Nghiên cứu hiệu quả điều trị loét da mạn tính của cao TG

MINISTRY OF EDUCATION AND TRAINING

MINISTRY OF DEFENSE

MILITARY INSTITUTE OF TRADITIONAL MEDICINE

LUONG THI KY THUY

STUDY ON THE THERAPEUTIC EFFECTIVENESS
OF TG EXTRACT ON CHRONIC SKIN ULCERS
Speciality: Traditional medicine
Code: 62720201

DOCTOR THESIS ABSTRACT

HANOI – 2016


THIS STUDY WAS COMPLETED AT THE MILITARY
INSTITUTE OF TRADITIONAL MEDICINE

Science instructors:
Associate Professor, Medicine Doctor LE DINH ROANH
Associate Professor, Medicine Doctor PHAM VIET DU

Reviewer 1: Associate Professor, Medicine Doctor LE LUONG DONG
Reviewer2: Associate Professor, Medicine Doctor LE VAN DOAN
Reviewer3: Medicine Doctor PHAM THI VAN ANH

The thesis is protected by The National Council at The
Military Institute of Traditional Medicine

At (time)

date

month

2016

YOU CAN READ THIS THESIS IN:
-

National Library
Library of Central Medicine Information
Military Institute of Traditional Medicine Library


3
INTRODUCTION
1. Reason for choosing this subject
A chronic skin ulcer (CSU) is an injury in the form of a sore on the
skin or a mucous membrane. A CSU is defined as a wound lesion that
lasts more than four weeks without remarkable healing tendency or as a
frequently recurring wound. CSU seriously affects the quality of life of
patients and causes immense costs to the healthcare system. Although
the prevention and treatment of CSU has improved greatly, CSU still
remains a great challenge for modern medicine.
According to traditional medicine theories, CSU has various symptoms
such as local sores, chronic pain and itching, it is also prone to relapse.
Traditional medicine has been used for the prevention and treatment of CSU
for many years and it has now expanded and is used worldwide.

TG extract or chicken egg yolk oil which is mentioned in
“Miraculous Effects of Southern Medicine” (Tue Tinh, 14 th century) and
“Compendium of Medical Material” (Shizhen Li, 1518-1593), is used to
treat ulcers because of its functions: qing-re (clearing heat), antiinflammatory, pain relief, moisturizing and proliferation.
2. Objectives of thesis
1) Determine the chemical composition and safety of TG extract.
2) Evaluate the effects of TG extract on CSUs in vitro rabbits.
3) Evaluate the clinical therapeutic efficacy of TG extract on CSUs
stage II and III.
3. Scientific and practical significances
- Determine the basic components of TG extract.
- The results show that TG extract delivers effective treatment of
CSUs: increases fibroblast migration, proliferation and activity, boosts
the extracellular matrix (ECM) biosynthesis, increases angiogenesis and
epithelial proliferation, and decreases MMP9, reduced destruction of
the ECM, therefore CSUs heal faster.
- Give a hypothesis of action mechanism of TG extract in the
treatment of CSUs.
- Provide a simple remedy which is easy to produce and can be
used across-the-board in the treatment of CSUs.


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4. The structure of the thesis
Our thesis consists of 129 pages: Introduction (3 pages);
Background (33 pages); Method (25 pages); Results (37 pages);
Discussion (29 pages); Conclusion (1 page); Request (1 page). There
are 158 references (Vietnamese: 21, English: 119, Chinese: 18); 29
tables, 1 chart, 68 figures, 14 appendixes in the thesis.
Chapter 1: BACKGROUND

1.1. Skin histology
The most common structural components within the dermis is the
extracellular matrix (ECM). Understanding the structure and function of
the ECM is the key to successful treatment of CSUs.
1.2. Pathophysiology of acute wound healing
Acute wound repair process can be divided into 4 continuous and
overlapping phases: coagulation, inflammation, proliferation and ECM
remodeling.
1.3. Pathophysiology of chronic skin ulcers
CSUs do not heal in normal phases and time scales. Despite intensive
treatment, the wound is unable to close. The pathological disorders: (1)
Proliferation reduction; (2) Imbalance between enzymes for protein
hydrolysis and inhibitors; (3) The appearance of old and weak cells.
1.3.5. Treatment for chronic skin ulcers
Systemic therapy (according to etiology)
Topical treatment: debridement, anti-inflammatory, anti-infection,
maintaining moisture balance, biological agents, skin grafts and other
forms of treatment. However, CSUs which are still a challenge for
modern medicine, still don’t have a specific drug to treat them.
1.4. Traditional medicinal opinion on chronic skin ulcers
Traditional medicine considers that CSU belongs to the “ulcer”
branch of the Ulcer and Sores diseases.
In traditional medicine, the pathogenesis of CSU is theoretically
caused by “Re (heat)”. The pathogenic process of CSU was described
first in “Lingshu: yongju” as follows: “harmful cold accumulates in the
meridian and the results in a stiffness in blood flow and obstruction,
which inhibits the circulation of defensive energy, leading to


