Nghiên cứu độc tính và tác dụng điều trị suy giảm sinh dục đực của viên hoàn cứng TD0014 trên thực nghiệm tt tiếng anh

MINISTRY OF EDUCATION
AND TRAINING

MINISTRY OF HEALTH

HANOI MEDICAL UNIVERSITY

MAI PHUONG THANH

EXPERIMENTAL STUDY ON THE SAFETY AND
EFFECTIVENESS OF TD0014 HARD PILLS
FOR THE TREATMENT OF
MALE SEXUAL DYSFUNCTION
Specialty: Pharmacology and Toxicology
Code: 62720120

SUMMARY OF DOCTORAL THESIS

HANOI – 2019


The study was completed at:
HANOI MEDICAL UNIVERSITY

Scientific supervisors:
1. Assoc. Prof. Ph.D Pham Thi Van Anh
2. Assoc. Prof. Ph.D Nguyen Trong Thong

Reviewer 1:
Reviewer 2:
Reviewer 3:

The doctoral thesis was publicly defended in front of the University Council at
Hanoi Medical University on

The thesis can be found at:
-

National Library of Vietnam

-

The Library of Hanoi Medical University


1

INTRODUCTION
Male sexual dysfunction includes disorders of desire, erectile dysfunction,
abnormal ejaculation, orgasmic dysfunction and failure of detumescence; these
conditions may appear alone or in combination with each other. Researching and
seeking drugs for the treatment of male sexual dysfunction originating from
medicinal herbs are of great interest to physicians. TD0014 is a preparation of
herbal medicines which comprises thirty-two medicinal plants and deer velvet. The
composition of TD0014 has several medicinal herbs that have been studied and
used since ancient times in traditional folk medicine as an aphrodisiac. However, no
studies have provided reliable shreds of evidence of their effects on reproductive
functions, or toxicity when combining them in TD0014. Therefore, the study titled
"Experimental study on the safety and effectiveness of TD0014 hard pills for
the treatment of male sexual dysfunction" was carried out to:
1. Determine acute and subchronic toxicity of TD0014 in experimental animals.

2. Evaluate androgenic activity and effects on erectile function of TD0014 in
experimental animals.
3. Evaluate the effects of TD0014 on sodium valproate-induced reproductive
decline in male rats.
Necessity of the thesis
Male sexual dysfunction is a common disorder associated with a wide range of
physical and psychological conditions. Although this disease is not fatal, does not
require emergency management but greatly affects the morale and quality of life of
patients. Research and development of preventive and therapeutic agents for male
sexual dysfunction have been becoming a special concern of world medicine.
Treatment of male sexual dysfunction according to modern medicine is highly
effective in improving symptoms. However, one must be conscientious of its
adverse effects. Following the current trends in medicine, the treatment approach
based on the theory of traditional medicine that has many advantages such as
reducing the economic burden, convenience, friendliness, and fewer side effects has
deployed in many countries around the world, including Vietnam, a country with
long-standing traditional medicine and is considered to have great potential for
medicinal materials in Southeast Asia and in the world with the source of rich and
diverse resources. The widespread use of herbal medicines, nevertheless, requires
scientific verification of their indications and effects by use of modern medical
analysis. Therefore, studies of toxicity and effects of medicinal herbs, herbal
remedies or products derived from medicinal plants such as TD004 which has used
in the market as dietary supplement are necessary, scientific and practical.
New contributions from the thesis


2

TD0014 is a preparation derived from over thirty natural ingredients. When
combining many medicinal ingredients in a preparation, the most worrying issue is

the interaction between the ingredients in the preparation process as well as in the
metabolism in the body, which can increase toxicity, decrease or loss of effects. The
study has shown positive results with high safety and good effectiveness in treating
male sexual dysfunctions of TD0014 hard pills on the experiment, which considered
a new contribution of the thesis. These results are the basis for further preclinical
and clinical trials which will provide the scientific foundation for using TD0014 in
treating male sexual dysfunction.
Tests of androgenic activity have been conducted by a number of domestic
research institutes with a variety of preparations, but mostly on castrated male rats.
Evaluation of the androgenic activity of TD0014 in weanling male rats can also be
considered a new contribution of the thesis.
Thesis outline
The thesis consists of 153 pages, including introduction (2 pages), overview
(44 pages), object and methods of research (12 pages), results (43 pages which
comprises 37 tables, 16 figures, 10 charts), discussion (49 pages), conclusion (2
pages), proposal (1 page). There are 194 English and Vietnamese references.
Chapter 1
OVERVIEW
1.1. Overview of male sexual dysfunction according to modern medicine
Male sexual dysfunction includes disorders of desire, erectile dysfunction,
abnormal ejaculation, orgasmic dysfunction and failure of detumescence; these
conditions may appear alone or in combination with each other. According to
modern medicine, methods used to treat male sexual dysfunction include
testosterone replacement therapy and treatments for one of the most common
symptoms of the disease which is erectile dysfunction.
1.1.1. Testosterone replacement therapy
Testosterone treatment aims to restore testosterone levels to the physiological
range in men with consistently low levels of serum testosterone and associated
symptoms of androgen deficiency. The aim is to improve quality of life, sense of
well-being, sexual function, muscle strength and bone mineral density. Current

testosterone therapies include implants, intramuscular injections, oral formulations,
transdermal delivery systems, transbuccal delivery systems, and intranasal
testosterone.
The risks of testosterone replacement therapy depend on age, life
circumstances and other medical conditions, include exacerbation of prostate
cancer, male breast cancer, worsening benign prostatic hyperplasia, hepatotoxicity


3

and liver tumor, polycythemia, an increased risk of obstructive sleep apnea and
congestive heart failure, infertility, and skin diseases. With these risks, TRT is
contraindicated in men with known or suspected androgen-dependent carcinoma of
the prostate or of the male mammary gland, severe lower urinary tract symptoms
due to benign prostatic hyperplasia, past or present liver tumors, severe chronic
cardiac failure (NYHA IV), severe sleep apnoea, hematocrit > 54%, and male
infertility-active desire to have children.
1.1.2. Drugs for erectile dysfunction
Lifestyle modifications, oral phosphodiesterase-5 (PDE5) inhibitors,
intracorporal injections, topical medication, surgery, vacuum devices, and
acupuncture are some of the treatments available today for erectile dysfunction,
among which PDE inhibitors are the first-line treatment in patients with erectile
dysfunction.
PDE5 inhibitors are indicated for treatment of men with erectile dysfunction,
which is the inability to achieve or maintain a penile erection sufficient for
satisfactory sexual performance. In order for them to be effective, sexual
stimulation is required. There are currently four widely approved PDE5 inhibitors
and are the first choice in treating erectile dysfunction, including sildenafil,
tadalafil, vardenafil, and avanafil; in addition, a number of other PDE5 inhibitors
are licensed in certain countries, such as udenafil and mirodenafil designated in

