Part A: INTRODUCTION 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 OBJECTIVES OF THE RESEARCH Studying the acute and semi-chronic toxicity of Balanoxi on the experimental animals; Assessing the effect of Balanoxi on sperm decline caused models; 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 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 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, pictures, 19 appendices, and 118 references (44 in Vietnamese, 71 in English, and 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 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 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 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 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 groups, each group of 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 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 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 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 groups received the medication and solvent continuously in weeks Mice were given a dose of ml/100g body weight, times/day, every hours After weeks, the male mice in groups mated with female mice in the same cage for weeks * Studying the recovery effects of Balanoxi on sperms reduced by Natri valproat model Adult male rats were randomly divided into groups of 15 each - Sperm reduced by Natri valproat: + Group (control group): did not drink Natri valproat, only drink Natri chloride 0.9% 20ml/kg/day + Groups and 3: Natri valproat dose of 500mg/kg/day in weeks - After weeks of taking Natri valproat, rats in the groups were given medicine in 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 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, 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) There were no general pathological changes in macroscopic morphology liver images in study group 1s and 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 >0.05 > 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 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 (p 0.05) Table 2.Balanoxi's protective effects on blood testosterone concentrations 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 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 increased compared to the group (p 0.05) Table 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 was notsignificantly differentfrom that of the control group (p> 0.05) The sperm vitality rate increased significantly compared to the control group (p 0.05 < 0.05 Comments:The sperm motility speed of mice in group decreased significantly compared to the group (p 0,05 < 0,01 Comments:Testosterone blood concentration ofthe group decreased significantly compared to that of the group 1, the testosterone concentrations ofthe group increased compared to that of the group (p 0.05 > 0.05 > 0.05 > 0.05 p3-2 < 0.05 > 0.05 > 0.05 > 0.05 Comments: The group had a significantly higher rate of rapidprogressive spermsthanthe group (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 decreased significantly compared to the group (p