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inflammation. Subsequently, harmful cold changes into harmful heat,
which causes tissue damage and then pus formation.”
The pathologic mechanisms underlying the refractory skin ulcers
are that “long term disease results in a deficiency and stasis in both qi
and blood, leading to a disorder of ying (nutrition) and wei (immunity)
and skin dystrophy.” The “Xu (deficiency)” and “Yu (stasis)” are two
major pathologic factors of the development of CSUs.
Y. J. Wang and H. F. Que (2009) divided CSU into three syndrome
types of blood stagnation with dampness-heat, qi deficiency with blood
stasis and spleen deficiency with dampness encumbrance.
Treatment of a CSU follow the principle of “Qing-Hua-Bu” method
to treat for the syndrome differentiation: 1) Qing-re (clearing heat), zhishi (remove dampness), hua-yu (resolve stasis), sheng-ji (stimulate the
growth of new skin) to treat dampness-heat syndrome; 2)Tonify qi, huayu, sheng-ji to treat qi deficiency with blood stasis syndrome; 3) Jian-pi
(strengthen spleen), zhi-shi, sheng-jitotreatspleen deficiency with
dampness encumbrance syndrome; and the last target is “qu-fu-sheng-ji,
ji-ping-sheng-zhang” (removing necrotic tissues to stimulate the growth
of new skin).
1.5. Experimental chronic skin ulcer models
Rudolph R. (1979) introduced an animal model of Adriamycin
ulceration. More than 50 days (rats) and 45-65 days (rabbits) of
prolonged testing is suitable for experimental research.
1.6. TG extract
TG extract or chicken egg yolk oil mentioned in “Miraculous
effects of Vietnamese medicine – Ten therapies” (Tue Tinh, 14 th
century), can be used for treating ulcers, sores and burns. Chicken egg
yolk oil is made by a traditional heating method and has a sweet, sour,
warm, non-toxic characteristic. This remedy is useful for treating
venereal diseases, ulcers and burns.
Rastegar F. et al (2011) studied the effect of egg yolk oil in the healing
rd

of 3 degree burn wounds in rats which showed that burn wounds healed
faster and had abundant re-epithelialization without scarring.


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Chapter 2: SUBJECTS AND METHODS
2.1. Drugs and Chemistries
TG extract which was made in the Pharmacy Department of The
Military Institute of Traditional Medicine is a topical concentration of 6
grams of medicine per milliliter (Figure 2.1).
2.2. Subjects
Determine the chemical composition of TG extract: 6 samples were
taken at random from a batch of new products.
Determine the safety of TG extract.
Skin irritation: 03 New Zealand White rabbits.
Acute toxicity of subcutaneous and oral administrations: Swiss
white mice (National Institute of Hygiene and Epidemiology), at least
10 mice per batch.
Dermal acute toxicity: 36 New Zealand White rabbits.

Figure 2.1. TG extract packaged form
Dermal subchronic toxicity: 30 New Zealand White rabbits.
Experimental therapeutic effects: 30 New Zealand White rabbits.
Evaluate clinical therapeutic effects: 64 chronic skin ulcers stage II
and III of 59 inpatients and outpatients at Departments of Military
Central Hospital 108 (A1, A2, A6, A7, A10, A15, B1A, B1B, B1C)
from 10/2012 to 04/2015.
Included criteria: stage II and III chronic skin ulcers.
Excluded criteria: stage IV skin ulcers, syphilis sores, tuberculosis,
leprosy; patients with systemic immunosuppression, systemic disease,

mental illness, blood disease, life-threatening, patients not cooperating
or dropped out of the treatment.


