1 INTRODUCTION The urgency of the study Gout is a common disease, due to the deposition of monosodium urate (MSU) crystals in synovial fluid or tissues Between - 4% of the adult population worldwide has gout In developed countries, the incidence of gout in men is between - 6% of the population, and between - 2% of the population In some developing countries, the incidence of gout may increase to 10% Gout usually develops in stages About 25% of gout patients have joint damage, irreversible joint deformity The quality of mental and physical life of gout patients decreased, due to the frequent affects of acute gout attacks, the number of swollen, painful joints increased over time, the progression and spread of tophus, using colchicin, corticosteroids To research into new herbal medicine for gout treatment that can limit unwanted effects is meaningful and necessary Therefore, over the past decade, scientists have studied a variety of herbal and traditional medicines for the gout treatment Vietnam traditional medicine has many effective formulas in treating gout, Si Miao Yan formula is one of them The composition of Si Miao Yan formula consists of Semen Coicis, Phellodendron sp Rutaceae, Achyranthes bidenta, Rhizoma Atractylodis Some experimental studies have shown that polysaccharides in Rhizoma Atractylodis and baicalein, oroxylin A in Cortex Oroxyli (replaced Phellodendron sp Rutaceae) have anti-inflammatory effect Stigmasterol and p-coumaric acid in Semen Coicis are involved in the elimination of uric acid We adjusted the dosage, the drug mix, the drug form then evaluated the effects of Tu dieu tan granule on two objectives: Study objectives Evaluating the effectiveness of Tu dieu tan granule on acute and sub-chronic toxicity, anti-inflammatory, analgesic and lowering serum urate level in experimental Evaluating anti-inflammatory, analgesic and lowering serum urate level and unexpected effects of Tu dieu tan granule in patient with chronic gout Practical implications and new contributions of the thesis This scientific study based on the effect evaluation of a new herbal product called Tu dieu tan granule which was used in chronic gout treatment According to the clinical experience, we reduce the traditional formula Si Miao to suit Vietnamese climate, physiological characteristics of the people and the purpose of treatment: replacing Phellodendron sp Rutaceae and Achyranthes bidenta with Cortex Oroxyli and Radix Achyranthes asperae, changing the dose of Si Miao formula, changing to granule form We had given the scientific evidence to explain the anti-inflammatory, analgesic and hypouricemic effects of Tu dieu tan granule in experimental and clinical The experimental results showed that Tu dieu tan granule has not shown the acute and subchronic toxicity Tu dieu tan granule has antiinflammatory, analgesic and hypouricemic effects by excreting uric acid through urinary system in the experimental model After 30 days of treatment, the clinical results showed that the combined of Tu dieu tan granule with allopurinol reduced serum uric acid (SUA) levels were 200,42 ± 100,14μmol/l; the effectiveness of lowering SUA decreased by 98,33%; decreased the number of swollen, painful joints, improved mean pain intensity on VAS scale and physical activities function according to VAS2, VAS3 Improving median function assessed by HAQ was 0,96 ± 0,32 point; Its efficacy of improving traditional symptoms was 98,33% The improvement of these indicators in the study group were statistically significantly higher compared to the control group (p < 0,05) Tu dieu tan granule had effect in treating both the wind damp heat impediment pattern and the phlegm blood stagnation pattern with SUA levels decreased 222,26 ± 114,84μmol/l; 194,97 ± 96,69μmol/l and Nimodiping score improvement was 10,92 ± 2,31 points; 10,62 ± 2,83 points; the difference between two patterns was not statistically significantly No adverse effects of Tu dieu tan granule in clinical or subclinical were observed These findings are scientific indications of the safety, antiinflammatory and analgesic, hypouricemic effects of Tu dieu tan granule in treating chronic gout 4.Thesis structure Apart from the Introduction and Conclusions chapters, the thesis consists of four chapters Chapter 1: Literature review 35 pages Chapter 2: Study subjects and methods 25 pages Chapter 3: Results 37 pages Chapter 4: Discussion 34 pages And 38 tables, graphs, images, 13 appendixes, and 151 references (34 Vietnamese references, 93 English