Nghiên cứu tính an toàn và tác dụng của viên nang balanoxi trên thực nghiệm và lâm sàng ở bệnh nhân suy giảm tinh trùng tt tiếng anh

Part A: INTRODUCTION
1. INTRODUCTION
According to the World Health Organization, the percentage of
infertility couples accounts for 12-18%, varying in countries, an average
of 15%. Sperm decline has been increasing and occupying a high
proportion. According to WHO studies, the minimum sperm density
decreased from 40 million/ml (1980) to 20 million/ml (1999), and 15
million/ml (2010). The progressive sperms declined from 50% (1999) to
32% (2010).
The treatment of sperm decline still faces many difficulties, mainly
because sperm decline has many complicated causes. Modern medicine
has many methods of treating sperm decline, but the results are lower
than the expectations of physicians and patients, due to unwanted effects
and long time treatment which have great effects on patients’ health and
economy.
Balanophora sp. of Balanophora genus, three species of Balanophora
fungosa indica, Balanophora latisepala, Balanophora laxiflora have
been found in Tuyen Quang, Hoa Binh, Lao Cai and Yen Bai provinces.
Balanophora sp. is often decocted or soaked in wine to support the
treatment of male diseases as impotence, unconscious semen leakage,
nocturnal emission infertility, and has shown very good results.
The pharmacological effects of Balanophora sp. has currently been
being researched on the treatment of reproductive malediseases.
Balanoxi hard capsules are formulated from total dry extract of
Balanophora indica.
2. OBJECTIVES OF THE RESEARCH
1. Studying the acute and semi-chronic toxicity of Balanoxi on the
experimental animals;
2. Assessing the effect of Balanoxi on sperm decline caused models;
3. Research the effect of Balanoxi on infertility patients due to sperm
decline.

PRACTICAL MEANING AND NEW CONTRIBUTIONS OF THE
RESEARCH
The dissertation is a scientific work conducted systematically from
experimental to clinical levels on Balanophora indica, a valuable
Vietnamese medicinal herb that has long been used in the folk.
Research results showed that Balanoxi has no oral toxicity, has
protective effects, recovers reproduction, increases serum testosterone,
increases the quantity and quality of sperm. The results of the study can
1


be applied on male fertility treatment,its contributions are, therefore,
very practical.
THE STRUCTURE OF THE DISSERTATION
In addition to the introduction and conclusion, the dissertation has 4
chapters:
Chapter 1: Overview
36 pages
Chapter 2: Materials, subjects, and research methods
16 pages
Chapter 3: Research results
45 pages
Chapter 4: Discussion
36 pages
The dissertation has 54 tables, 13 charts, 6 pictures, 19 appendices, and
118 references (44 in Vietnamese, 71 in English, and 3 in Chinese).
Part B: CONTENT
CHAPTER 1: OVERVIEW
1.1. VIEWS OF MODERN MEDICINE ON SPERMS, SPERM
DECLINE, AND TREATMENT OF SPERM DECLINE

1.1.1. The causes of sperm decline
- Sperm decline due to Gonadotropin Releasing Hormone
(GnRH) disorders:
Certain reproductive hormones play a crucial role in sperm production
and development in general as well as in the differentiation stages of
sperm development. Pathological disorders of reproductive hormones
lead to a huge change in the number and quality of sperm:
+ GnRH deficiency
+ Excessive endocrine excretion
- Sperm decline due to disorders of sperm production in the testicles
+ Sperm decline due to genetic diseases
+ Sperm decline due to testicular damages
+ Sperm decline due to a number of other causes
+ Sperm decline due to diet, bacterial infections, high
temperatures, immune factors, living conditions, X-rays, illness, stress,
etc.
1.1.2. The methods of sperm decline treatment of modern medicine
As the causes of sperm decline are complicated and they often coexist
together, it is necessary for physicians to prescribe reasonable treatment
such as a combination of medical, surgical, and fertility treatments to
increase the rate of pregnancy.
- By medication: medication treatment should be indicated for patients
with sperm decline due to unknown etiology.Commonly used
2


medications: Antioxidants (Glutathion, L-arginin), hormones
(Gonadotropin, Androgen, and estrogen receptor antagonists)
- Surgery: surgery should be indicated for patients with impaired
spermatozoa that affect the sex life, such as varicose veins, ectopic

testicles, testicular water or inguinal hernia. Connective surgery should
be indicated for patients with no sperms by connecting vas deferens.
The 3 main assisted reproductive technologies for treating male
infertility due to sperm decline include IUI, IVF, and ICSI.
1.2. VIEWPOINTS OF TRADITIONAL MEDICINE ON SPERMS,
SPERM DECLINE, AND TREATMENT OF SPERM DECLINE
1.2.1. Conceptions of traditional medicine on the process of Jing
production and the factors affecting the Jing production
- Jing, according to traditional medicine consists of two types (inborn
Jing and after-born Jing), it is the basic material for body composition
and body nutrition. During body development, Jing is always consumed
and also regularly replenished by spleens to maintain living activities.
Both types of Jing are stored in the kidneys.
1.2.2. Disease types and the treatment of sperm decline according to
traditional medicine
* Jin Kidney deficiency type: darkish face, dizziness, ringing in the ears,
weak and tired back and knees, loose or falling teeth, growing gray,
tiredness, forgetfulness, impaired memory, dullness, weak limbs, slow
movements, rapid aging, impotence, no conception, low semen volume,
sperm deficiency. Taut deep no force Chi pulse, if there is Yin
deficiency Fire excess, there will be five center restlessness and fever,
reddish tongue, less moss, taut pulse. Strategies: Reinforcing Qi and
Nourishing and Tonifying Yin Kidney. Remedies: ‘Zuogui wan’ and
‘Wuziyanzong wan he liuwei wan’
* Yang Kidney deficiency type: darkish face, fear of cold, cold feet,
back and knees are tired and cold; Patients feel tired, uninterested; large
amount of urine, urinating many times, loose stools in the early
morning; Low sperm count, weak sperm motility, impotence, premature
ejaculation, deep fine pulse or deep slow pulse, pale tongue, white moss.
Strategies: Warming Kidney and enhancing Yang, tonifying Jing and