Korea, or lodenafil used in Brazil. All PDE5 inhibitors are contraindicated in
patients with a known hypersensitivity to any component of the agents; patients
who are taking any form of organic nitrates, regularly or intermittently; patients
who have loss of vision in one eye because of non-arteritic anterior ischaemic optic
neuropathy (NAION), regardless of whether this episode was in connection or not
with previous PDE5 inhibitor exposure. They should not be used in men for whom
sexual activity is inadvisable (e.g. patients with severe cardiovascular disorders
such as unstable angina or severe cardiac failure), severe hepatic impairment,
hypotension (blood pressure < 90/50 mmHg), recent history of stroke or myocardial
infarction and known hereditary degenerative retinal disorders such as retinitis
pigmentosa (a minority of these patients have genetic disorders of retinal
phosphodiesterases).
1.2. Overview of the mode of action of medicinal herbs for male sexual
dysfunction
A great number of traditional herbal medicine have been used to treat male
sexual dysfunction, especially erectile dysfunction, applied singly or in the form of
compound formulas. Most of the herbal remedies are used empirically and thereby
are not convincing. In this condition, many studies are being conducted to
investigate the underlying mechanism of those herbs using modern biotechnology.


4

Figure 1. The effects of male sexual stimulation of the main bioactive compounds
of medicinal plants
1.3. Herbal formula TD0014 preparation
The major ingredients of the herbal formula are obtained from thirty-three
natural products: Tribulus terrestris (4.00g), Chrysanthemum sinense (1.83g),
Prunus persica (1.14g), Vigna cylindrica (1.14g), Eurycoma longifolia (0.69g),
Sophora japonica (0.57g), Dioscorea persimilis (0.43g), Dioscorea tokoro (0.40g),

Polygonum multiflorum (0.40g), Citrus deliciosa (0.34g), Polyscias fruticosa
(0.34g), Tinospora sinensis (0.29g), Chaenomeles lagenaria (0.29g), Passiflora
foetida (0.29g), Zizyphus sativa (0.29g), Rehmannia glutinosa (0.23g), Angelica
sinensis (0.23g), Alisma plantago-aquatica L. var. orientalis Samuelsson (0.23g),
Achyranthes bidentata (0.23g), Schizandra chinensis (0.23g), Morinda offcinalis
(0.23g), Rosa laevigata (0.23g), Allium sativum (0.20g), Lycium sinense (0.17g),
Glycyrrhiza uralensis (0.14g), Panax ginseng (0.11g), Ligusticum wallichii (0.11g),
Cistanche tubulosa (0.11g), Atractylodes macrocephala (0.11g), Radix Codonopsis


5

(0.11g), Cuscuta sinensis (0.11g), Psoralea corylifolia (0.06g), Cornu Cervi
parvum (7.2mg). The composition of TD0014 has several medicinal herbs that have
been shown to enhance sexual activity in many preclinical and clinical studies,
including Tribulus terrestris, Eurycoma longifolia, Angelica sinensis, Morinda
offcinalis, Lycium sinense, Panax ginseng, Ligusticum wallichii, Cistanche
tubulosa, Cuscuta sinensis, Psoralea corylifolia.
Chapter 2
OBJECTS AND RESEARCH METHODS
2.1. Polyherbal formula TD0014 preparation
TD0014 was manufactured as hard pills according to the quality standard of
Sao Thai Duong Joint Stock Company, Vietnam. The preparation was packed in 7.5
grams per sachet. The predicted human dose of TD0014 is 2 sachets in divided
doses (equivalent to 15 grams of raw medicinal materials/day).
2.2. Animals: Swiss mice, Wistar rats.
2.3. Research methods
2.3.1. Acute and subchronic toxicity of TD0014 in animals
2.3.1.1. Acute oral toxicity
The acute oral toxicity study of TD0014 was conducted according to the

general guidelines for methodologies on research and evaluation of traditional
medicine of WHO and determined lethal dose of 50% by the Litchfield-Wilcoxon
method.
Adult male mice were separately divided into groups of 10 animals. After an
overnight fast, each dose of TD0014 was administered orally from the highest nonlethal dose to the lowest one that killed 100% mice. The animals were observed for
signs of toxicity and mortality for the first critical 72 hours and thereafter daily for 7
days. The oral median lethal dose (LD50) was calculated according to the number
of mice mortality within the first 72 hours.
2.3.1.2. Subchronic oral toxicity
The subchronic oral toxicity study of TD0014 was carried out according to the
general guidelines for methodologies on research and evaluation of traditional
medicine of WHO. The toxicity study was carried out using thirty-three adult male
rats. The rats were divided into three groups of 11 animals per group. Group I
served as the vehicle control and received 1 ml/100 g b.wt. sterile distilled water
daily while groups II and III were administered TD0014 at the dose of 1.8 and 5.4
g/kg b.wt. daily in appropriate volume of distilled water for 90 days.
On the first day (D0) and at the end of day 30 (D30), D60, and D90, rats in all
group were weighed and collected blood samples from femoral vein for determining
haematological parameters (red blood cell, haemoglobin concentration, hematocrit,


6

mean capsular volume, total white blood cells and white blood cell differential,
platelet count) and biochemical parameters (alanine aminotransferase (ALT),
aspartate aminotransferase (AST), albumin, total cholesterol, total bilirubin,
creatinine).
At the end of the experiment, 30% of rats of each group were made
unconscious by cervical dislodgement, then the internal organs (liver, kidney) were
removed and observed for any gross lesions. These organs were preserved in a

fixation medium of 10% buffered formalin for histopathological study.
2.3.2. Androgenic activity of TD0014 in immature male rats
The androgenic activity of TD0014 evaluated by the Hershberger assay has
been adapted and standardized according to OECD guidelines.
2.3.2.1. Androgenic effects of TD0014 in the castrated male rats
For the experimental, 45 peripubertal male rats were used with aged between
42 and 50 days. After castration by removing both testes and epididymides and full
recovery in seven days, the rats were separated into 5 groups of 9 animals each.
- Group I: Intact + distilled water
- Group II: Castrated + distilled water
- Group III: Castrated + testosterone at dose of 0.4 mg/kg
- Group IV: Castrated + TD0014 at dose of 1.8 g/kg
- Group V: Castrated + TD0014 at dose of 5.4 g/kg).
The rats received testosterone by s.c injection or TD0014 by oral gavage, once
daily for 10 consecutive days. 24 hours after the last administration, the rats were
sacrificed by exsanguination. Blood samples from the carotid artery were collected
for testosterone measurement. The five androgen-dependent tissues: ventral
prostate, seminal vesicles, levator ani-bulbocavernosus muscle (LABC),
bulbourethral glands (Cowper), and glans penis were harvested and weighed.
2.3.2.2. Androgenic effects of TD0014 in the weanling male rats
Forty weanling 21-day old male rats were separated into 4 groups of 10
animals each, treated daily for 10 consecutive days with distilled water (control; 10
mL/kg b.w orally), testosterone propionate (TP) s.c. (androgenic control; 1
mg/kg/day s.c.), or TD0014 at 1.8 and 5.4 g/kg by gavage. The animals were killed
24 h after the last dose, followed by exsanguination. Blood samples from the carotid
artery were collected and stored until analyzed for testosterone concentration. At
necropsy, the reproductive organs (testis, epididymis, seminal vesicles, ventral
prostate, Cowper's glands and levator ani/bulbocavernosus muscles [LABC]) were
excised, trimmed free of fat and connective tissues, and weighed.
2.3.3. Effects of TD0014 on erectile function