references, 24 Chinese references) 3 Chapter LITERATURE REVIEW 1.1 The concept of gout according to modern medicine 1.1.1 Cause and pathogenesis The pathogenesis of gout is due to hyperuricemia When SUA level rises over saturation, uric acid deposits in the form of MSU in joint or tissues, triggering inflammatory reactions that relating acute gout attacks Between gout flares, MSU are deposited and formed tophus The more uric acid is deposited the more inflammatory reaction becomes a continuous inflammation circle 1.1.2 Diagnosis of chronic gout According to Bennett and Wood criteria (1968) 1.1.3 Treatment of chronic gout Besides reducing the symptoms of pain and inflammation, the most important target is reducing SUA, which allows patients to prevent complications of chronic renal failure, organ damage Treatment Principle: Patients maintain a diet of chronic gout, combined with hypouricemic drug Start at low doses, gradually increasing to therapeutic doses and maintain continuously, uninterrupted, which can be combined with NSAIDs or colchicin to prevent acute gout attacks Target is lowering SUA levels less than 6mg/dl (360μmol/l) in chronic gout or 5mg/dl (300μmol/l) in chronic gout with tophus Common drug are xanthin oxidase inhibitors (allopurinol, febuxostat ) which inhibit xanthin oxidase According to American College of Rheumatology, starting dose of allopurinol should be no greater than 100mg/day and start at 50mg/day in stage or severe CKD Dosage can be raised above 300mg daily, even with renal impairment Febuxostat is indicated for patients with allopurinol-resistant or allergic allopurinol Febuxostat inhibits vasoactive agents and prevents inflammation In addition, other urate lowering therapy such as uricosuric therapy (probenecid, benzbromarone), uricolytic drugs (Uricozym, Pegloticase), urine alkalinization (sodium bicarbonate solution) may be considered 1.2 The concept of gout according to traditional medicine 1.2.1 Cause and pathogenesis According to traditional medicine, gout has the name is “Tong Feng” Tong Feng is used to refer the pain causes by wind Gout is cateogrised as “Tong bi” according to traditional medicine (Bi means standstill, not cleared) The causes of “Tong bi” include six external climatic factors, seven emotional factors, and other pathogenic factors 4 1.2.2 Treatment of gout Depending on the clinical types, there are appropriate treatments principles and specific formulas According to Hao Xian Gou and Jie Tian Ping – China (2008), Tong Feng is divided into five patterns: Wind cold damp impediment pattern, wind damp heat impediment pattern, phlegm blood stagnation pattern, spleen and kidney yang deficiency pattern, liver and kidney yin deficiency pattern For the wind cold damp impediment pattern, the principles include of dispelling wind, scattering cold, eliminating dampness, circulating meridians; the appropriate herbal formula are Tigao Tai Tang or Fangjian Fengge Tang For the wind damp heat impediment pattern, the principles include of dispelling wind, clearing heat, eliminating dampness; the appropriate herbal formula are Er Miao San combined with Bai Hu Gui Zhi Tang; for the damp heat pattern include numbness, the principles include of clearing heat, eliminating dampness, circulating meridians, analgesic, the appropriate herbal formula are Si Miao San combined with Long Dan Da Gan Tang For the phlegm blood stagnation pattern, the principles include of circulating blood, dispelling stasis, transforming phlegm; the appropriate herbal formula are Fu Yuan Huo Xue Tang combined with Er Chen Tang For the spleen and kidney yang deficiency pattern, the principles include of tonifying spleen qi, supplementing kidney yang; the appropriate herbal formula is Wu Tou Gui Zhi Tang For the liver and kidney yin deficiency pattern, the principles include of nourishing yin, supplementing kidney and liver yin; the appropriate herbal formula is Du Huo Ji Sheng Tang 1.3 Review on experimental models evaluating toxicity and the effectives of herbal medicine in treating gout Tu dieu tan granule is derived from the traditional formula Si Miao Although the formula composition does not change, but due to the reduction of each dose in the formula compared to the old and converted formulation, Tu dieu tan granule should be determined toxicity to ensure safety for testing the next phase Tu dieu tan granule was evaluated acute and sub-chronic toxicity It was evaluated antiinflammatory, analgesic and hypouricemic effects in experimental models such as