7


8
2.3. Method
2.3.1. The chemical composition analysis and the safety of TG extract
2.3.1.1. Chemical composition analysis
Determine fatty acids in Gas Chromatography Mass Spectrometry
(GC/MS). Quantitate some metals in Atomic Absorption Spectroscopy
(ASS). Determine pH in Seven Easy device.
2.3.1.2. Determine the safety of TG extract
* Evaluate acute toxicity: oral, subcutaneous (WHO guidelines)
and topical administration (the guidance of the Organization for
Economic Co-operation and Development (OECD).
* Evaluate chronic toxicity: topical administration on rabbit for 90
days (the guidance of the OECD number 411, 1981).
2.3.2. The therapeutic efficacy of TG extract on experimental chronic
skin ulcers
Method: Controlled experimental study on animals.
Use the model of Rudolph R et al: Experimental skin ulcers
produced by Adriamycin on rabbits.
Experimental treatment model of TG extract
Each rabbit was treated with 2 parallel therapies: Controlled ulcers:
Group 1 (10 rabbits): Change the dressing, wash and cover sores with
gauze soaked in NaCl 0.9% solution. Group 2 (20 rabbits): Silver
Sulfadiazine grease (SS). The study ulcers (30 rabbits): topical TG

extract. Change the dressing once every 2 days and photograph the ulcers.
Location: Laboratory - National Institute of Burns.


9
Figure 2.5. Measure the size of CSUs using the Image Pro Plus software
Evaluate the therapeutic efficacy in general anatomy
Measuring size: perform on ulcer photographs (with standard ruler
(cm): Askina, B. Braun) on the 1 st day, 14th day and 22nd day of
treatment using polygon mode of the Image Pro Plus 4.5 software
(America) (Figure 2.6).
Evaluate the effectiveness of TG extract by rate of ulcer size
reduction:
Percentage change in wound area of a CSU (%) = (initial size –
inspection time size)/initial size x 100
Evaluate the effectiveness of experimental treatment on scoring
system by Sanada DESIGN H et al (2004) (Table 2.2.).
Histopathology
Biopsy the ulcers 3 times: 1st day, 14th day, 22nd day of the
treatment.
Histopathological test: H&E (hematoxylin eosin) staining, scanned
by light microscope.
Immunohistochemistry: Randomly selected biopsy samples of 5
animals, immunohistochemical staining (ABC method) to represent
CD34, Vimentin and MMP9.
Ultrastructure: Template biopsies were scanned on electron
microscope (JSM 5410LV, JEOL, Japan), and transmission electron
microscope (JEM 1400, JEOL, Japan).
2.3.3. Evaluate clinical therapeutic effectiveness of TG extract
Method: clinical trial, longitudinal and cross-sectional study.

Systemic therapy: follow to protocol.
Site treatment: prepare ulcers bed: clean the surface, excision. All
lesions had their dressing changed, cleansed and ointment was applied
once per day.
Medication: Apply TG extract on ulcer surface once per day.
Dosage: 0.5 to 1 ml of TG extract per 1000 mm2 of skin. Cover and
hold a thin piece of gauze in place.
Method of evaluating the clinical therapeutic effectiveness
Measuring ulcer size: Measure the size of ulcers on photographs
taken at the moments: the start, the middle and the end of treatment
using polygon mode of Image Pro Plus 4.5 software (US).
Evaluate the ulcer size changing:


10
Percent of ulcer size reduction (%) = (initial size – inspection time
size)/initial size x 100
Table 2.2. Ulcers scoring based on the DESIGN criteria
DESIGN criteria
Values
Score
Depth
I, II, III, IV stage
Exudate
- No; - Less to medium; - Much
0; 3; 6
0;
<400;
400
to<1600;

1600
to
<
3600;
3600
to
0;
3;
6; 8; 9;
Size (mm2)
< 6400; 6400 to< 10000; ≥ 10000
12; 15
Infection
No; obvious symptoms or localized infection
0; 3
Unknown (closed wounds)
0
Granulation tissue ≥50% area of injury
3
< 50% area of injury
6
Necrotic tissue
No; Yes
0; 3
Pocketing
No; Yes
0; 6

Evaluate the clinical effectiveness of treatment based on DESIGN
scoring system of Sanada H et al (2004) (Table 2.2).