The experiment was performed according to the method of Gajbhiye et al.
(2015). Adult male rats were randomly separated into 3 groups of 6 animals each: 1)


7

control, 2) sildenafil treatment, and 3) TD0014 treatment. In each group, animals
were administered per os one-time with either distilled water (10 ml/kg b.w.), or
sildenafil (6 mg/kg b.w.), or TD0014 (1.8 g/kg b.w.).
At 2 hours after treatment, rats were anaesthetized with an intraperitoneal
injection of ketamine at a dose of 25 mg/kg. The corpus cavernosum were exposed.
To measure intracavernosal pressure (ICP), a heparinized 23-gauge butterfly needle
was inserted into the proximal portion of the corpus cavernosum. A bipolar
electrical stimulator was placed on the ganglion to stimulate the cavernosal nerve
for 60 seconds at 5 V and 20 Hz for 2 millisecond periods. The cavernosal nerve
stimulation was conducted 3 times with a 10-minute interval between stimulations.
The mean systemic arterial pressure (MAP) was monitored simultaneously with ICP
monitoring. The right carotid artery was dissected and then PE-50 tubing was
inserted into the carotid artery. The catheter was connected to both a pressure
transducer and an amplifier unit which was connected to a data acquisition module.
Before and after each electrical stimulation, ICP and MAP was recorded on a
computer by Powerlab system record software.
Erectile function was evaluated by the following parameters: ICP before and
after the electrical stimulation of the cavernous nerve (basal ICP and maximal ICP),
time to the maximal ICP, response time to the elec- trical stimulation of the
cavernous nerve, total ICP (ICP vs stimulation time, area under curve), MAP and
maximal ICP/MAP ratio.
2.3.4. Effects of TD0014 on sodium valproate-induced reproductive decline in
male rats
The experiment used sodium valproate (SVP) to cause reproductive toxicity in

male rats according to a model described by Nishimura et al. (2000).
2.3.4.1. Protective role of TD0014
The adult male rats were randomly divided into the four groups. The control
group (Group 1, negative control) was given distilled water orally. Another group
(Group 2, posittive control) received SVP (500 mg/kg, oral) for 7 weeks and served
as toxic control. The remaining two groups (Group 3 and 4) received TD0014 orally
at doses of 1.8, and 5.4 g/kg respectively along with SVP for 7 weeks. On 5th week,
one male rat was randomly coupled with two untreated virgin females for 2 weeks.
At the end of 7th week, all rats were weighed and killed by exsanguination.
Following parameters were calculated:
- Male rats: the weights of testes and accessory sexual organs (glans penis,
epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator
ani/bulbocavernosus muscles [LABC]), serum testosterone level, semen analysis
(sperm counts, sperm motility, sperm viability and sperm morphology),
histopathology of testis.


8

- Female rats: pregnancy rate.
2.3.4.2. Restorative role of TD0014
Adult male rats were randomly divided into four groups. The animals were
given SVP at the dose of 500 mg/kg/day for 7 weeks to cause reproductive toxicity,
then distilled water or TD0014 was continuously administered orally for 10 days:
- Group 1: not given SVP for 7 weeks, distilled water 10 mL/kg/day for 10 days.
- Group 2: given SVP for 7 weeks, distilled water 10 mL/kg/day for 10 days.
- Group 3: given SVP for 7 weeks, TD0014 1.8 g/kg/day for 10 days.
- Group 4: given SVP for 7 weeks, TD0014 5.4 g/kg/day for 10 days.
After 10 days of treatment, one male rat was randomly coupled with two
untreated virgin females for 2 weeks. At the end of pairing period, parameters of

male and female rats were determined similarly to the study of protective effects of
TD0014.
2.4. Statistical analysis
Data were analyzed by Excel 2010 and SPSS 22.0 software, using appropriate
statistical algorithms (Student's t-test, Paired t-test, Mann-Whitney U test, Chisquare test). A p value<0.05 was considered as statistically significant.
Chapter 3
RESULTS
3.1. Acute and subchronic toxicity of TD0014 in animals
3.1.1. Acute toxicity of TD0014
Table 3.1. Correlation between TD0014 dose and mice mortality
Group
(n = 10)

Dose of TD0014
(g/kg)

Percentage of
dead (%)

Other abnormal
findings

Group 1
22.50
0
None
Group 2
33.75
0
None

Group 3
45.00
0
None
Group 4
56.25
0
None
In all-male testing animals, signs of neither toxicity nor death among the mice
were observed within the critical 72 hours post-administration and to the end of the
seventh day. Hence the LD50 evaluated by the Litchfield - Wilcoxon method could
not be determined (orally).
3.1.2. Subchronic toxicity of TD0014
3.1.2.1. Body weight and clinical observation
Neither deaths nor obvious clinical signs of toxicity in the rats were observed
for all groups. Physical observation of the treated rats throughout the experimental
period indicated that none of them showed signs of toxicity in their skin, eyes, or


9

behavioral changes, diarrhea, tremors, sleep, and coma. Normal body weight gains
were observed during the study period compared to the control group.
3.1.2.2. Hematological analysis
Daily oral administration of TD0014 for 90 days produced no effect on all
hematological parameters. The results show that none of the groups differed
significantly when compared to the control for all parameters.
3.1.2.3. Biochemical analysis
None of the biochemical parameters were affected by the oral administration
of TD0014 for 90 days. There was no statistical difference in the concentration of

the indicator enzymes of liver cell damage (AST, ALT) in the group treated with
TD0014 compared to the group treated with distilled water. No significant changes
in liver function parameters (albumin, total cholesterol, total bilirubin) and
glomerular filtration function parameters (creatinine) were noted.
Table 3.2. Effects of TD0014 on serum transaminase levels

D0
D30
p (paired t-test)
D60
p (paired t-test)
D90
p (paired t-test)

Control
(n = 11)
112.91 ±
25.04
110.27 ±
10.62
> 0.05
101.55 ±
7.06
> 0.05
104.73 ±
22.00
> 0.05

AST (UI/L)
1.8 g/kg

(n = 11)
108.64 ±
15.52
103.82 ±
26.04
> 0.05
113.36 ±
20.21
> 0.05
106.73 ±
17.70
> 0.05