the carrageenin-induced rat paw oedema, rat peritonitis inflammatory, rat arthritis after intrasynovial injection of sodium urate; hot plate, hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test Instrument; potassium oxonat induced hyperuricemia in rat, diuretic and uricosuric activity in mice, xanthin oxidase inhibatory activity in vitro and in vivo models 1.2 Review on gout treatment studies 1.2.1 Review on gout treatment studies according to Western medicine Currently, almost studies have focused on reducing SUA levels and maintaining SUA levels below 6mg/dL ( 420μmol/l for male and > 360μmol/l for female According to traditional medicine: Patient was diagnosed wind damp heat impediment pattern or phlegm blood stagnation pattern 8 * Exclusion criteria: Subjects with hepatic failure, renal failure, myocardial infarction, cerebral hemorrhage; Long-term gastrointestinal disturbances affect the process of drug absorption and metabolism; mental illness, malnutrition, infectious diseases; contraindications for allopurinol; Tong Feng symptoms not belong to the wind damp heat impediment pattern or phlegm blood stagnation pattern; uncooperative in the studies; pregnant or lactating women 2.3 Study methods 2.3.1 Experimental studies: We conducted an experimental controlled study with the following stages 2.3.1.1 Investigating the acute and sub-chronic toxicity effects * Acute toxicity: was assessed in white mice using Litchfield Wilcoxon method based on OECD and WHO guidelines The mice received the medication under investigation with increasing doses through oral use The aim was to identify the non-lethal highest dose (0%), the lethal lowest dose (100%) and the intermediate doses The mice were observed for 72 hours to monitor their mortality rate and overall health condition; and were observed for days since medication administration to monitor mortality rate * Sub-chronic toxicity: According to WHO guideline, Wistar rat were devided into groups: Controll group (distilled water); Treatment group (Tu dieu tan granule 1,8g/kg/day - corresponding to the clinical dose); Treatment group (Tu dieu tan granule 5,4g/kg/day - three times higher than the clinical dose) Rats were drunk water and Tu dieu tan granule for weeks continuously The rats were observed to monitor their weight, eating, sleeping, activity, digestion, hematology, biochemistry, liver and kidney function, liver and kidney histopathology Comparisons were made between preand post-treatment, and between the treatment and control groups 2.3.1.2 Acute anti-inflammatory effect Evaluating anti-inflamatory effect in experimental models such as the carrageenin-induced rat paw oedema, rat peritonitis inflammatory, rat arthritis after intrasynovial injection of sodium urate The carrageenininduced rat paw oedema model was conducted according to Winter method The rat peritonitis inflammatory model was conducted according to Patel method The rat arthritis after intrasynovial injection of sodium urate model was conducted according to Faires and McCarty method 2.3.1.3 Analgesic effect Evaluating analgesic effect in experimental models such as the hot plate model (evaluation the central analgesic effect), the hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test Instrument (evaluation the peripheral analgesic effect) The hot plate model was conducted according to Vogel method The hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test Instrument was conducted according to Randall-Selitto method 2.3.1.4 Hypouricemic effect Evaluating hypouricemic effect in experimental models such as the potassium oxonat induced hyperuricemia in rat (evaluating hypouricemic effect), diuretic and uricosuric activity in mice (evaluating the urinary uric acid excretion), the xanthin oxidase inhibatory activity in vitro and in vivo models (evaluating the synthesis inhibition effect of uric acid) The potassium oxonat induced hyperuricemia in rat was conducted according to Starvic method The xanthin oxidase inhibatory activity in vitro and in vivo model was conducted according to Nguyen Thi Thanh Mai method The diuretic and uricosuric activity in mice was conducted according to Starvic method 2.3.2 Clinical studies Study design: Clinical intervention, prospective placebo-controlled double-blind study 2.3.2.1 Study procedure After clinical examination, the patients who met the inclusion criteria were enrolled into the study An independent study staff divided the study subjects into groups, each group has 60 patients Patients were