reinforcing Qi. Remedies: ‘Jin guishen Qi wan’, ‘wuziyanzong’, and
‘Yougui Yin’
* Spleen deficiency Jing damage type: fatigue, pale face, dizziness,
headache, forgetfulness, poor appetite, bloating, low sperm count, sperm
vitality weakness, impotence, premature ejaculation, tongue quality,
rough, white tongue moss, forceless pulse. Strategies: reinforcing Qi and
3


invigorating Spleens, nourishing blood and promoting the production of
Jing. Remedies: ‘Gui pi tang’ (sheng fang ji) and ‘bazhen sheng jing
tang’
*Liver Qi stagnation and Qi stagnation blood stasis type: weak sperm,
high death rate, low sperm count, painful testicles, testicular vein,
impotence, feeling of bloating in chest and stomach, darkish tongue with
blood stasis spots, sluggish taut pulse or rapid taut pulse. Strategies:
dispersing the stagnated liver Qi; alleviating mental depression,
nourishing spleens and regulating nutrients from food; promoting blood
circulation, removing blood stasis and restoring Jing flow. Remedies:
‘Heijiaoyao san’ (he jiju fang) and ‘xuefuzhuyu tang’ (yi Lin gaituo).
* Wetness heat in lower jiao type infertility: backache, tired legs,
fatigue, feeling bitter in mouth, anorexia, dizziness, dry mouth without
wanting to drink water; itching or severe genital irritation, soreness and
obstruction in perineum or testes, dense semen with foul odor, which can
be inferred semen contains many red and white blood cells; low sperm
count, high dead sperm rate, cloudy urine, burning sensation in the
urethra during urination or ejaculation; reddish tongue, yellowish moss,
rapid smooth pulse or soft smooth pulse.Strategies:Clearing heat,
promoting diuresis to eliminate wetness evil, and removing toxic
substances. Remedies: ‘Pi jie fen Qing Yin’ (dan xi xinfa) and ‘Long tan

zuogan’ (yi sun jinjian)
1.2.3. Overview of Balanophora sp.
There are about 20 species of Balanophora sp. in the Balanophora
genus. In Vietnam, only three species of Balanophora sp. have been
found namely Balanophora fungosa indica, Balanophora latisepala, and
Balanophora laxiflora. Balanophora indica is found in moist or
broadleaf limestone forests. Its samples were collected in December
2015 at the summit of Hong Mountain in Khuon Man, Binh Yen, Son
Duong, Tuyen Quang. The scientific name is assessed at the Vietnam
Institute of Medicinal Materials.
In Vietnam this medicinal plant is often used as a tonic for postpartum
women to stimulate appetite. In particular, this medicinal plant is often
soaked in winefor men to enhance and endure erections. In Malaysia, all
Tinosporacapillpes Gagep is also used as aphrodisiac.
Effects according to traditional medicine
- Pharmacological properties: Sweettaste has the effects of warming
channels, liver, kidneys, and colon. Balanophora sp. has two main
therapeutic effects: notifying kidneys and supporting kidneys, which are
suitable for treating impotence, infertility, tired legs, and exhaustion; and
4


laxative, suitable for treating fluid deficiency disorders which lead to dry
intestines, and constipation.
- Mainly treating physiological weakness, impotence, frigidity,
backache, knee pain, and anorexia.
CHAPTER 2: MATERIALS, SUBJECTS,
AND RESEARCH METHODS
2.1. MATERIALS FOR RESEARCH
Balanophora indica samples were collected in December 2015 at the

summit of Hong Mountain in Khuon Man, Binh Yen, Son Duong, Tuyen
Quang. The scientific name is assessed at Vietnam Institute of Medicinal
Materials.
Balanophora indica is produced at the Research and Application Center,
the Military Institute of Traditional Medicine, under its standards.
2.2. RESEARCH SUBJECTS
The white mouse, Swiss strain, both sexes, weighing 18-22 grams, used
to study acute toxicity and the effect of the medication on fertility and on
chromosomes.
Newzealand White rabbits (semi-chronic toxicity test), white fur,
healthy, both sexes, weighing 1.8 -2.5kg; supplied by theBreeding
center, Testing Institute
2.3. RESEARCH METHODS
2.3.1. Study the toxicity of Balanoxi on experimental animals
2.3.1.1 Acute toxicity study
* Principles of implementation
The acute toxicity of Balanoxi by oral route was determined mice by
Litchfied-Wilcoxon method. The research was conducted at the
Department of Experimental Research, Military Institute of traditional
medicine.
* Procedure
Male white mice, weighing 18-22 grams, were divided into 8 groups,
each group of 8 mice. Each group took Balanoxi with increasing doses,
from the highest dose that did not kill mice to the lowest dose that
caused all mice dead.
Mice were monitored continuously in 24 hours after taking the
medication.Their general conditionswere monitored in 7 days after
medicationtaking. The number of dead mice was counted in the groups
within 72 hours after taking the medication to determine the LD50.
2.3.1.2. Semi-chronic toxicity

5


The semi-chronic toxicity of Balanoxi was determined on white
Newzeland White rabbits, white, healthy, both sexes, weighing 1.8 2.5kg; supplied by theBreeding center, Testing Institute. The study was
conducted at the Department of Experimental Research, Military
Institute of traditional medicine in accordance with WHO guidelines and
regulations of the Ministry of Health of Vietnam.
2.3.2. Study on the effects of Balanoxi on the sperm declined by Natri
valproat model
* Studying the protective effects of Balanoxi on male white mice whose
sperms were declined by Natri valproat
Male white rats were randomly divided into 3 groups of 15:
- Group 1: Oral 0.9% sodium chloride + solvent.
- Group 2: Natri valproat 500mg/kg/day + solvent.
- Group 3: Natri valproat 500mg/kg/day + Balanoxi 0.7g/kg body
weight.
Male rats in all 3 groups received the medication and solvent
continuously in 7 weeks. Mice were given a dose of 2 ml/100g body
weight, 2 times/day, every 2 hours. After 5 weeks, the male mice in
groups mated with 2 female mice in the same cage for 2 weeks.
* Studying the recovery effects of Balanoxi on sperms reduced by Natri
valproat model.
Adult male rats were randomly divided into 3 groups of 15 each.
- Sperm reduced by Natri valproat:
+ Group 1 (control group): did not drink Natri valproat, only drink Natri
chloride 0.9% 20ml/kg/day
+ Groups 2 and 3: Natri valproat dose of 500mg/kg/day in 7 weeks.
- After 7 weeks of taking Natri valproat, rats in the groups were given
medicine in 4 weeks as follows:

+ Group 1: solvent 20ml/kg body weight.
+ Group 2: solvent 20ml/kg body weight.
+ Group 3: Balanoxi 0.7g/kg body weight.
2.3.3. Study on the therapeutic effects of Balanoxi on the treatment of
infertility patients due to sperm decline
- Research design: prospective study, open clinical trial, comparing
differences before and after the treatment.
- Sample size: Sample size 30, male patients aged 20-60 with sperm
decline Yang Kidney deficiency type.
- Selection criteria:
According to modern medicine: Age from 20 to 60; Infertility I and
infertility II due to sperm decline; Voluntarily participated in the
6


research; fully abide orders and instructions of the doctor during the
research procedure.
According to Traditional Medicine: Patients with sperm decline Yang
kidney deficiency type
- Dosages and administration: 10 Balanoxi500mg daily, 2 times (in the
morning and afternoon), after eating, continuously in 10 weeks.
* Tests
- Blood biochemistry before the treatment: Urea, Creatinin, AST, ALT
to assess and exclude patients with liver and kidney pathologies. After
the treatment, the treated patients were re-tested these values to assess
liver and kidney function status to determine if they were affected by
Balanoxi.
- Quantification of serum testosterone before the treatment: assessing the
quantity of serum testosterone in selected patients after the treatment.
- Seminogram before and after the treatment: seminogramis the most

important test to assess the fertility of men. The testing standards and
assessment criteria are under the guidance of WHO (1999) and (2010)
[2], [3].
- Testicular and testicles veinsultrasoundbefore the treatment to rule out
other causes of sperm decline.
Seminogram,
biochemistry,
hematology,
and
hormones
Testosteronetests were done at the labo of Military Institute of
traditional medicine.
2.4. Data processing: The research data was processed statistically by
the Student's t-test method. The data is expressed in the form: X ±SD
. The difference is significant when p <0.05.
2.5. Ethical issues in the research: After the study, the toxicity and
effects of Balanoxi on experimental animals to prove their safety and
effectiveness, the medication was tested on voluntary patients under the
permission of the Scientific Council, the Military Institute of traditional
medicine.
CHAPTER 3: RESEARCH RESULTS
3.1. Results of the toxicity test
3.1.1. The Acute toxicity study
The white mice took the medication at the highest dose of 20g/kg of
body weight but no mice died, and there were no abnormal symptoms
within 72 hours after taking the drug and during the next 7 days.
3.1.2. Results of the semi-chronic toxicity study
7

During the experiment, the rabbits in all 3 groups were eating and acting
normally, were agile, had bright eyes, silky hair, and firm stools. No
significant symptoms were observed in all three groups of the rabbits
during the study period. After 4 and 8 weeks of the treatment with
Balanoxi, red blood cell count, Hemoglobin content, Hematocrit, white
blood cell count, white blood cell count and platelet count, AST, ALT,
GGT, total bililubin creatinine concentration, Ure in the rabbit blood in
study groups 1 and 2 were notdifferent from that of the control group
and there were no differences between the research groups at the same
testingtime (p> 0.05).
There were no general pathological changes in macroscopic morphology
liver images in study group 1s and 2.
3.2. Results of the protective and restorative effects of Balanoxi on
the experimental models
3.2.1. Protective effects
Table 1.Balanoxi's protective effects on genitals weight ofsperm decline
induced by Natri valproat male rats
Groups
Group
1:
Natri clorid
+ water
Group
2:
Valproat +
water
p2-1
Group
3:
Valproat +

Balanoxi
p3-1
p3-2

Genitals weight (mg/100g body weight)
Seminal
Cowper’
Testicles
Prostate
Glans
vesicle
s gland
1.157
0.270
0.144
0.041
0.054
±
±
±
±
±
0.086
0.057
0.021
0.007
0.006
0.991
0.207
0.119

0.036
0.049
±
±
±
±
±
0.092
0.070
0.013
0.006
0.005
<0.05
<0.01
< 0.05
< 0.05
> 0.05
1.063
0.302
0.137
0.039
0.052
±
±
±
±
±
0.093
0.051
0.022

0.006
0.007
< 0.05
>0.05
>0.05
> 0.05
> 0.05
> 0.05
<0.05
< 0.05
>0.05
> 0.05

Levator
muscles
0.359
±
0.047
0.350
±
0.036
> 0.05
0.352
±
0.029
> 0.05
> 0.05

Comments: Rats in group 3 increased testicles weight compared to
group 2, but the difference was not statistically significant (p> 0.05).

The weight of seminal vesicles and prostate increased significantly
compared to group 2 (p <0.05). The weight of Cowper’s gland, glans
and levator muscles in group 3 were not significantly different from
group 2 (p> 0.05).
Table 2.Balanoxi's protective effects on blood testosterone
concentrations ofsperm decline induced by Natri valproat male rats
8


Groups
Group 1: Natri clorid + water
Group 2: Valproat + water
p2-1
Group 3: Valproat + Balanoxi
p3-1
p3-2

Testosteron (nmol/l)
8.91 ± 1.38
6.09 ± 1.20
< 0.01
8.50 ± 1.08
> 0.05
< 0.01

Comments:Testosterones concentration of group 3 increased compared
to the group 2 (p <0.01) and no significant difference compared to the
group 1 (p> 0.05).
Table 3. The effects of Balanoxi on sperm density and rate of of sperm
decline induced by Natri valproat male rats

Groups
Group 1: Natri clorid +
water
Group 2: Valproat +
water
p2-1
Group 3: Valproat +
Balanoxi
p3-1
p3-2

Sperm density and rate of male
Spermvitality
Sperm density/ml
rate(%)
126.91 ± 18.30