5.4 g/kg
(n = 10)
121.00 ±
24.31
117.36 ±
30.40
> 0.05
112.55 ±
17.46
> 0.05
107.90 ±
17.04
> 0.05

Control
(n = 11)
60.00 ±

18.07
50.64 ±
7.12
> 0.05
53.45 ±
9.13
> 0.05
66.00 ±
12.24
> 0.05

ALT (UI/L)
1.8 g/kg
(n = 11)
62.18 ±
13.55
51.00 ±
11.74
> 0.05
58.45 ±
14.08
> 0.05
60.91 ±
11.78
> 0.05

5.4 g/kg
(n = 10)
59.00 ±
7.76

50.91 ±
8.84
> 0.05
61.64 ±
10.68
> 0.05
60.40 ±
14.71
> 0.05

3.1.2.4. Histopathology study
- Gross pathologic observations: Liver and kidney did not show any abnormal
changes in texture, shape, size or color compared to the control. There was no
sign of necrosis or lesion was appreciated on the organs of all treated groups.
- Light microscopy of liver: There were no significant histopathological
presentations observed in the groups treated with distilled water and treatment
groups. The microscopic examination of liver sections of rats showed the normal
architecture of structural units of the liver, the hepatic lobules, formed by cords
of hepatocytes separated by hepatic sinusoids. No portal inflammation was seen.
- Light microscopy of kidney: There were no adverse histopathological
presentations observed in all the treatment groups. The microscopic architecture


10

of sections of kidney in treated groups had a similar appearance to that of the
controls in which renal corpuscles maintaining their normal size of urinary space
and normal tubular structures are examined. No necrosis was observed.
3.2. Androgenic activities of TD0014 in immature male rats
Table 3.3. Effects of TD0014 on weights of accessory sexual organs and serum

testosterone levels of the castrated male rats
Groups (n = 9)
Intact + Castrated Castrated + Castrated Castrated
Parameters
distilled + distilled testosterone + TD0014 + TD0014

Glans
penis
Seminal
vesicles
Weight
Ventral
(mg/100g
prostate
b.wt)
Cowper's
glands
LABC
Testosterone
(nmol/L)

water

water

28.2 ±
7.8
19.7 ±
5.7
22.2 ±

6.4
8.4 ±
1.9
104.1 ±
19.2
1.817 ±
0.491

21.0 ±
5.8*
8.7 ±
2.1***
5.0 ±
1.6***
1.3 ±
0.2***
44.6 ±
9.0***
0.120 ±
0.038***

0.4 mg/kg

1.8 g/kg

42.2 ± 4.2### 22.3 ± 4.5
81.6 ±
19.2###

9.3 ± 2.4

34.2 ± 8.3### 8.9 ± 2.8##
10.7 ± 2.6###
139.6 ±
30.2###
3.098 ±
0.975†††

5.4 g/kg
22.6 ± 6.3
8.2 ± 2.6
17.7 ±
4.2###

1.9 ± 0.6#

1.6 ± 0.3#

39.8 ± 9.2

36.5 ± 9.8

0.166 ±
0.031†

0.366 ±
0.113†††

*p<0.05; ***p<0.001 compared with group I (intact + distilled water) (Student’t-test)
#

##
###
p<0.05; p<0.01; p<0.001 compared with group II (castrated + distilled water) (Student’t-test)

With regard to castrated controls, rats exposed to the plant extracts showed a
significant increase in the relative weights of the ventral prostate and the Cowper’s
glands, and a significant increase in the serum testosterone levels.
On the weanling animals, TD0014 at the dose of 5.4 g/kg caused a significant
increase in the weights of Cowper’s glands, LABC, and prostate of rats, while
TD0014 at the dose of 1.8 g/kg caused only a significant increase in the weight of
Cowper’s glands. TD0014 treatment resulted in a significant increase in the serum
testosterone levels.


11

Table 3.4. Effects of TD0014 on weights of reproductive organs and serum
testosterone levels of the weanling male rats
Groups (n = 10)
Parameters
Testosterone TD0014
TD0014
Control
1.0 mg/kg
1.8 g/kg
5.4 g/kg
891.6 ±
893.7 ±
Testis
901.8 ± 180.9 786.5 ± 159.1

117.9
162.9
Seminal
215.8 ±
21.9 ±
24.1 ± 6.7
19.5 ± 5.6
***
vesicles
48.4
5.7
252.2 ±
130.1 ±
123.8 ±
Epididymis 127.6 ± 24.0
Weight
***
35.4
30.0
22.6
(mg/100g
Ventral
100.2 ±
37.6 ±
b.wt)
24.4 ± 8.0
21.7 ± 5.1
***
prostate
17.6

7.9**
Cowper's
3.4 ± 0.7
21.2 ± 3.0*** 4.8 ± 1.3** 4.3 ± 0.8*
glands
194.6 ±
46.0 ±
70.8 ±
LABC
55.0 ± 11.0
***
23.4
10.2
15.4*
15.343 ±
0.235 ±
0.293 ±
Testosterone (nmol/L)
0.087 ± 0.002
1.939***
0.089*** 0.062***
*p<0.05; **p<0.01; ***p<0.001 compared with control (Student’t-test)

ICP (mmHg)

3.3. Effects of TD0014 on erectile function
70
60
50
40

30
20
10
0

**
Control

***

Sildenafil

*

Before stimulation

TD0014

After stimulation (maximal ICP)

* p < 0.05; ** p < 0.01; *** p < 0.001 compared with control (Student’t-test)

Chart 3.1. Intracavernous pressure (ICP) before and after electrical stimulation of
the cavernous nerve in rats from each experimental group
Before stimulation: TD0014 elevated the basal ICP level which was
statistically different compared to the control animals.
After stimulation: TD0014 group exhibited an increase in maximal ICP, total
ICP, and response time to the electrical stimulation, but they were not statistically
significant compared with distilled water group. There was no statistical

12

differences in MAP and maximal ICP/MAP values between the TD0014 group and
the control group.
Table 3.5. Effects of TD0014 on time to the maximal ICP and response time to the
electrical stimulation of the cavernous nerve, and maximal ICP/MAP ratio
Maximal
Groups
Time to the
Response time to
MAP
(n = 6)
maximal ICP (s)
stimulation (s)
ICP/ MAP
38,833 ± 20,614