comparable regarding age, sex, disease level (the number of painful and swollen joints, SUA level), traditional pattern Each patient’s identity was encoded using a code attached to his/her medical record The information whether the patients were in the study or control group was entirely unknown to all the patients and healthcare staff who were directly involved in the treatment of the patients The medication under investigation and the placebo were presented in the same format During 30 treatment days, patients in the study group used allopurinol and Tu dieu tan granule, patients in the control group used allopurinol and placebo Patient was treated on the first day of the study (D0): Allopurinol (01 tablet taken once per day, after meal); Tu dieu tan granule (1 package per time taken twice per day, hour before meal); placebo (1 package per time taken twice per day, hour before meal) During the study, patients did not use other medicines In case of patient with gout flare was painful and ask for analgesic 10 medicine, he/she would be allowed to use Mofen 400mg in days maximum (01 tablet per time, taken twice or three times per day) The patients’ identities were only revealed after the course of treatment was completed, then data collection, analysis and report were carried out 2.3.2.2 Study indicators and evaluation methods - Describe characteristics of the study subjects: Age, gender, historical symptoms (disease level, risk factors, traditional patterns) - Clinical indicators: Study indicators was used to evaluate the effectiveness of Tu dieu tan granule in clinical and subclinical and compare the effectiveness between the control and study group, preand post-treatment These indicators were assessed in the first day (D0) and the 30th day (D30) of the treatment course: Anti-inflammatoy effect (reduced the number of swollen joints); analgesic effect (reduced the number of painful joints, improved VAS scores); improved active function (improved VAS2, VAS3, HAQ scores); improved traditional symptoms according to Nimodiping score Evaluating the treatment effective in sub-clinical (D0, D30); evaluating SUA levels reduction in the study and control groups, in two traditional patterns - Unexpected effects were monitored during the whole course of treatment: The unexpected symptoms in clinical; hematological indicators in subclinical 2.3.2.3 Statistical analyses: Data analyses were performed using SPSS 22.0 A p-value of ≤ 0.05 was considered statistically significant 2.3.2.4 Research ethics: The study was approved by the Medical Ethics Committee of Hanoi Medical University Permission for the study to be conducted at MITM was retrieved from the hospital’s General Sciences Council Chapter STUDY RESULTS 3.1 Results of experimental studies 3.1.1 Investigating the acute and sub-chronic toxicity effects * Acute toxicity: After oral administration of Tu dieu tan granule from to hours, all rats showed normal function, agility, smooth fur, normal eating activities and dry stool Rats drank Tu dieu tan granule at the dose 62,5g and 78,1g were diarrhea on the last days There were no adverse symptoms, no rats died within 72 hours of administration and for days Acute toxicity and LD 50 of Tu dieu tan granule have not been determined 11 * Subchronic toxicity: During the experiments, the rats in all groups showed normal function There were no difference in overall conditions and weight Tu dieu tan granule at the dose of 1,8g/kg after weeks of continuous administration and 5,4g/kg after weeks of continuous administration showed no haematopoietic toxicity and the treatment did not change the rats’ liver and kidney function indicated by biochemiscal as well as histopathological tests Tu dieu tan granule at the dose of 5,4g/kg after weeks of continuous administration showed the changes in hematological indicators but still in normal standards 3.1.2 Acute anti-inflammatory effects * The carrageenin-induced rat paw oedema model: Tu dieu tan granule at the dose of 1,8g/kg and 5,4g/kg had no anti-inflammatory effect at all times after causing inflammation * The rat peritonitis inflammatory model Table 3.1 Acute anti-inflammatory effects of Tu dieu tan granule on the volume and components of rat peritonitis inflammatory Tu dieu tan Tu dieu tan Biological Aspirin control granule granule Indicators 200mg/kg group (n = 10) (n = 10) 1,8g/kg (n = 10) 5,4g/kg (n = 10) Volume of peritonitis 1,33 ± 1,53 ± inflammatory 2,81 ± 0,79 1,32± 0,79*** 0,76*** 0,38*** fluid (ml/100g) Number of 66,57±45,6 