82.917 ± 5.10

80.167 ± 11.49

71.75 ± 6.95

< 0,001

< 0.001

88.58 ± 13.24

81.67 ± 5.36

< 0.001
> 0.05

> 0.05
< 0.01

Comments: Sperm density of group 3 was notsignificantly
differentfrom that of the control group (p> 0.05). The sperm vitality rate
increased significantly compared to the control group (p <0.01).
Table 4.Balanoxi's protective effects on sperm motility of of sperm
decline induced by Natri valproat male rats
Progressive rate (%)
NonFast
Slow
Non-motile
progressive
Group 1: Natri
56.25 ±
33.42 ± 4.48 4.92 ± 1.44 5.42 ± 1.16
clorid + water
5.19
Group 2: Valproat
64.25 ±
20.58 ± 3.67 7.25 ± 2.05 7.92 ± 1.31
+ water
5.08
p2-1
< 0.001
< 0.01

< 0.001
< 0.01
Group 3: Valproat
55.20 ±
34.42 ± 16.30 6.08 ± 2.06 4.33 ± 2.06
+ Balanoxi
15.95
p3-1
> 0.05
> 0.05
> 0.05
> 0.05
p3-2
< 0.01
> 0.05
< 0.001
> 0.05
Groups

9


Comments: The group 3 had a significantly higher rate of rapid
progressive sperms than that of the group 2 (p <0.01) and there were no
differences compared to group 1 (p> 0.05); meanwhile, the percentage
of non-progressive sperm rate significantly decreased compared to group
2 (p <0.001) and no difference compared to the group 1 (p> 0.05). The
slow progressive rate and the motility ratewas not different from that of
the control group (p> 0.05).
Table 5.Balanoxi's protective effects on sperm motility in sperm decline

induced by Natri valproat male rats
Groups
Group 1: Natri clorid + water
Group 2: Valproat + water
p2-1
Group 3: Valproat + Balanoxi
p3-1
p3-2

Sperm motility speed (µm/s)
44.48 ± 6.43
36.16 ± 8.87
< 0,05
43.59 ± 5.91
>0.05
< 0.05

Comments:The sperm motility speed of mice in group 2 decreased
significantly compared to the group 1 (p <0.05). The figure ofthe group
3 increased significantly compared to group 2 (p <0.05).
- Evaluation of testicular histopathology (protective effect)
* Density of spermatomy tube: Density of spermatomy tube of 3 groups
was high, there were no differences between groups.
* The thickness of the epithelial layer: The thickness of the epithelial
layer of the semen of the group 1 (control group) and group 3 (Balanoxi)
were mostly of medium and thick, 92.33% and 83.97% respectively,
while that of the group 2 (the Natri valproat) was 55.4%.
* Sertoli cell density: The Sertoli cell density of the group 1 was 100%
within the normal limit, the average of the group 3 was 84.77%, both
higher than that of group 2 with only 50.37% in the average limit.

Typically, group 2 had only 44.97% of Sertoli cells.
* Sperm density: Sperm densities of the groups 1 and 3 were both of
medium and, 91.93%, (high: 51.47%, average 40.47%) and 84 , 9%
(high 49.27%, average 35.63%)respectively, while that of the group 2
was only 54.7%, (high 20.6%, average 34.1%)
- The protective effects of Balanoxi on the reproductive indices in female
rats mated with decline induced by Natri valproat male rats
Table 6.Balanoxi's protective effects on reproductive indices in female
mice subjected to surgery to observe
Groups
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Indices
Pregnancy rate
Number of corpus
luteum/ 1 female
mouse
Number of fetus/ 1
female mouse
Number of health
fetus / 1 female
mouse

Group
1

Group
2

p2-1

Group
3

p3-1

p3-2

75.50

33.33

<
0.05

58.33

<0.0
5

<
0.05

10.33
± 1.56

10.67
± 1.50

>
0.05

10.50
± 1.00

>
0.05

>
0.05

10.00
± 1.22

8.25
± 0.96

<
0.05

9.00
±0.82

>
0.05

>
0.05

9.78
± 1.09

7.00
± 1.15

<
0.01

8.57
± 0.98

<
0.05

<
0.05

Comments: In the group 3, the pregnancy rate, the average number of
of health fetus / 1 female mouse increased significantly compared to
group 2 (p <0.05). The number of corpus luteum/ 1 female mouse was
not significantly different in comparison with the other groups (p> 0.05).
3.2.2. Recovery effects
The recovery effects of Balanoxi on male white rats caused reproductive
impairment by sodium valproat.
Table 7.The recovery effect of Balanoxi on sperm decline induced by
Natri valproat male rats
Weights of genital organs
(mg/100g body weight)
Groups

Testicles
Group 1: Natri
clorid + water
Group
Valproat
water
p2-1

2:
+

Group
Valproat
Balanoxi

3:
+

p3-1
p3-2

0.041
±
0.002
< 0.05

Levator
muscle
s
0.05

0.330
±
±
0.003
0.023
0.048
0.316
±
±
0.003
0.018
> 0.05 > 0.05

0.127
±
0.007

0.042
±
0.002

0.049
±
0.002

0.323
±
0.021

< 0.05

> 0.05

> 0.05
> 0.05

> 0.05
> 0.05

> 0.05
> 0.05

Seminal
Cowper’
Prostate
vesicle
s gland

1.004
±
0.112
0.845
±
0.115
<0.01

0.251
±
0.038
0.202
±

0.031
< 0.01

0.136
±
0.010
0.122
±
0.099
< 0.01

0.901
±
0.102

0.221
±
0.027

<0.05
>0.05

< 0.05
> 0.05

0.044
± 0.003

Glans

Comments: The weights of testices, seminal vesicles and prostate
weight mice inthe group 3 increased compared to that of the group 2, but
11


the difference was not statistically significant (p> 0.05). The weight of
glans and levator muscles in the group 3 was not significantly different
from that of the group 2 (p> 0.05).
Table 8.Balanoxi's recovering effects on blood testosterone
concentrations of sperm decline induced by Natri valproat male rats
Groups

Testosteron (nmol/l)

Group 1: Natri clorid + water
Group 2: Valproat + water
p2-1
Group 3: Valproat + Balanoxi
p3-1
p3-2