Use the following formula to determine the change of detail scores:
Changing in detail scores according to DESIGN:
Changing in detail score of ulcers = (inspection time score) (initial score)
Proportion of effective treatment for ulcers
Efficiency ratio (%) = (Number of effective ulcers) / (number of
effective ulcers + number of inefficient ulcers) x100
Effective: reducing ≥50% of total score compared to the initial score.
Ineffective: reducing<50% of the total score compared to the initial score.
Percentage of complete healing (%) = (number of < 1 cm2ulcers at
the end of the treatment) * 100 / total number of ulcers.
2.4. Statistical Analysis.
Use Epi Info 2008 and SPSS 13.0 software and statistical analysis
methods in medical research and evaluate the results.


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Chapter 3: RESULT
3.1. The chemical composition and the safety of TG extract
3.1.1. The chemical composition of TG extract: shown in Table 3.1.
Table 3.1. Fatty acid composition of TG extract
No.

Composition

n

1
2
3
4

5
6
7
8

Palmitic acid (C16:0)
Palmitoleate (C16:1)
Stearic acid (C18:0)
Oleic acid (C18:1)
Linolenic acid (18:2)
Arachidonic acid (C20:4)
Docosahexaenoic acid (C22:6)
Cholesterol

6
6
6
6
6
6
6
6

Content (mg/ml) (
SD)
21,01±0,63
1,72±0,16
15,27±1,55
38,13±3,20
6,79±0,64

1,14±0,09
1,77±0,30
21,01±0,63

X

±

Essential components of TG extract are the long-chain fatty acids:
oleic acid (omega-9), linolenic acid (omega-3, omega-6),
docosahexaenoic acid (DHA), arachidonic acid (a type of omega-6) and
cholesterol. Magnesium (Mg) (3,28±0,63), zinc (Zn) (3,95±1,93) are
presented in TG extract. pH of TG extract was 5.2 ± 0.2, acidic.
3.1.2. The safety of TG extract
Determine the acute toxicity of TG extract
Identify the acute toxicity of subcutainous administration: With
volume, the highest concentration of TG extract 60g medicine per kg in
rats, there were no signs of acute toxicity. LD50 of subcutainous TG
extract was unidentified.
Determine acute toxicity of oral administration: maximum dose of
450 mg per kg in rats did not show signs of acute toxicity, after a week
of tracking. LD50 of oral TG extract was unidentified based on the
Litchfield – Wilcoxon method.


12
Identify the acute toxicity of topical administration: The lethal dose
50% of the animals (LD50) of TG extract with the highest dose (14,4g
per kg) was not able to be determined.

13
The sub-chronic toxicity of TG extract
After 6 weeks and 12 weeks of topical TG extract, no special
expression was figured out in the control group and two study groups.
The hematopoietic function, liver and kidney functions tests in both 2
study groups have no significant difference compared to the control
group and before treatment (p > 0.05). Liver, kidney and skin
histopathology did not show any pathological lesions. TG extract had
no experimental sub-chronic toxicity.
3.2. Therapeutic efficacy of TG extract on experimental chronic
skin ulcers
3.2.1. Experimental chronic skin ulcers in rabbits
2 skin ulcers were produced successfully on each of 30 rabbits with
similar sizes and characteristics. Average time to produce ulcer was 21.3
± 2.6 days, average size was 11.1 ± 3.5 cm2.
3.3.2. The therapeutic effectiveness of TG extract
Treatment duration:
Table 3.13. Compare treatment duration between groups (day(s))
Group
NaCl 0,9%(1)
SS (2)
TG extract (3)

n

X

10
20

30

29.9±2.4
26.7±2.0
22.3±2.9

± SD

p
p1-2<0,01
p2-3<0,001
p1-3<0,001

Healing time of the treatment group was significantly shorter than the 2
control groups (p <0.001), (t-test).
Changes in ulcer size:
Table 3.14. Changes in ulcer size after 14 and 22 days of treatment
Time
After 14
days of
treatment
After 22
days of
treatment

Group
TG extract (1)
NaCl (2)
SS (3)
TG extract (1)

NaCl (2)
SS (3)

n

X

30
10
20
30
10
20

2.99±1.31
5.48±2.11
4.95±1.57
0.52±0.55
3.44±1.31
2.15±0.67

± SD

p
p1-2<0.05
p1-3<0.05
p2-3>0.05
p1-2<0.01
p1-3<0.01
p2-3<0.05

14
The ulcer size of TG extract group decreased faster than the 2
control groups did (p <0.01).
The proportion of complete healing is calculated by the ratio of
ulcer which completely closed or size <1cm2 on the 22nd day.
Table 3.15. Compare effectiveness of completely closed ulcer
on the 22nd day
Group
Effectiveness Ineffectiveness Total
p
TG extract (1)
29 (96.7%)
1 (3.3%)
30
p1-2<0.001;
NaCl 0,9%(2)
1 (10%)
9 (90%)
10
p1-3<0.001;
SS (3)
11 (55%)
9 (45%)
20
p2-3<0.05
Total
41
19