109,441 ± 50,721

106,34 ± 18,08

Sildenafil

40,016 ± 18,290

158,902 ±
47,607**

96,43 ± 13,76

TD0014

38,819 ± 14,245

114,196 ± 17,298

110,67 ± 5,05

Control

0,40 ± 0,11
0,56 ±
0,14***
0,44 ± 0,09

**p<0.01; ***p<0.001 compared with control
Total ICP 2500
(mmHg*s)
2000

*

1500
1000
500
0
Control

Sildenafil

TD0014

*p<0.05 compared with control

Chart 3.2. Effect of TD0014 on total ICP (ICP vs stimulation time, area under curve)
3.4. Effects of TD0014 on sodium valproate-induced reproductive decline in
male rats
3.4.1. Protective effects
3.4.1.1. Effects on the weight of organs
The weight of organs, including testes, accessory sexual organs (glans penis,
epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator
ani/bulbocavernosus muscles [LABC]), and several other organs (liver, kidney,
adrenal glands) in the SVP treated group were significantly reduced as compared to
the control group. A daily oral administration of TD0014 for 7 consecutive weeks
was followed by a significant increase in relative weights of the testes, some
accessory organs (TD0014 at the dose of 5.4 g/kg caused a significant increase in
the weights of glans penis, seminal vesicles, epididymis, and Cowper’s glands of
rats; TD0014 at the dose of 1.8 g/kg caused only a significant increase in the weight


13

of glans penis and epididymis), and adrenal glands when compared to SVPintoxicated control group.
3.4.1.2. Effects on sperm analysis
Table 3.6. Protective effects of TD0014 on the size of seminiferous tubule, sperm
count and viability in SVP-intoxicated rats
Size of seminiferous
Sperm count
Sperm viability

Groups
6
tubule (pixell)
(10 /mL)
(%)
Negative control
452.74 ± 55.12
144.74 ± 18.73
93.71 ± 2.50
Positive control
326.09 ± 38.81***
3.83 ± 1.17***
59.83 ± 12.73***
▲
▲▲▲
TD0014 1.8 g/kg
371.97 ± 25.62
18.33 ± 5.85
51.67 ± 15.11
106.20 ±
TD0014 5.4 g/kg
462.20 ± 58.25▲▲▲
88.70 ± 6.18▲▲▲
▲▲▲
33.13
***p<0.001 compared with negative control (Student’t-test)
▲
p<0.05; ▲▲▲p<0.001 compared with positive control (Student’t-test)

Table 3.7. Protective effects of TD0014 on sperm morphology in SVP-intoxicated rats

Abnormal (%)
Groups
n Normal (%)
Head
Midpiece
Tail
Negative control
8 59.00 ± 6.98 14.57 ± 4.20 10.00 ± 1.63 16.43 ± 5.32
29.17 ± 1.17 31.33 ± 6.98 20.83 ± 3.76
Positive control
6
18.67 ± 3.44
***
***
***
37.83 ±
14.50 ±
TD0014 1.8 g/kg
6
24.17 ± 5.38
23.50 ± 6.41
11.07
3.39▲
40.90 ±
18.70 ± 5.03
16.00 ±
TD0014 5.4 g/kg 10
24.40 ± 7.12
▲
▲▲▲

12.62
4.64▲
***p<0.001 compared with negative control (Student’t-test)
▲
p<0.05; ▲▲▲p<0.001 compared with positive control (Student’t-test)
TD0014 5.4 g/kg
TD0014 1.8 g/kg
Positive control
Negative control
0%
Immotile

20%
In situ motility

40%

60%

Slow motility

80%

100%

Rapid motility

Chart 3.3. Protective effects of TD0014 on sperm motility in SVP-intoxicated rats
When compared with positive intoxicated rats, TD0014 with all tested doses
significantly increased the size of seminiferous tubule and sperm count, while the

14

percentage of sperm viability and abnormality were only improved at the dose of
5.4 g/kg TD0014.
The percentage of immotile sperm was 100% in SVP-intoxicated rats. The
semen of male rats received TD0014 at the dose of 1.8 g/kg had motile sperms,
however, the spermatozoa have non-progressive motility, i.e it moved only in situ.
The male rats exposed to multiple oral dose of TD0014 at 5.4 g/kg/day were
significantly improved the speed of movement of spermatozoa with the presence of
rapid and slow progressive sperm.
3.4.1.3. Effects on serum testosterone level and histopathological of testis

Negative control

Positive control

Normal histological structure of seminiferous Atrophied seminiferous tubules and
tubules filled with mature sperms
edema with absence of sperms

TD0014 at 1.8 g/kg

TD0014 at 5.4 g/kg

Partial improvement of the germinal epithelium Seminiferous tubules with a narrow
of seminiferous tubules, but their lumen still lumen filled by sperm cell lineage
wide and not full of sperm cell lineage
Figure 3.1. Protective effects of TD0014 on photomicrographs of testes

Table 3.8. Protective effects of TD0014 on serum testosterone in SVP-intoxicated rats
Groups
n
Testosterone (nmol/L)
Group 1: Negative control
8
4,17 ± 1,22
Group 2: Positive control (SVP)
6
1,35 ± 0,44***
Group 3: SVP + TD0014 (1.8 g/kg)
6
4,30 ± 1,10▲▲▲
Group 4: SVP + TD0014 (5.4 g/kg)
10
5,64 ± 1,03▲▲▲≠
***p<0.001 compared with negative control (Student’t-test); ▲▲▲p<0.001 compared with positive
control (Student’t-test); ≠p<0,05 compared with TD0014 at low dose (Student’t-test)

Oral administration of SVP (500 mg/kg) to rats during 7 weeks of the
experimental period significantly decreased serum testosterone when compared


15

with the normal control group. Co-administration of TD0014 with SVP
significantly increased serum testosterone when compared with the intoxicated
control group, in a dose-dependent fashion.
3.4.1.4. Effect on pregnancy rate of female rats
The positive control group had only 1/20 pregnant female rats (pregnancy rate

was 5%) and a marked decrease compared to the negative control group (60%).
There was no difference in the pregnancy rate of the low-dose TD0014 group (10%)
and the positive control group. The pregnancy rate of the high-dose TD0014 group
(30%) was significantly higher than that of the positive control group.
3.4.2. Restorative effects
3.4.2.1. Effects on the weight of organs
The weight of organs, including testes, accessory sexual organs (glans penis,
epididymis, seminal vesicles, ventral prostate, Cowper's glands and levator
ani/bulbocavernosus muscles [LABC]), and several other organs (liver, kidney, adrenal
glands) in the SVP treated group were significantly reduced as compared to the control
group. A daily oral administration of TD0014 at the dose of 5.4 g/kg for 10 consecutive
days was followed by a significant increase in relative weights of the testes and 3
accessory organs (seminal vesicles, epididymis, and LABC) of rats; TD0014 at the
dose of 1.8 g/kg caused only a significant increase in the weight of 2 accessory sexual
organs (seminal vesicles, LABC) when compared to SVP-intoxicated control group.
TD0014 at both doses tended to increase adrenal gland weight.
3.4.2.2. Effects on sperm analysis
Table 3.9. Restorative effects of TD0014 on the size of seminiferous tubule, sperm
count and viability in SVP-intoxicated rats
Size of
Sperm count
Sperm
Sperm speed
Groups
seminiferous
6
(10 /mL)
viability (%)
(μm/s)
tubule (pixell)

161.78 ±
Negative control
429.70 ± 20.00
71.67 ± 6.67 54.46 ± 4.91
24.15
380.87 ±
60.44 ±
58.22 ±
38.91 ±
Positive control
15.20***
16.48***
10.03**
6.56***
98.44 ±
58.89 ±
49.97 ±
TD0014 1.8 g/kg 405.82 ± 21.57▲
▲▲▲
19.82
14.42
12.55▲
107.00 ±
67.00 ±
47.01 ±
TD0014 5.4 g/kg 405.70 ± 15.84▲
▲▲▲
▲
25.62
4.21

9.15▲
***p<0.001 compared with negative control (Student’t-test)
▲
p<0.05; ▲▲▲p<0.001 compared with positive control (Student’t-test)

TD0014 at both doses significantly increased size of seminiferous tubule,
sperm count and sperm motility, simultaneously decreased sperm abnormality.