8.42 ± 1.10
6.87 ± 1.14
< 0,01
8,30 ± 1,00
> 0,05
< 0,01

Comments:Testosterone blood concentration ofthe group 2 decreased
significantly compared to that of the group 1, the testosterone

concentrations ofthe group 3 increased compared to that of the group 2
(p <0.01).
Table 9.Balanoxi's recovering effects on sperm densities and ratios of
sperm decline induced by Natri valproat male rats
Sperm density and rate
Sperm density /ml
Alive sperm rate (%)

Groups
Group 1: Natri clorid +
water
Group 2: Valproat +
water
p2-1
Group 3: Valproat +
Balanoxi
p3-1
p3-2

110,67 ± 27,28

80,33 ± 9,01

75,25 ± 18,78

66,67 ± 6,27

< 0,01

< 0,001

80,67 ± 15,34

77,08 ± 6,41

< 0,01
> 0,05

> 0,05
< 0,01

Comments:The sperm density of the group 3 (Balanoxi) was not
different from that of the control group (p> 0.05). The percentage of
alive sperm increased significantly compared to that of the control group
(group 2) (p <0.01). The percentage of alivesperm in group 3 did not
change significantly in comparison with the group 1 (p> 0.05).
Table 10.Balanoxi'srecovering effects on sperm motility of white rats
caused reproductive impairment by Natri valproat.
Groups

Mobility & Progressive (%)
Rapid
Slow
NonNon-motile
progressive progressive progressiv
12


e
Group 1: Natri clorid

34.42 ± 7.87 5.08 ± 1.50 5.58 ± 1.44 54.92 ± 8.03
+ water
Group 2: Valproat +
22.08 ± 6.95 7.33 ± 2.01 7.42 ± 2.02 63.17 ± 7.11
water
p2-1
< 0.01
< 0.01
< 0.05
< 0.05
Group 3: Valproat +
32.17
55.17 ±
6.17 ± 1.85 6.50 ± 1.88
Balanoxi
±13.54
13.67
p3-1
> 0.05
> 0.05
> 0.05
> 0.05
p3-2
< 0.05
> 0.05
> 0.05
> 0.05

Comments: The group 3 had a significantly higher rate of
rapidprogressive spermsthanthe group 2 (p <0.05) and there were no

differences between the group 3 and 1 (p> 0.05). ). The rates of slow
progressive, non-progressive, and non-motile sperms were not different
from the control group (p> 0.05).
Table 11. Balanoxi's recovering effects on sperm motility of sperm
decline induced by Natri valproat male rats
Groups

Motility speed (µm/s)

Group 1: Natri clorid + water

42.50 ± 7.53

Group 2: Valproat + water
p2-1

34.17 ± 8.90
< 0.05

Group 3: Valproat + Balanoxi

41.67 ± 6.32

p3-1
p3-2

> 0.05
< 0.05

Comments:The sperm motility speed of the group 2 decreased

significantly compared to the group 1 (p <0.05). The sperm motility
speed of the group 3 increased significantly compared to the group 2 (p
<0.05).
- Evaluate the recovering effects of Balanoxi on the histopathology of
the testicles
* Density of spermatomy tube: Density of spermatomy tube of 3 groups
was high; there were no differences between groups.
* The thickness of the epithelial layer: The thickness of the seminal
epithelial layer of groups 1 and 3 were of medium and thick (97.67%
and 98.23%), while the figure was75.9% in the group 2.
* Sertoli cell density: Sertoli cell density within the normal limit of the
group 1 was 100%, the ratio for the group 3 and 2 was 92.37%
and72.87%, respectively.
13


* Sperm density: The sperm density of groups 1 and 3 were of high and
average (98.07% and 97.03%), higher than that of the group 2 (72.73%).
Table 12.Balanoxi'srecovering effects on the reproductive indices in
female mice subjected to surgery to observe
Indexes
Pregnancy rate
Number of corpus
luteum/ 1 female
mouse
Number of fetuses /
1 female mouse
Number
of
healthyfetuses / 1

female mouse

Groups
Group
p2-1
1
<
50.00
0,05

p3-1

p3-2

<
0.05

<
0.05

9.67
± 1.56

>
0.05

9.70
± 1.70

>

0.05

>
0.05

9.00
± 1.40

6.60
± 1.67

<
0.05

7.80
±0.80

>
0.05

>
0.05

8.7
± 1.09

5,80
± 1.30

<

0,01

7,20
± 1.20

<
0.05

<
0.05

Group
1

Group
1

83.33

33.33

9.30
± 1.70

Comments: The pregnancy rate, the average number of healthy fetuses/
1 mother mouse of the group 3increased significantly compared to the
group 2. The number of corpus luteum/1 female mouse in groups was
not statistically different. (p> 0.05).
3.3. Results of the study on the therapeutic effects of Balanoxi on
infertility patients due to sperm decline

3.3.1. . Results of the study on the therapeutic effects of Balanoxi on
infertility patients due to sperm decline
Table 13. Comparison of serum testosterone concentrationsbefore and
after the treatment
Time
Before the treatment
After the treatment

Testosterone indices (nmol/l)

X´ ± SD
16.50 ± 4.79
18.20 ± 4.48

Max- Min
8.25 - 28.5
9.88 - 27.8

p
0.0038

Comments:Before the treatment, the average serum testosterone
concentration was 16.50 ± 4.79 (nmol / l), after the treatment the figure
increased to 18.20 ± 4.48 (nmol / l). The difference was statistically
significant (p <0.05).
Table 14. Comparison of the seminogram test results before and after
the treatment

14

Indices

Before
the
treatmen
t (1)
After
the
treatmen
t (2)

X´ ±
SD
Max
Min
X´ ±
SD
Max
Min

p* 1-2

Alive
sperm
(%)

Progressiv
e (%)

Rapid
progressiv
e (%)

Normal
-shaped
(%)