60
TG extract group had healing rates significantly higher than control
groups (p <0.001) (Fisher test).
Evaluate ulcers according to DESIGN system
Table 3.16. Compare DESIGN total score between the groups
Group

DESIGN total score

0 day
NaCl 0,9% (n=10)
21.01
SS (n=20)
21.3±0.72
TG extract (n=30) 21.2±0.63
p
p1-2-3>0.05

th

14 day
16.8±2.9
16.5±3.2
6.3±0.9
p1-2,3<0.01

p

nd

22 day
11.1±3.51
12.5±3.12
0.9±1.63
p1-2,3<0.01

pn0-n22<0.05
pn0-n22<0.05
pn0-n14,n22<0.01

After 14 and 22 days of treatment, total score of TG extract group
decreased significantly faster compared to two control groups (p <0.01)
(t-test).
The therapeutic efficacy according to the rate >50% of reduction
on the DESIGN total score
Table 3.17. Compare therapeutic efficacy between the groups according
to DESIGN
Ineffectivenes
p
Group
Effectiveness
Total
s
TG extract (1)
29 (96.7%)
1 (3.3%)
30
p1-3<0.05
NaCl 0,9%(2)
4 (40%)

6 (60%)
10
p1-2<0.001
SS (3)
15 (75%)
5 (25%)
20
p2-3>0.05
Total
45
15
60


15
The effective treatment proportion of TG extract group was significantly
higher compared to control groups (p <0.05, p <0.001) (Fisher test).
Histopathology
The similar ulcers were made in rabbits: frozen necrotic tissue,
inflammatory cell infiltration (Figure 3.6).

a
b
Figure 3.6 and 3.10. Histopathology of skin ulcers which were treated
with TG extract.
a - 3.6. Initial treatment, frozen necrotic tissue (**), inflammatory cells
(→). b – 3.10. TG extract group on the 14th day of treatment: Fibroblasts
proliferation (→), angiogenesis (←- -), epithelial cells (**) .
The control groups still showed remarkable inflammatory process
after 14 days of treatment. There was moderate proliferation of

fibroblast and blood vessel after 22 days.
In the TG extract group, there were dominant fibroblast
proliferation, angiogenesis, some samples showed epithelial cells on the
14th day (Figure 3.10). Extracellular matrix structure similar to normal
skin, a thin epidermis covered after 22 days of treatment:
Immunohistochemistry staining
CD34 staining


16
Figure 3.14.CD34 staining of skin samples which was treated with TG
extract at 14th day. Positive blood vessels with dark brown CD34
(Rabbit 22). X400 (cited).
The positive level with CD34 of the groups was different over time. It
was slightly positive with CD34 of control groups on the 14th day, strongly
positive on the 22nd day. The TG extract group had a strong positive with
CD34 on the 14th day and decreased after 22 days of treatment.
Vimentin antibody staining

Figure 3.15. Vimentin antibody staining, TG extract group, 14th day, X400
At the beginning, all skin ulcer samples were negative with
Vimentin. On the 14th day, the control groups samples had light
positive with Vimentin, increased on the 22 nd day. The TG extract group
samples had strong positive with Vimentin on the 14 th day and had
decreased on the 22nd day.
MMP9 antibody staining

Figure 3.16. 0.9% NaCl control group on the 14th day, strong positive
with antibodies MMP9. X400.

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At the beginning, all ulcers were strongly positive with MMP9.
After 14 days, the control groups ulcer had strong positive with MMP9
and reduced on the 22nd day. The TG extract group had strong positive
with MMP9 on the 14th day and only scattered on the 22nd day.
Ultrastructure
Ultra-structural characteristics of chronic skin ulcers before
treatment: infiltration of inflammatory cells, decreasing of the
extracellular matrix density, disruption, swelling and disintegration of
collagen fiber bundles, degeneration of fibroblasts.

