Research results showed that Balanoxi has no oral toxicity, hasprotective effects, recovers reproduction, increases serum testosterone,increases the quantity and quality of sperm.. Patho
Trang 1The treatment of sperm decline still faces many difficulties, mainlybecause sperm decline has many complicated causes Modern medicinehas many methods of treating sperm decline, but the results are lowerthan the expectations of physicians and patients, due to unwanted effectsand long time treatment which have great effects on patients’ health andeconomy.
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 theexperimental animals;
2 Assessing the effect of Balanoxi on sperm decline caused models;
3 Research the effect of Balanoxi on infertility patients due to spermdecline
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, hasprotective effects, recovers reproduction, increases serum testosterone,increases the quantity and quality of sperm The results of the study can
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Trang 2be 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 4chapters:
Chapter 2: Materials, subjects, and research methods 16 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 productionand development in general as well as in the differentiation stages ofsperm development Pathological disorders of reproductive hormoneslead 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, hightemperatures, 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 coexisttogether, it is necessary for physicians to prescribe reasonable treatmentsuch as a combination of medical, surgical, and fertility treatments toincrease the rate of pregnancy
- By medication: medication treatment should be indicated for patientswith sperm decline due to unknown etiology.Commonly used
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Trang 3medications: Antioxidants (Glutathion, L-arginin), hormones(Gonadotropin, Androgen, and estrogen receptor antagonists)
- Surgery: surgery should be indicated for patients with impairedspermatozoa that affect the sex life, such as varicose veins, ectopictesticles, 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 maleinfertility 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 (inbornJing and after-born Jing), it is the basic material for body compositionand body nutrition During body development, Jing is always consumedand 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, slowmovements, rapid aging, impotence, no conception, low semen volume,sperm deficiency Taut deep no force Chi pulse, if there is Yindeficiency Fire excess, there will be five center restlessness and fever,reddish tongue, less moss, taut pulse Strategies: Reinforcing Qi andNourishing 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; largeamount of urine, urinating many times, loose stools in the earlymorning; Low sperm count, weak sperm motility, impotence, prematureejaculation, 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, spermvitality weakness, impotence, premature ejaculation, tongue quality,
rough, white tongue moss, forceless pulse Strategies: reinforcing Qi and
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Trang 4invigorating 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 withoutwanting to drink water; itching or severe genital irritation, soreness andobstruction in perineum or testes, dense semen with foul odor, which can
be inferred semen contains many red and white blood cells; low spermcount, high dead sperm rate, cloudy urine, burning sensation in theurethra 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, SonDuong, Tuyen Quang The scientific name is assessed at the VietnamInstitute of Medicinal Materials
In Vietnam this medicinal plant is often used as a tonic for postpartumwomen to stimulate appetite In particular, this medicinal plant is oftensoaked in winefor men to enhance and endure erections In Malaysia, allTinosporacapillpes 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 aresuitable for treating impotence, infertility, tired legs, and exhaustion; and
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Trang 5laxative, suitable for treating fluid deficiency disorders which lead to dryintestines, 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, TuyenQuang The scientific name is assessed at Vietnam Institute of MedicinalMaterials
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 onchromosomes
Newzealand White rabbits (semi-chronic toxicity test), white fur,healthy, both sexes, weighing 1.8 -2.5kg; supplied by theBreedingcenter, 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 byLitchfied-Wilcoxon method The research was conducted at theDepartment of Experimental Research, Military Institute of traditionalmedicine
* 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 thatcaused all mice dead
Mice were monitored continuously in 24 hours after taking themedication.Their general conditionswere monitored in 7 days aftermedicationtaking The number of dead mice was counted in the groupswithin 72 hours after taking the medication to determine the LD50
2.3.1.2 Semi-chronic toxicity
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Trang 6The semi-chronic toxicity of Balanoxi was determined on whiteNewzeland White rabbits, white, healthy, both sexes, weighing 1.8 -2.5kg; supplied by theBreeding center, Testing Institute The study wasconducted at the Department of Experimental Research, MilitaryInstitute of traditional medicine in accordance with WHO guidelines andregulations 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 whosesperms 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 bodyweight
Male rats in all 3 groups received the medication and solventcontinuously in 7 weeks Mice were given a dose of 2 ml/100g bodyweight, 2 times/day, every 2 hours After 5 weeks, the male mice ingroups mated with 2 female mice in the same cage for 2 weeks
* Studying the recovery effects of Balanoxi on sperms reduced by Natrivalproat 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 Natrichloride 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 givenmedicine 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, comparingdifferences before and after the treatment
- Sample size: Sample size 30, male patients aged 20-60 with spermdecline Yang Kidney deficiency type
- Selection criteria:
According to modern medicine: Age from 20 to 60; Infertility I andinfertility II due to sperm decline; Voluntarily participated in the
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Trang 7research; fully abide orders and instructions of the doctor during theresearch procedure.