18.2 ±
10.60

33.83
±
9.74

22.07 ±
7.92

1.23 ±
1.94

22.43
± 7.28

0 - 43

0 - 49

0 - 40

0-7

0 - 35

32.57 ±
13.84

48.17
± 7.90

38.67 ±
9.07

9.63 ±
4.52

30.8 ±
6.59

7 - 58

34 63

14 - 55

0 - 20

20 - 44

<0.001

<0.001

<0.001

16.6 ± 6.25

8.4 ± 4.16

8.37 ±
6.30

2 - 27

0 - 19

0 - 26

14.37 ±
10.91

<0.00
1
14.33
± 8.96

4 - 44

4 - 37

<0.001

X´ ±
Changes

Density
(milion/ml
)

SD
Max
-Min

Comments:Before the treatment, the average sperm density was 18.2 ±
10.6 (%), after this treatment, the density increased to 32.57 ± 13.84 (%)
(p<0.001). The average survival rate of sperm before treatment was
33.83 ± 9.74 (%) increased to 38.67 ± 9.07 (%) after treatment (p
<0.001).
Other indices of semen, such as progressiveness, motile and normalshaped sperm increased significantly after the treatment (p <0.001).
Before the treatment, there were 14 patients with sperm density ≥ 20
million/ml with an average density of 27.57±6.85 million/ml (the lowest
was 20 million/ml and the highest was 43 million/ml). After the
treatment, there were 23 patients with sperm density ≥ 20 million/ml
with an average density of 38.48±9.63 million/ml (the lowest of 20
million/ml and the highest of 58 million/ml). (p<0.001).
Before and after the treatment, no patients had ≥ 75%alive sperms.
However, the average survival rate beforethe treatment was 33.83±9.74
(%) (the lowest was 0% and the highest was 49%); After the treatment
the figure increased to 48.17±7.90 (%). Before and after the treatment,
no patient had ≥ 25% rapid progressive sperms. The average progressive

rate before the treatment was 1.23±1.94 (%) (the lowest was 0% and the
15


highest was 7%), the figure increased to 9.63±4.52 (%) after the
treatment (the lowest was 0% and the highest was 20%) (p <0.05).
Table 15.Changes in sperm count and quality after the treatment
Indices
Increase in sperm count and quality
Increase only in sperm count (sperm
density)
Increase only in sperm quality (A,
PR+NP, DD,)
No changes
Total

Number of
patients (n)
29
0

Percentage %

0

0

1

3.33

30

100

96.67
0

Comments: After finishing the treatment, 29 patients had rises in their
sperm count and quality, accounting for 96.67%; Only 1 patient had no
changes (3.33%). No patients increasedonly in either sperm count or
quality.
Table 16. Proportion of wives getting pregnant after the treatment
Indices
Wives getting pregnant
Wives not getting pregnant
Total

Number of
patients (n)
9
21
30

Percentage %
30.0
70.0
100

Comments: After the treatment, there were 9 wives of the patients got

pregnant, accounting for 30.0%
Table 17. Changes in seminogram test results before and after the
treatment between two groups of patients whose wivesgot pregnant and
did not

Wife pregnant

Indices
Before
´ ±
the test X
SD
(1)
MaxMin
After
X´ ±
the test
SD
(2)
MaxMin
p*1-2

Density
(million/
ml)

Alive
sperm
(%)

Progressive
(%)

Rapid
progressive
(%)

Normal
-shaped
(%)

12.44 ±
7.32

32.56 ±
13.12

21.33 ±
9,22

0.33 ± 1.0

21.33 ±
10.45

0 - 26

0 - 45

0 - 31

0-3

0 - 35

33.11 ±
16.02

46,89 ±
9.10

37,33 ±
12.33

8.78 ± 4.15

33,89 ±
5.16

7 - 58

34 - 62

14 - 53

0 - 16

26 - 40

0.002

0.0026

<0.001

<0.001

<0.001

16


Wife not pregnant

´
Changes X
(3)
SD
MaxMin
Before
´
the test X
SD
(1)
MaxMin
After
X´
the test
SD
(2)

MaxMin
p*4-5
´
Changes X
(6)
SD
MaxMin
p**3-6

±

±

±

±

20.67 ±
14.74

14.33 ±
11.34

16.0 ± 7.30

8.44 ± 4.16

12.56 ±
7.65

2 - 44

-4 - 37

2 - 26

0 - 16

2 - 26

20.67 ±
10.97

34.38 ±
8.23

22.38 ±
7.53

1.62 ± 2.13

22.90 ±
5.69

4 - 43

19 - 49

10 - 40

0-7

11 - 30

32.33 ±
13.22

48.71 ±
7.50

39.24 ±
7.57

10.0 ± 4.72

29.48 ±
6.79

10 - 52

36 - 63

26 - 55

0 - 20

20 - 44

<0.001
11.67 ±

7.76

<0.001
14.33 ±
8.06

<0.001
16.86 ±
5.93

<0.001

<0.001
6.57 ±
4.78

-4 - 31

0 - 30

5 - 27

0 - 19

0 - 16

0.018

1.000

0.737

0.970

0.007

8.38 ± 4.26

Comments: Before the treatment, the average sperm density was
12.44±7.32 (million/ml) in the group of patients whose wives who got
pregnant, the figure increased to33.11±16.02 (million/ml) after the
treatment. The average sperm density was 20.67±10.97 (million/ml)),
the group of patients whose wives who did not get pregnant before the
treatment, the figure increased to 32.33±13.22 (million/ml) after the
treatment.
There was an increase of20.67 ± 14.74 (million / ml) in the sperm
density after the treatment in the group of patients whose wives who got
pregnant. Also, there were growth in the number of alive, progressive,
rapid progressive, and normal-shaped sperms in the 2 groups of patients
after the treatment in comparison with before the treatment, the
difference was statistically significant (p <0.05; p <0.001). The value of
increase the number of alive, progressive, and rapid progressive sperms
in the group of patients whose wives got pregnant compared to those
whose wives did not get pregnant was not significantly different (p>
0.05), The value of increase the number normal-shaped sperms in the
group of patients whose wives got pregnant was higher than that of the
group whose wives did not get pregnant (p> 0.05) (p <0.01).
- Changes in the traditional medicine disease type symptoms
Table 18.Changes in the traditional medicine disease type symptoms
after the treatment

17


Symptoms

Before the
treatment

After the
treatment

p

n
11

%
36.67

n
8

%
26.67

<0.05

Pale tongue, whitish
moss

30

100

4

13.33

<0.05

Pain and back and
knees

30

100

17

56.67

<0.05

Premature ejaculation

15

50.0

2

6.67

<0.05

Erectile dysfunction

15

50.0

4

13.33

<0.05

Dark greenish face

Comments: Before the treatment, there were 11 patients with darkgreenish face (36.67%), 30 patients with pale tongue, white moss, and
backache (100%); 15 patients had premature ejaculation (40.0%); 15
patients with erectile dysfunction (50.0%). After the treatment, the
figures decreased to 26.67%, 13.33%, 67.67%, 6.67%, and 13.33%,
respectively (p <0.05).
- Unwanted effects of Balanoxion patients:After the treatment, no
patients experienced undesirable clinical signs (nausea, bloating, rash,
insomnia, headache, dizziness).
CHAPTER 4: DISCUSSION
4.1. The toxicity of the extract of Balanophora indica
- Acute toxicity

The acute toxicity dose of Balanoxi LD50 was not determined in the
white mice by oral route by the method of Litchfield - Wilcoxon. The
maximum dose that the mice can tolerate was 20g/kg of body weight
which was 16.66 times of the expected clinical dose.
- Semi-chronic toxicity
The results of the semi-chronic toxicity study of Balanoxi made from
Balanophora indica showed that it had non-chronic toxicity in the dose
equivalent to clinical dose of 0.3g/kg/day 3 times the clinical equivalent
dose of 0.9 g/kg/day in 8 consecutive weeks. The results showedthe
medication can be used for long-term use in humans, which is also
consistent with the folk experience of using Balanophora indica for
healing.
18


4.2. The effects of Balanoxi on the sperm declined model
The effects of Balanoxi on spermatogenesis and testicular function may
all be related to the androgen activity of the herbal medication. Balanoxi
may have stabilized the blood testosterone levels before the depleting
agent in some ways; it might be from an exogenous Androgen or
impacting the Testosterone metabolism from Cholesterol, or stimulating
enzymes to participate into the metabolism and synthesis of
testosterones such as dehydrogenase or hydroxylase. This synthesis and
metabolism can occur in the testices, liver, adrenal cortex, etc.
According to the study of Nguyen Thanh Huong (2017) on the effects of
the philosophy of Balanophora laxiflora dose o0.28g/kg and 1.4g/kg
body weight of rats showed clear Androgen activity. Balanoxi was made
from Balanophora indica which may also have similar androgen
activity, which is consistent with studies on chromatographic diagrams
of the chemical composition Balanophora sp. by Cam ThiLinh, which

isolated mainly compounds including β-sitosterol, a plant original
steroid. According to previous studies, β-sitosterol has stimulating
effects on immune system, relieves pain, reduces inflammation and
enhances sexual function. This may be a key factor leading to Balanoxi's
protective effects against reproductive toxins on the white rats in this
study.
The recovery effects of Balanoxi on the weight of reproductive organs
was unclear, it is possible that the treatment duration was not long
enough to assess the effects. The shortage of time might due to the fact
that the reproductive organs were operated when they were most
severely damaged, they,thus, may not be able to recover by themselves
while the new reproductive cell layers were not born to replace them yet.
The effect on sperm quality may be due to the fact that Balanoxi acts
through the Androgen activity to the sperms, causing them to continue to
be produced from undamaged sperm cells. Balanoxi restores and
promotes their activity more strongly through Androgen activity, which
is consistent with the results of the Testosterone concentration of mouse
blood. It may be from an exogenous Androgen in Balanoxi capsules or
acting on the pathway of Testosterone metabolism from Cholesterol, or
stimulating enzymes involved in the metabolism and synthesis of
testosterone such as dehydrogenase or hydroxylase. This synthesis and
metabolism can occur in the testes, liver, adrenal cortex, etc.
Hard Balanoxi capsules have the effect of restoring reproductive cells
very clearly, reproductive cells on the histopathology of male rat testes.
Comparing the correlation between the histopathological rehabilitation
19


and the results of sperm quality and quantity recovery in trial group 3
can be judged that due to the time of using Balanoxi 4 weeks after

stopping using Natri valproat 7 weeks are not enough to fully express
the recovery effect of hardened Balanoxi capsules in experiment, so
although the start of the recovery of some cells involved in sperm
production, the sperm density in sperm increased statistically
significantly compared to that of the group 2 (the control group). But the
results also showed a partial recovery effect of Balanoxi capsules and its
Androgen activity on the testicular histopathology images and sperm
quality and especially increased the conception rate and the number of
mice born.
4.3. The effect of hard capsules Balanoxi in the treatment of
infertility due to sperm decline
- Quantity and quality of sperm by infertility before and after the
study.
Average sperm density and percentage of sperm progressing to average
increased after treatment in both infertility group I and infertility II (p
<0.05). The increase rate in 2 infertility groups is quite similar.
Balanoxi capsules did not only increase sperm density and the
percentage of motile sperms in infertility type II, but it also improvedthe
figures in infertility type I. The reason why infertile patients II are those
who previously had the minimum number and quality of sperm are
sufficient for a pregnant wife, but later on due to the impact of life's
factors such as polluted environment, polluted food, extending mental
stress, and infection, etc, have sperm decline because of the imbalance in
the body. Infertility I is often congenital or due to serious spermdepleting diseases that lead to poor sperm quality and make it difficult to
conceive. According to the results of experimental research in this
thesis, Balanoxi has the effects of protecting and recovering damaged
sperm and sperm cells, stimulating sperm and sperm cells to be more
active.
This result is consistent with the relationship between quantity, sperm
quality and fertility. Hard Balanoxi works well in decline sperm patients

both infertility I and II.
- The improvement of Yang deficiency symptoms according to
traditional medicine.
According to modern medicine, testosterone has effects on protein
metabolism and muscle structure, one of the most important male
characteristics is that muscle mass develops strongly after puberty, under
the effect of mass testosterone. The muscles can increase by more than
20


50% compared to women. Testosterone is also used for the elderly as an
"anti-aging" hormone to increase metabolism, and increase the strength
of the body. With the amount of testosterone secreted daily in men, basal
metabolism increased by 5-10% compared with the absence of the effect
of testosterone. If the basal concentration of testosterone increased by
15%, the number of erythrocytes in blood 1 mm 3 increased by about
20%, for this reason, the number of red blood cells in men is usually
higher than that of women, about 700,000 cells / mm3.
Balanoxi, according to the research results of this thesis, stimulates the
increase of excretion of testosterone, in addition to its effect on basal
metabolism; it also has a positive effect on the process of making red
blood cells and the process of increasing protein synthesis. Because the
Balanoxi can also work on other endocrine glands like the adrenal gland,
the thyroid gland, the pituitary gland, it can improve symptoms of fear
of cold, dark blue face, pale tongue; increased libido; reduce symptoms
of semen, stimulate erections.
In the Yang deficiency syndrome, the trace elements Cu, Zn, Ca, and
Mg all decrease, while the Balanoxi made from the Balanophora indica
medicine contained trace elements (Ca, P, Fe, Zn), so the medication
works to promote the activity of enzymes that increase protein synthesis

and increase energy, which can increase the heart rate, increase
myocardial contractility, dilate vessels and thus increase the rate of
blood circulation and enhance perfusion for the muscles. Therefore,
Balanoxi can improve the symptoms of darkish face, pale tongue, moss
of white tongue, cold hands and feet, and fatigue.
According to traditional medicine, kidney Yang is also known as Fire of
the gate of life or original Yang. Kidney Yang deficiency impairs the
ability to warm the body, causing Wetness Water dominates inside and
the muscles weaken. The disease is mainly due overwork, weak Kidney
and old age, prolonged illness negatively affects kidneys, or congenital
diseases.
Therefore, the symptoms of the dark greenish face, pale tongue, whitish
moss, pain in back knees, erectile dysfunction, preferring to eat warm,
cold back, fear of cold, cold feet, weak deep pulse are due to Yang
kidney deficiency, which leads to the reduction of Yang Qi circulation
and Qi transmission function. The unfavorable opening and closing
cause blood stasis and phlegm stagnation which are evil Qi causing dark
greenish face. Since Kidney stores the inborn Yang Qi, so the Yang part
of the five organs must be borne of the YangQi in the kidneys to be
born. The unbalance of the Kidney Fire leads to the fear of cold, cold
21


limbs. This is because of the fact that Yang belongs to Fire, Fire is hot
and outside, while Water is cold and inside, the Yang deficiency causes
the cold expose to the outside. Kidneys determines bones, back belongs
to Kidneys, Yang Kidney deficiency leads to pain in back. The Gate of
Life has direct impact on the genital organs, belongs to Yin. If the Fire
of the gate of life is deficient, knees will be cold. The unbalance
originalYang and weak Jing Qi lead impotence, premature ejection, and

cold Jing. Chi pulse belongs to Kidney, as a result, Yang Kidney
deficiency causes the Chi pulse deep or slow. The Yang Kidney
deficiency fails to make Spleen Earth, so Fire deficiency leads to Earth
deficiency, which reduces the transmission of the Sea of Water, blood,
and after-born Jing. As a result, face is pale and fatigue. The Kidney
Tonifying and Yang Strengthening strategy, hence, should be utilized.
Bananoxi made from Balanophora indica extract has the effects
tonifying Fire, blood sufficiency will lead to bright face. As Yin and
Yang are balance the above symptoms will decrease.
- Unwanted effects of Balanoxi on patients
After the treatment, there were no patients with undesirable clinical
signs (nausea, bloating, rash, insomnia, headache, dizziness).
CONCLUSION
1. The Acute toxicity and semi-chronic toxicity of Balanoxi on
experimental animals.
- The acute toxicity: The medicationhas no acute toxicity on oral route.
The highest amount of medication that can be transferred to the white
mouse's stomach, 20g/kg of body weight, did not cause death in mice.
- Semi-chronic toxicity: The medication did not have a semi-chronic
toxicity. Doses of 0.3 g/kg body-weight/day (human dose) and 0.9g/kg
body-weight/day (3 times of human dose) in 8 weeks did not change the
general condition, weight, hematopoietic function, liver function, kidney
function, and liver and kidney histopathology.
2. The effects of Balanoxi on the sperm declineinduced models
- Protective effects
Male rats used Balanoxi dose 0.7g/kg body weight in 7 weeks with Natri
valproat 500mg/kg body weight. The results showed that the medication
increased the weight of seminal vesicles, prostate gland, content of
Testosterone, rate of alive sperms, sperm motility speed, rapid
progressive sperms; reduced the percentage of non-progressive sperms,

and increased conception rates, and the number of mice born.
- Recovery effects
22


Male rats used Balanoxi dose 0.7g/kg body weight in 4 consecutive
weeks, right after taking Natri valproat 500mg/kg body weight in 7
consecutive weeks. The results showed that the medication increased the
concentration Testosterone, sperms rate, progressive sperms, conception
rate,and the number of mice born.
- Assessing the effects of Balanoxi on the histopathology of the testices
In both studies evaluating the protective and restorative effect of
Balanoxi capsules, the drug increased the thickness of the epithelial
layer, density of Sertoli cells, density of sperm compared to the
reproductive impairment.
3. The effects of Balanoxi in the treatment of infertility patients due
to sperm decline
Therapeutic effects of the dose 05g/day, in 10 consecutive weeks:
- increases the excretion of serum testosterone.
- increases the sperm count and quality.
- The seminogram test indices of 09/30 patients increased that led
to their wives got pregnant, accounting for 30%.
- The sperm count and quality of 29/30 patients increased,
accounting for 96.67%. Meanwhile, only 1/30 patients had semen
unchanged, accounting for 3.33%.
* Undesirable effects:The dose of 05g/day, in 10 weeks of the
treatment, did not cause any clinical, hepatic or renal function of the
patients.
RECOMMENDATIONS
1. It is suggested that more patients and longer treatment time should be

applied to acquire more reliable results.
2. Some other effects of Balanophora indica such as effects on erectile
ability, digestive system, and genetics, etc. should be further studied.
3. The Balanoxi can be used in the treatment of sperm decline patients.

23