Figure 3.18. Extracellular matrix before treatment: disruption, swelling
and disintegration of collagen fiber bundles (→). TEMx10000.
Ultra-structural characteristics of 2 control groups: After 14 days of
treatment, the inflammation was still dominant. SS greased group
appeared migratory fibroblasts, increased collagen synthesis activity.
After 22 days of treatment, there is increased migration and activity of
fibroblasts in both groups but stronger in SS group.

A
B
Figure 3.30. Ultrastructure of 0.9% NaCl group ulcers (A): fibroblasts
(**), extracellular collagen (→) TEM x20000. SS group (B, 3.33) at
22nd day. B-fibroblasts, micro collagen (**), granular endoplasmic
reticulum (*). TEM x25000


18

A
B
Figure 3.25 and 3.26. Ultrastructure of TG extract group ulcer on the
14th day. A, new extracellular collagen (→). X15000 TEM. B,
fibroblasts, granular endoplasmic reticulum (**), microfilaments near
cell membrane (→). TEM X2500.

A
B
Figure 3.34 and 3.36. Ultrastructure of ulcers which were treated with
TG extract, 22nd day. A, epithelial (**). SEM X2000. B - New collagens
(**). TEM x10000
th
On the 14 day, the TG extract group showed the proliferation and
migration of active fibroblast, strong biosynthesis and secretion of
collagen. New extracellular collagens, strong angiogenesis.
On the 22nd day of the TG extract group, epithelial cells covered the
surface of ulcers. Collagen structured and arranged similarly to normal
dermis. Most of fibroblast reduced activity.
3.3. Evaluate the clinical therapeutic efficacy of TG extract for
chronic skin ulcers
3.3.1. Characteristics of patients and ulcers
There were 59 patients with stroke, nerve damage, cancer
metastases, depletion accounting for 59.32% (35). The injury patients
accounted for 27.12% (16) and 8.47% of patients due to complications


19
of treatment, 5.08% diabetes. 5 (8.5%) patients had 2 ulcers, 54 (91.5%)
patients had one ulcer.

Table 3.19. Site and rate of skin ulcers were treated with TG extract
Diagnosis
Number
Ratio
Leg ulcers
7
10.9%
Forearm and hand ulcers
4
6.3%
Arm ulcers
1
1,6%
Buttock ulcers (tailbone, ischium,
29
45.3%
trochanter)
Thigh ulcers
3
4.7%
Knee ulcers
4
6.3%
Heel ulcers (heel, ankle)
13
20.3%
Instep ulcers
3
4.7%
Total

64
100.0%
Tailbone, ischial, trochanteric ulcers accounted for 45.3%. The heel
ulcers accounted for 20.3% and leg ulcers were 10.9%.
Table 3.20. Ulcer classification before treatment (NPUAP / EPUAP)
Before treatment
classification

Number

Ratio

Stage II

9

14.1%

Stage III

55

85.9%

Total

64

100.0%

Stage III ulcers are the majority with 55/64 (85.9%).
3.3.2. Clinical effectiveness of TG extract on chronic skin ulcers
Duration of treatment: Average duration of treatment was 25.67 ±
14.34 day, the shortest: 7 days, the longest: 68 days.
Changing in ulcer size
Table 3.23. Compare ulcer size (mm2) before and after treatment and
the rate of complete healing (test t)


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Area (mm2)

n

<400
400-<1600
1600-<3600
3600-<6400
6400-<10000
>10000
Total

7
36
15
3
2
1
64

Before
treatment1
X

End of
treatment2

Complete
healing

X

( ± SD)
( ± SD)
264.1 ±70.8
23.2 ± 18.2
7 (100%)
797.5 ± 298.2
99.7 ± 95.7
34 (94.4%)
2399.6 ± 544.2 683.0 ± 574.5 8 (53.3%)
4374.4 ± 255.1 1115.0 ± 1057.3 1 (33.3%)
7375.1 ± 597.2 2046.8 ± 2681.3
19029.5
318.6
50 (78.1%)

p
p1-2<0.01

p1-2<0.01
p1-2<0.01
p1-2<0.01
p1-2<0.01

Ulcer size from 400 to <1600 mm 2 are the most common, followed
by ulcers size 1600 to <3600 mm2 and <400 mm2. The ulcer size
reduced significantly after treatment (p<0.01). The average rate of
complete healing is 78.1%. Ulcer size <400 mm 2 has the highest rate
with 100%. The rate of ulcer size from 400 to <1600 mm2 is 94.4%.
Other larger ulcers have lower complete healing rates.
Effective treatment:
Effective if ulcer size reduced ≥ 50%, ineffective if ulcer size
reduced<50% compared to before treatment.
Table 3.24. Effectiveness of treatment according to the area of the ulcer
Ulcer size
Number Ratio
Reduced<50%