According to Traditional Medicine: Patients with sperm decline Yangkidney deficiency type
- Dosages and administration: 10 Balanoxi500mg daily, 2 times (in themorning 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 Afterthe treatment, the treated patients were re-tested these values to assessliver and kidney function status to determine if they were affected byBalanoxi
- Quantification of serum testosterone before the treatment: assessing thequantity of serum testosterone in selected patients after the treatment
- Seminogram before and after the treatment: seminogramis the mostimportant test to assess the fertility of men The testing standards andassessment criteria are under the guidance of WHO (1999) and (2010)[2], [3]
- Testicular and testicles veinsultrasoundbefore the treatment to rule outother causes of sperm decline
Seminogram, biochemistry, hematology, and hormonesTestosteronetests were done at the labo of Military Institute oftraditional 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 andeffectiveness, the medication was tested on voluntary patients under thepermission of the Scientific Council, the Military Institute of traditionalmedicine
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 ofbody weight but no mice died, and there were no abnormal symptomswithin 72 hours after taking the drug and during the next 7 days
3.1.2 Results of the semi-chronic toxicity study
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Trang 8During the experiment, the rabbits in all 3 groups were eating and actingnormally, were agile, had bright eyes, silky hair, and firm stools Nosignificant symptoms were observed in all three groups of the rabbitsduring the study period After 4 and 8 weeks of the treatment withBalanoxi, red blood cell count, Hemoglobin content, Hematocrit, whiteblood cell count, white blood cell count and platelet count, AST, ALT,GGT, total bililubin creatinine concentration, Ure in the rabbit blood instudy groups 1 and 2 were notdifferent from that of the control groupand there were no differences between the research groups at the sametestingtime (p> 0.05).
There were no general pathological changes in macroscopic morphologyliver 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
Genitals weight (mg/100g body weight)
Testicles Seminalvesicle Prostate Cowper’s gland Glans musclesLevatorGroup 1:
Natri clorid
+ water
1.157
±0.086
0.270
±0.057
0.144
±0.021
0.041
±0.007
0.054
±0.006
0.359
±0.047Group 2:
Valproat +
water
0.991
±0.092
0.207
±0.070
0.119
±0.013
0.036
±0.006
0.049
±0.005
0.350
±0.036
p2-1 <0.05 <0.01 < 0.05 < 0.05 > 0.05 > 0.05Group 3:
Valproat +
Balanoxi
1.063
±0.093
0.302
±0.051
0.137
±0.022
0.039
±0.006
0.052
±0.007
0.352
±0.029
p3-1 < 0.05 >0.05 >0.05 > 0.05 > 0.05 > 0.05
p3-2 > 0.05 <0.05 < 0.05 >0.05 > 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 significantlycompared to group 2 (p <0.05) The weight of Cowper’s gland, glansand levator muscles in group 3 were not significantly different fromgroup 2 (p> 0.05)
Table 2.Balanoxi's protective effects on blood testosterone
concentrations ofsperm decline induced by Natri valproat male rats
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Trang 9Groups Testosteron (nmol/l)
Group 1: Natri clorid + water 8.91 ± 1.38
Group 3: Valproat + Balanoxi 8.50 ± 1.08
Comments:Testosterones concentration of group 3 increased compared
to the group 2 (p <0.01) and no significant difference compared to thegroup 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
Group 1: Natri clorid +
Comments: Sperm density of group 3 was notsignificantly
differentfrom that of the control group (p> 0.05) The sperm vitality rateincreased 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
+ water 20.58 ± 3.67 7.25 ± 2.05 7.92 ± 1.31 64.25 ±5.08
p2-1 < 0.001 < 0.01 < 0.001 < 0.01Group 3: Valproat
+ Balanoxi 34.42 ± 16.30 6.08 ± 2.06 4.33 ± 2.06
55.20 ±15.95
p3-1 > 0.05 > 0.05 > 0.05 > 0.05
p3-2 < 0.01 > 0.05 < 0.001 > 0.05
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Trang 10Comments: The group 3 had a significantly higher rate of rapid
progressive sperms than that of the group 2 (p <0.01) and there were nodifferences 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) Theslow progressive rate and the motility ratewas not different from that ofthe control group (p> 0.05)
Table 5.Balanoxi's protective effects on sperm motility in sperm decline
induced by Natri valproat male rats
Group 1: Natri clorid + water 44.48 ± 6.43
Group 2: Valproat + water 36.16 ± 8.87
Group 3: Valproat + Balanoxi 43.59 ± 5.91
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 groupswas high, there were no differences between groups
* The thickness of the epithelial layer: The thickness of the epitheliallayer 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%, bothhigher 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 ofmedium and, 91.93%, (high: 51.47%, average 40.47%) and 84 , 9%(high 49.27%, average 35.63%)respectively, while that of the group 2was 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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Trang 11Indices Group1 Group2 p2-1 Group
3 p3-1 p3-2
Pregnancy rate 75.50 33.33 0.05< 58.33 <0.05 0.05<Number of corpus
luteum/ 1 female
mouse
10.33
± 1.56 ± 1.5010.67 0.05> ± 1.0010.50 0.05> 0.05>Number of fetus/ 1
female mouse ± 1.2210.00 ± 0.968.25 0.05< ±0.829.00 0.05> 0.05>Number of health
fetus / 1 female
mouse
9.78
± 1.09 ± 1.157.00 0.01< ± 0.988.57 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 togroup 2 (p <0.05) The number of corpus luteum/ 1 female mouse wasnot 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 reproductiveimpairment by sodium valproat
Table 7.The recovery effect of Balanoxi on sperm decline induced by
Natri valproat male rats
clorid + water
1.004
±0.112
0.251
±0.038
0.136
±0.010
0.044
± 0.003
0.05
±0.003
0.330
±0.023
0.202
±0.031
0.122
±0.099
0.041
±0.002
0.048
±0.003
0.316
±0.018
p2-1 <0.01 < 0.01 < 0.01 < 0.05 > 0.05 > 0.05Group 3:
Valproat +
Balanoxi
0.901
±0.102
0.221
±0.027
0.127
±0.007
0.042
±0.002
0.049
±0.002
0.323
±0.021
p3-1 <0.05 < 0.05 < 0.05 > 0.05 > 0.05 > 0.05
p3-2 >0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05
Comments: The weights of testices, seminal vesicles and prostate
weight mice inthe group 3 increased compared to that of the group 2, but
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