16

Sperm viability tended to increase compared to positive control group with the
presence of TD0014, however a statistically significant difference was only
observed in the high dose TD0014 group.
Table 3.10. Restorative effects of TD0014 on sperm morphology in SVP-intoxicated rats
Abnormal (%)
Groups
Normal (%)
(n = 9)
Head
Midpiece
Tail
Negative control
56.83 ± 4.12
19.67 ± 1.21 10.33 ± 2.07 13.17 ± 1.17
26.43 ±
Positive control
44.14 ± 3.67***
14.29 ± 2.63* 15.14 ± 1.86*
2.88***

TD0014 1.8 g/kg 51.67 ± 4.59▲▲ 22.50 ± 4.93 11.00 ± 1.41▲ 14.83 ± 1.94
TD0014 5.4 g/kg
49.75 ± 5.12▲ 23.88 ± 2.36 11.75 ± 1.67▲ 14.63 ± 2.45
*p<0.05; ***p<0.001 compared with negative control (Student’t-test)
▲
p<0.05; ▲▲p<0.01 compared with positive control (Student’t-test)

3.4.2.3. Effects on serum testosterone level and histopathological of testis

Negative control

Positive control

Normal histological structure of Mild fluid retention in the interstitial tissue,
seminiferous tubules filled with fewer sperms in the germinal epithelium of
mature sperms
the seminiferous tubule

TD0014 at 1.8 g/kg

TD0014 at 5.4 g/kg

Mild fluid retention in the interstitial Thick germinal epithelium of the
tissue, fewer sperms in the seminiferous tubule containing a full range
seminiferous tubule lumen
of sperm cell lineage
Figure 2. Restorative effects of TD0014 on photomicrographs of testes
TD0014 with all tested doses significantly increased serum testosterone level,
in a dose dependent manner, when compared with positive intoxicated rats.

17

Table 3.11. Restorative effects of TD0014 on serum testosterone in SVP-intoxicated rats
Groups
n
Testosterone (nmol/L)
Group 1: Negative control
9
4.17 ± 1.22
Group 2: Positive control (SVP)
9
1.35 ± 0.44***
Group 3: SVP + TD0014 (1.8 g/kg)
9
4.30 ± 1.10▲▲▲
Group 4: SVP + TD0014 (5.4 g/kg)
9
5.64 ± 1.03▲▲▲≠
***p<0.001 compared with negative control (Student’t-test); ▲▲▲p<0.001 compared with positive
control (Student’t-test); ≠p<0.05 compared with TD0014 at low dose (Student’t-test)

3.4.2.4. Effect on pregnancy rate of female rats
There were no pregnant female rats in the positive control group after the
pairing period. The pregnancy rate in the low- and high-dose TD0014 group was
11.1% and 16.7%, respectively. No differences in pregnancy rate were observed in
TD0014-treated groups.
Chapter 4
DISCUSSION
4.1. Acute and subchronic toxicity of TD0014 in animals

4.1.1. Acute toxicity
The acute toxicity study of TD0014 indicated no changes in the behavior and
in the sensory nervous system responses in the animals. Also no adverse
gastrointestinal effects were observed in the male mice used in the experiment with
up to 56.25 g/kg b.wt. The median acute toxicity value (LD50) of the extract was
estimated to be more than 56.25 g/kg body weight. According to Ghosh (1984) and
Klaasen et al. (1995), TD0014 could be classified as being non toxic, since the
LD50 by oral route was found to be above 15 g/kg body weight.
4.1.2. Subchronic toxicity
4.1.2.1. Body weight and clinical observation
Daily treatment with both doses of TD0014 to male Wistar rats for a period of 90
days did not show any toxicity related morbidities and mortalities. The body weight
changes serve as a sensitive indicator of the general health status of animals. The
weight gains were observed in all animals administered with TD0014. It can be stated
that TD0014 did not interfere with the normal metabolism of animals as corroborated
by the nonsignificant difference from animals in the vehicle control group.
4.1.2.2. Hematological analysis
Evaluation of hematological parameters can be used to determine the extent of
the deleterious effect of TD0014 on the blood of an animal. A blood count test was
undertaken for all the TD0014-treated and control groups and the results show no
significant effects. The insignificant effect of TD0014 on red blood indices (total
red blood cells, hemoglobin concentration, hematocrit, mean corpuscular volume),


18

total white blood cells and white blood cell differential, platelet count indicates that
TD0014 did not affect functions of the hematopoietic system.
4.1.2.3. Biochemical analysis
Liver and kidney function analysis is very important in the toxicity evaluation

of drugs and plant extracts as they are both necessary for the survival of an organism.
Liver is a primary destination for any toxic substance entered to the body,
especially through gastrointestinal route, the liver suffers first. Because of its wide
range of functions, any abnormal change in the liver will definitely affect complete
metabolism of an animal. High levels of transaminases are reported in liver diseases
or hepatotoxicity. The non-significant change of these enzymes between the control
and treated group animals after 90 days administration indicated that TD0014 did
not cause adverse toxic effect or hepatic damage on the liver. Any abnormal change
in total bilirubin, albumin, and total cholesterol might be due to reduced functions
of the liver. Thus, the insignificant change in serum concentration of total bilirubin,
albumin and total cholesterol in the TD0014-treated and control group further
confirmed that TD0014 did not impair the hepatocellular functions at any of the
doses tested.
Kidney is a sensitive organ, whose function is known to be affected by a
number of factors such as drugs including phytochemicals of plant origin that
ultimately lead to renal failure. Creatinine is excreted by glomerular filtration and
the clearance is dependent on the rate at which it is removed from the blood by the
kidneys, therefore, an increase in the plasma creatinine level suggests kidney
damage specifically renal filtration mechanism. In the present study, change in
plasma creatinine level TD0014-treated groups showed non-significant differences
indicating a normal renal function.
4.1.2.4. Histopathology study
Histopathological examinations of liver and kidneys harvested from treated
and control animals provide information to strengthen the findings on biochemical
and heamatological parameters. The microscopic examination revealed that none of
the organs from TD0014-treated rats showed any alteration in cell structure,
inflammation or any unfavourable effects when viewed under the light microscope
using multiple magnification powers. Since there are no significant increases
observed in liver and kidney parameters, therefore strongly suggests that there are
no obvious detrimental effects or morphological disturbances caused by the daily

oral administration of TD0014 for 90 days.
In light of these findings, we may conclude that TD0014 is not toxic in all the
doses studied herein and did not produce any toxic signs or evident symptoms at
acute and subchronic oral toxicity. Looking for the toxicity information of the
natural ingredients of TD0014, we found that most of these herbs had an LD50