6

9,4%

Reduced ≥ 50% or closed

58

90,6%

Total

64

100,0%

There was 90.6% (58/64) ulcers with ulcer size reduction ≥ 50% or
completely closed, only 9.4% (6/64) ulcers with ulcer size
reduced<50%.
Results of treatment according to DESIGN
Table 3.25. The results of clinical treatment according to DESIGN
Criteria

n

Thời gian

p


21

Exudation
Size
Infection
Granulation
Necrosis
Pocketing
Total
p

64
64
64
64
64
64
64

Before
treatment1
5.12±1.38
6.48±1.97
2.91±0.52
5.86±0.63
2.72±0.88
3.05±3.02
26.14± 5.881

Middle
2.68±1.20
4.48±1.99
0.83±1.35
3.23±0.96
0.69±1.27
1.29±2.49
13.20±6.79
p1-2<0.01

End of
treatment2

0.78±1.33
2.15±2.53
0.23±0.81
1.34±1.59
0.32±0.94
0.09±0.74
4.92± 6.252

p1-2<0.01
p1-2<0.01
p1-2<0.01
p1-2<0.01
p1-2<0.01
p1-2<0.01

The results showed that: all the details and total scores before and
at the end of treatment were statistical significant differences (p <0.01).


22
Table 3.26. Therapeutic efficiency of TG extract according to DESIGN
DESIGN

Number

Ratio

DESIGN total reduced <50%

7

10.9%

DESIGN total reduced ≥50%

57

89.1%

64

100.0%

Total

Treatment for chronic ulcers with TG extract showed that there
were 57/64 ulcers (89.1%) decreased in total score ≥50% DESIGN,
7/64 ulcers (10.9%) decreased in total score DESIGN <50% compared
to before treatment.

Figure 3.40.The ulcer of patient Luong Van G, prior to treatment with
TG extract.
Figure 3.42. At the end of treatment with TG extract (52nd day).
Comment: In general, the ulcers which were treated with TG extract not
only closed completely but also had characteristics such as a soft skin,
no scars, most of them had structures, morphology similar to normal
skin. In some cases, the surface is covered incompletely with a thin
layer of skin epithelium (Figure 3.42).
Chapter 4: DISCUSSION
4.1. The components and the safety of TG extract

4.1.1. The chemical components of TG extract
TG extract contains saturated and unsaturated fatty acids of omega3, omega-6, omega-7 and omega-9 groups which are particularly
interesting in modern medicine due to their biological activities . Oleic
acid (omega-9): as an antioxidant can prevent the effects of free


23
radicals. Linolenic acid has antioxidant effects, especially used in the
treatment for eczema. Arachidonic acid (AA) (a type of omega-6) is the
most common eicosanoid precursor, is metabolized in the cell to form
prostaglandins, leukotrienes that are essential signaling molecules. They
control for multiple systems of the body, such as inflammation and
immunity and also is a component of cell membrane phospholipids,
participate in the repair process and proliferation of cells.
Docosahexaenoic acid (DHA) promotes the immune response, the
chemical elements of cellular response, treating for autoimmune
diseases.
In addition, TG extract contains cholesterol which is not favorable
environment for the growth of bacteria. Magnesium and Zinc
participate in components of proteolytic enzymes depending on metals,
helps balance in ulcers.
4.1.2. Evaluate the safety of TG extract
Determine the acute toxicity of TG extract
Under the guidance of the Organization for Economic Co-operation
and Development (OECD), TG extract can be applied directly to the
skin lesions so it should be tested oral acute, subcutaneous (usage
likely) and topical administration toxicity.
With all three acute toxicity tests, there were unidentified the lethal
dose 50% of the experimental animals (LD50) of TG extract. Thus, TG
extract has no acute oral, subcutaneous and topical toxicity.

Determine the sub-chronic toxicity of TG extract
Based on the guidance of the OECD (1981), we evaluated the subchronic toxicity of TG extract in 90 days on rabbit skin. The study
results showed that TG extract applied on skin continuously for 12
weeks at the highest dose (14,4g / kg / day) did not cause negative
impact on general condition such as movement, liver, kidney and
hematopoietic functions compared to the control groups and before
treatment (p> 0.05). The histopathological images of the liver, kidney,
skin also showed no signs of macroscopic and microscopic disease or
injury compared to control samples.


