, liver,
kidney and blood function tests. Microscopic examination of liver,
kidney and skin also found no signs of damage.
2. The effectiveness of LT extract in wound treatment
The effect of LT extract on experiment
Antibacterial effect of testing: At the ratio of 1: 4, LT extraxt has
bactericidal effect on S. aureus and P. aeruginosa, bacteriostatic
effect with E. coli after 24 hours of exposure.
In experimental wound model, LT extract does not cause any
undesirable effects on experimental rabbits after 14 days of
treatment.
Wounds treated with LT extract has a healing speed of 1.4 ±
0.17 cm2 / day, faster than 0.9% NaCl treatment (1.34 ± 0.075 cm 2 /
day) with p <0.05, and the equivalent of treatment with 1% silver
sulphadiazine (1.44 ± 0.16 cm2 / day) with p> 0.05.


25
LT extract wounds had a positive bacterial culture rate and local
bacterial density decreased significantly compared to 0.9% NaCl
wounds (p <0.05), the level of reduction was similar to silver
sulphadiazine group, with p> 0.05. Histology also showed a decrease
in inflammatory cells, an increase in the density of fibroblasts, an
increase in the density of neovascular in the LT extract group.
Clinical trials of wound treatment
The study process did not detect any undesirable effects of LT
extract, hematological tests and serum biochemistry test changes
were not statistically significant compared to before the study.
The group of acute wounds after 14 days of treatment with LT
extract, narrowing area was 43.71 ± 7.32%, equivalent to 1%
sulphadiazine silver group (38.63 ± 18.35%) . Bacterial density was
222.78 ± 210.35 bacterial / cm2, lower than before study (601.67 ±
444.72 bacterial / cm2) with p <0.05 and equivalent to 1% silver
sulphadiazine group ( 204.38 ± 263.72 bacterial / cm2) with p> 0.05.
The group of chronic wounds after 14 days of LT extract
treatment had a narrowing area was 34.01 ± 13.55%, equivalent to
the group of 1% sulphadiazine silver (30.24 ± 10.23%) with p> 0.05.
Bacterial density decreased from 889.24 ± 499.45 to 317.58 ± 388.98
bacterial / cm2, the level of reduction was equivalent to 1% silver
sulphadiazine. Histology and ultrastructure both show that the
healing process is going smoothly.

REQUEST
1. Continue to study the effects of treatment of acute and
chronic wounds on clinical with a larger sample size, especially with
complex acute wound groups.
2. Study the effect of treating wound of LT extract on patients
with burns and patients with fire wounds.


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