19

value above 2 g/kg and were classified by GHS as unlikely to present acute hazard,
simultaneously long-term use on experimental animals also did not cause adverse
effects on the structure and function of some internal organs.
4.2. Androgenic activities and effects of TD0014 on erectile function
4.2.1. Androgenic activities of TD0014
The Hershberger Bioassay is a short-term in vivo screening assay for androgen
agonists which is based on the changes in weight of several androgen-dependent
tissues on the day after treatment is ended. The Hershberger Bioassay achieves its
sensitivity by using males with minimal endogenous androgen production. This is
achieved either through the use of castrated males, provided an adequate time after
castration for the target tissues to regress to a minimal and uniform baseline weight
is allowed, or by the use of weanling males in which there is minimal endogenous
androgen production. Castration enhances the precision of the assay to detect weak
androgens. The intact stimulatory weanling version of the Hershberger bioassay
appeared to be able to consistently detect effects on androgen-dependent organ
weights from medium or highly potent androgens. According to OECD guidelines,
a statistically significant increase in two or more target organ weights of the test
substance groups compared to the vehicle control group indicates that the test
substance is positive for potential androgenic activity.
In the castrated male rats, the weights of the ventral prostate and Cowper’s
glands significantly increased in the TD0014 treatment groups as compared with the

non-treated castrated rats. In the weanling male rats, TD0014 at the dose 5.4 g/kg
significantly increased the weights of ventral prostate, Cowper’s glands and LABC,
meanwhile TD0014 at the dose of 1.8 g/kg altered only the weight of Cowper’s
glands as compared to control group. The above results have shown that TD0014
may have androgenic effects, which seems to be more potent with the dose 5.4 g/kg
than with the dose 1.8 g/kg. To explain the androgenic activity of TD0014 can be
based on the role of phytoandrogens present in some medicinal plants of this
preparation, including cognate or true phytoandrogens (protodioscin from the aerial
parts of Tribulus terrestris), and phytoandrogen-ogenic compounds that augment
the ability of the body to generate androgens were found in Tribulus terrestris
(protodioscin, protogracillin), Eurycoma longifolia (eurycomanone, eurypeptides),
Cistanche tubulosa (echinacoside), Morinda offcinalis (polysaccharides), Panax
ginseng (ginsenoside-Rb1, ginsenoside Rg3, polysaccharides), Lycium sinense,
Cuscuta sinensis, Psoralea corylifolia.
4.2.2. Effects of TD0014 on erectile function
Basal ICP was determined after 2 hours of treatment and before electrical
stimulation of the cavernous nerve. Observing in Chart 3.1, the administration of
TD0014 at 1.8 g/kg/day showed an improvement of ICP level before the start of


20

electrostimulation, which means the herbal preparation has a pro-erectile activity.
Subsequently TD0014 can improve the ICP value during erection. After
electrostimulating the cavernosal nerve, compared to the control group, TD0014
tended to have an elevation in the maximal ICP, total ICP, and a prolonged response
time to the stimulus, i.e prolong the duration of penile tumescence, however, these
results showed no signitificant differences (p > 0.05). Thus, in the current study,
TD0014 given once before nerve stimulation markedly increased baseline ICP,
slightly increased both the maximal ICP and the response time to the stimulus.

These results suggested that TD0014 may be beneficial for patients with erectile
dysfunction, and can improve the patient's sexual life. To explain partly the impacts
of this herbal formula on the enhancement of penile erection may refer to the role
functional mimetics of androgens which do not need to bind to an androgen receptor
to cause a similar functional response as the androgens, found in some medicinal
plants of this preparation, including echinacoside from Tribulus terrestris,
ginsenoside Rg1 from Panax ginseng, chuanxiongzine from Ligusticum wallichii,
and the active ingredients that need to be specifically identified in Eurycoma
longifolia, Angelica sinensis, and Cuscuta sinensis. In addition, medicinal herbs that
increase blood testosterone levels may also be involved in the trend of improving
erectile function of TD0014, including plants containing true phytoandrogens
(protodioscin from Tribulus terrestris) and plants containing phytoandrogen-ogenic
compounds (Tribulus terrestris, Eurycoma longifolia, Cistanche tubulosa, Morinda
offcinalis, Panax ginseng, Lycium sinense, Cuscuta sinensis, Psoralea corylifolia).
The effect of TD0014 on blood pressure was shown in Table 3.5. The results
showed that TD0014 could increase ICP without significantly impacting on MAP.
The data suggested that TD0014 could selectively enhance erectile function without
affecting systemic blood pressure. Therefore it could limit the side effects on blood
pressure and cardiovascular diseases, which are common undesirable effects of
drugs currently used to treat erectile dysfunction.
4.3. Effects of TD0014 on sodium valproate-induced reproductive decline in
male rats
Sodium valproate (SVP) is an antiepileptic drug commonly used in the
treatments of epileptic seizures including panic attack, anorexia nervosa, anxiety
disorder, posttraumatic stress disorder, migraine, psychiatric conditions, and bipolar
disorders. However, SVP has been reported to exert side effects on reproductive
systems in both men and experimental animals. In the epileptic men treated with
SVP, their testosterone levels and semen qualities have been significantly decreased
involving infertility. In animals, SVP significantly decreases FSH, LH, and
testosterone hormones with testicular damages. Results published by Nishimura T et

al. (2013) indicated that 7-week administration orally of TD0014 at the dose of 500