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Thus, TG extract did not cause subchronic toxicity on usage dose
used in clinical practice (4,8g / kg / day) and triple dose (14,4g / kg /
day) in 90-day trial based on the guidance of the OECD (1981).
4.2. Experimental therapeutic efficacy of TG extract on chronic
skin ulcers
4.2.1. Set up chronic skin ulcer model on rabbits
The ulcers on rabbits were created by the way of Rudolph R et al
(1979) and the macroscopy, histopathology, immunohistochemistry and
ultrastructure of injury were consistent with the pathological disorders
of CSUs. The total duration of the ulcers is 50 to 60 days. Thus, we
succeeded in creating CSUs on rabbits for research purposes.
4.2.2. Evaluate the effectiveness of treatment
The duration of treatment
Time to completely close a chronic wound is considered at the end
of the treatment and are defined as complete re-epithelialization of skin
and not require drainage or dressing, is identified be 2 times of dressing
changes for about 2 weeks.
The Table 3.13 shows that the ulcers on experimental animals were

healed with an average time 29.9 ± 2.4 days. Two granted mechanisms
are shrinkage and tissue regeneration. The SS group average time
shorter healing statistical significance compared to 0.9% NaCl group (p
<0.01). Recognized mechanism of SS is because silver's antibacterial
properties. Average time instant completely wound of the treatment
group was 22.3 ± 2.9 days shorter with statistical significance (p
<0.001) compared to self-healing time of about 1 week and faster
statistical significance compared to patients treated with SS. This result
allows us to think that the therapeutic efficacy of TG extract is due to
address the site pathological disorders - the main cause of CSUs.
The ulcer size
Ulcer size and its changes during the treatment are important
criteria. In our study, the healing time of ulcers which were treated with
TG extract is 22.3 ± 2.9 days. Therefore, based on the facts and the
"Guidance for Industry: Chronic cutaneous ulcer and burn wounds Developing products for treatment" of the US Food and Drug
Administration, we determine the final time of treatment is 22th day.


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The results showed that ulcers which were treated with TG extract
miniaturized faster statistically (p <0.01 and p <0.05) compared to the
control groups. At 22th day, there were 29 lesions (96.7%), with an area of
< 1cm2 and only 1 case (3.3%) with an area > 1 cm2. Meanwhile, the
average size of the control ulcers still is 3.44 ± 1.31cm2(NaCl group) and
2.15 ± 0.67 cm2(SS group), only 11 cases had area < 1cm2 (55%). Kotz P.
et al evaluated the therapeutic effect of ice containing silver (2009), this
percentage was 27% in 21 days of the average duration of treatment. Our
results also showed lower efficiency in SS group than TG extract.
Evaluate the effectiveness of treatment according to DESIGN scale
DESIGN scale which was launched by Sanada H et al in 2004 is

considered as a tool to evaluate therapeutic effect of products treating
CSU in clinical trials (Japanese Society of Pressure). The criteria shows
all the characteristics of CSU and is divided into detailed scale helping
quantify the changes in clinical practice (Table 2.2).
The results showed that TG extract reduced detailed scores faster on
compared to the control groups (p <0.01). On 22 nd day, 29/30 (96.7%)
lesions which were treated with TG extract reduced the total score ≥ 50%
compared to before treatment. This rate is 40% in NaCl 0.9% group and
75% in the SS group. Thus, TG extract reduced ulcer size, increased
granulation tissue, decreased necrosis and reduced the DESIGN total score
faster with statistical significance compared to the control groups (p <
0.01).
4.2.3. Activity mechanism of TG extract
In chronic skin ulcers, factors such as foreign bodies, necrotic
tissue, bacteria (biofilm membrane), and the free radicals prevent the
healing process. The authors around the world are focusing on:
increasing the level of growth factors; maintaining the balance between
proteolytic enzymes and their inhibitors; stimulates proliferation and
increase fibroblast activity. Trials evaluating the effectiveness of
traditional medical treatment of chronic skin ulcers are also based on
the same grounds. The results of histopathology, ultrastructural and
immunohistochemistry made, we believe that TG extract had the
following activity mechanisms: