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

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

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

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


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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

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:


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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;

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
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.


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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.


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29.9±2.4
26.7±2.0
22.3±2.9

± SD

p
p1-2


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The ulcer size of TG extract group decreased faster than the 2
control groups did (p
22 day
11.1±3.51
12.5±3.12
0.9±1.63
p1-2,3
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


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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.


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


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

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%



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

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
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
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


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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.

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