21

mg/kg produced the most pronounced reproductive decline in adult male rats.
Therefore, in this thesis, we have selected SVP at 500 mg/kg/day continuously
received for 7 weeks as a reproductive impairing agent for animals, based on which
assessed protective and restorative effects of TD0014.
4.3.1. Protective effects
The positive effects of the testicular structure is assessed through changes in
testicle weight and histology, and size of seminiferous tubule. TD0014 at both doses
increased significantly the testis weight and size of seminiferous tubule of SVPintoxicated rats. In the histopathological examination of the testis, this herbal remedy
ameliorated partially the testicular histopathological lesions seen. The greater protective
effect was observed for the group treated with TD0014 at the dose of 5.4 g/kg.
The testes have two functions – to produce sperm and hormones, particularly
testosterone. The analysis of semen samples which were collected from cauda
epididymis was used for evaluating the sperm production of testicles. Effects of
TD0014 on sperm quantity and quality were recorded in Table 3.6, Table 3.7, and
Chart 3.3. A dose-dependent increase in the sperm count was observed in the group
treated with different doses of TD0014 (1.8 and 5.4 g/kg) compared to the toxic
group. Meanwhile, the sperm quality index were only improved at the dose of 5.4
g/kg TD0014, with an increase in the sperm viability and motility, and a decrease in
the sperm abnormalities. The beneficial impacts of TD0014 on sperm quantity and
quality were also shown indirectly by the pregnancy rate of female rats after 2
weeks of mating. The pregnancy rate in the positive control group was only 5%,
while the rate of the low-dose TD0014 group was 10% and the high dose TD0014
group was 30%. These results were consistent with an increase in the serum
testosterone and adrenal weight observed in TD0014 groups. The sex accessory
organs are known to be sensitive to serum testosterone level. TD0014 elevated the

level of testosterone so it tended to increase the weights of androgen-dependent
accessory sex organs. There was a positive correlation between the increase in
serum testosterone level of each tested dose of TD0014 with the number of sex
accessory organs increased significantly weight compared to positive control group:
TD0014 at the dose of 1.8 g/kg caused a significant increase in the weights of glans
penis and epidydimis; TD0014 at the dose 5.4 g/kg, in addition to glans penis and
epidydimis, also significantly increased the weights of seminal vesicles and
Cowper’s glands.
From the above results, it can be seen that TD0014 expressed the protective
effects against SVP-induced reproductive toxicity, which seems to be more potent
with the dose of 5.4 g/kg than with the dose of 1.8 g/kg.
4.3.2. Restorative effects


22

Observing the data used to assess the restorative effects of TD0014 on
testicular damage caused by SVP, testicular weight in TD0014 groups tended to
increase higher than the toxic group, in which the increase in weight in the highdose TD0014 group was statistically significant (p <0.05). The size of seminiferous
tubule of the TD0014-treated rats also increased significantly compared to the
positive control group. The percentage of normal structural testicle samples of
TD0014 groups was 66.7%, higher than the toxic group (50%). The improvement in
testicular histology of TD0014 in the restoration model was not as clear as in the
protection model. This may be due to the short-term administration of TD0014 in
the recovery model (10 days), which is not enough to make significant changes in
testicular structure as in the protection model with long-term administration of
TD0014 (7 weeks). More lasting studies are needed to evaluate the long-term
effects of TD0014 on the restoration of testicular structure damage caused by SVP.
Impaired testicular functions after receiving SVP also had been restored
partially in the presence of TD0014. With sperm production, TD0014 helped

accelerate the recovery in both quantity and quality of sperms. TD0014 at all tested
doses significantly increased sperm density compared to the positive control group.
Sperm survival was also higher in TD0014 groups, where TD0014 at high dose
expressed a better effect with a statistically significant increase when compared to
the toxic group. Other indicators of sperm quality, including morphology and
motility of the sperms, all showed a clear recovery when using TD0014. Pregnancy
rates in female rats paired with male rats given orally TD0014 also indirectly
reflected the positive effects of TD0014 on sperm quantity and quality: the
pregnancy rate corresponding to low-dose and high-dose TD0014 was 11.1% and
16.7%, meanwhile no female rats coupled with male rats of the positive control
group were pregnant.
With the testicular endocrine function, TD0014 with all tested doses
significantly increased serum testosterone level, in a dose-dependent manner, when
compared with positive intoxicated rats. This result was consistent with the
restorative effects on testicular structural damage of TD0014 mentioned above. The
trend of increasing adrenal weight in the rats received the test product also
contributed to improving serum testosterone levels. Matching to the increase in
testosterone concentration was the gain in the weights of some secondary sex organs
in the TD0014 groups compared to the positive control group: TD0014 at the dose
of 1.8 g/kg significantly increased the weights of seminal vesicles and LABC; with
TD0014 at the dose of 5.4 g/kg, in addition to seminal vesicles and LABC, a
significant increase in epididymis weight also was detected.
From the above results, it can be seen that TD0014 expressed the restorative
effects on SVP-induced reproductive decline. Likewise the protection model, the


23

impacts seem to be more potent with the dose of 5.4 g/kg than with the dose 1.8
g/kg. The mechanism involved in the protective and restorative effects of TD0014

against sodium valproate induced reproductive toxicity in male rats is unknown.
However, based on the results of the individual studies of herbal components of
TD0014, it is possible to partly explain the effectiveness of the herbal remedy on
these research models.
SVP can inhibit the synthesis and secretion of testosterone through directly
affecting the testes or indirectly affecting the secretion of gonadotropins. Several
medicinal plants used in traditional medicine as the aphrodisiac agents, such as
Tribulus terrestris, Eurycoma longifolia, Cistanche tubulosa, Morinda offcinalis,
Lycium sinense, Panax ginseng, Cuscuta sinensis, and Psoralea corylifolia,
exhibited the ability to raise the concentration of sex hormones (testosterone, LH,
FSH) on many experimental and clinical studies. These effects of medicinal
materials may be due to active phytochemicals, phytoandrogen (Tribulus terrestris,
Eurycoma longifolia, Cistanches Herba, Morinda officinalis, Panax ginseng), or
other mechanisms (Lycium sinense, Cuscuta sinensis, Psoralea corylifolia) which
are continuing to be studied. Besides the increase in the synthesis and secretion of
sex hormones, some herbs such as Panax ginseng and Cuscuta sinensis are also
productive in increasing the sensitivity and ability to respond to these hormones in
the target organs through increasing the expression of androgen receptors, LH
receptors and FSH receptors.
Another important mechanism of SVP-induced organ injuries is the ability to
regulate the activity of many enzymes related to oxidative stress of SVP leading to
elevated free radical production causing cytotoxicity. Along with stimulating effects
on sexual activity, medicinal materials presented in TD0014, such as Tribulus
terrestris, Eurycoma longifolia, Cistanches Herba, Morinda officinalis, Panax
ginseng, Lycium sinense, Cuscuta sinensis, and Psoralea corylifolia also expressed
antioxidant activities in lots of testing. Many other medicinal herbs in the herbal
formula TD0014, although no studies have shown the effectiveness of these plants
in improving reproductive functions, but have been found anti-oxidant properties,
that prevent damage to reproductive organs involving the formation of free radicals
caused by SVP.

CONCLUSIONS
1. Acute and subchronic toxicity of TD0014 in animals
- Acute oral toxicity: the LD50 evaluated by the Litchfield - Wilcoxon method
could not be determined.
- Subchronic oral toxicity: the long-term oral administration of TD0014 for 90
days did not affect body weight and clinical observation, the parameters of