16,43±12,38* 21,96±13,47* 21,55±10,60* white blood * * * cells (G/l) Protein content of 37,63±16,4 15,12±9,12** 16,19 ± 20,20 ± peritonitis * 8,57** 11,10* fluid (mg/dl) ** p ≤ 0,01 and *** p ≤ 0,001 compared to control group The results showed that Tu dieu tan granule at the two doses reduced the volume and protein content of rat peritonitis inflammatory statistically significantly compared to the biological 12 control group (p < 0,001), reduced the number of white blood cells (p < 0,01); the effect was equivalent to aspirin 200mg/kg * The rat arthritis after intrasynovial injection of sodium urate - The effects of Tu dieu tan granule on the inflammatory levels, size and temperature of the knee joint: Tu dieu tan granule at the dose of 2,1g/kg had effect on reducing knee oedema, synovial fluid, lowering the temperature of the inflammatory site, reducing the infiltration of inflammatory cells after hrs and 24 hours The difference was statistically significantly compared to the biological group (p < 0,05) - Knee pathology in rats treated Tu dieu tan granule: The synovial fluid has a sloughed area which is easy to observe The synovial fluid had less oedema fluid, urate crystals and infiltration of inflammed cells than the biological control group 2 (HE x 40) (HE x 400) Picture 3.1 Knee pathology in rats treated Tu dieu tan granule (Rat number 25, treated Tu dieu tan granule) The synovial fluid had less edema fluid, urate crystals The infiltration of inflammed cells 3.1.3 Analgesic effects * The hot plate model: Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had effets on prolonging reaction time for temperature, the difference was statistically significantly compared to the biological control group (p < 0,05) * The hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test Instrument: Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had effets on increasing the endurance for painful force, increasing the response time for painful stimulation, the difference was statistically significantly compared to the biological control group and to pre-treatment (p < 0,05) 13 3.1.4 Hypouricemic effects * The potassium oxonat induced hyperuricemia in rat Table 3.2 Hypouricemic effect of Tu dieu tan granule on serum uric acid level in rats Groups n Induction control group allopurinol 20mg/kg group 10 10 Tu dieu tan granule 3,6g/kg group 10 Tu dieu tan granule 10,8g/kg group 10 The reduction compaired to control group Uric acid (mmol/L) 168,90 ± 33,59 39,40 ± 4,43∆∆∆ 54,80 ± 10,04∆∆∆ 50,80 ± 16,96∆∆∆ 76,67% 67,55% 69,92% ∆∆∆ U rinary uric acid (mmol/L) p < 0,001 compared to the induction control group Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had hypouricemic effects, the difference was statistically significantly compared to the induction control group (p < 0,001) The SUA levels reduction at the dose 10,8g/kg higher than the dose 3,6g/kg of 2,37% * The xanthin oxidase inhibatory activity in vitro and in vivo model: Tu dieu tan granule had negative result on inhibiting xanthin oxidase reaction in vitro In in vivo model, Tu dieu tan granule at the dose of 2,1g/kg decreased uric acid optical absorption, the difference was not statistically significantly compared to the control group (p > 0,05) Tu dieu tan granule had no effects on inhibiting xanthin oxidase in vivo and in vitro * The diuretic and uricosuric activity in mice: 20 15 10 11.8 17.8 18.2 9.4 Group Induction group Tu dieu tan 3,6g/kg Tu dieu tan 10,8g/kg Chart 3.1 The effects of Tu dieu tan granule on urinary serum uric acid level in mice Lô nghiên cứu 14 Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg increased uric acid excretion in mice, the difference was statistically significantly compared to the biological control group (p < 0,05) 3.2 Results of clinical studies 3.2.1 Characteristics of study subjects A comparison between the study group and the control group regarding age, sex, occupation, disease severity indicated (number of swollen and painful joints, average duration of chronic gout, VAS1, VAS2, VAS3, HAQ scores) at D0 showed no significant difference (p > 0,05) 3.2.2 Results of supporting gout treatment in clinical and subclinical 3.2.2.1 Supporting gout treatment in clinical and subclinical of Tu dieu tan granule according to Western medicine * Acute anti-inflammatory effects Table 3.3 Improvement in the number of swollen joints Average number of Study group Control group p study-control ± SD (n = 60) ± SD (n = 60) swollen joints D0 D30 1,05 ± 0,47 0,65 ± 0,48 1,08 ± 0,28 0,83 ± 0,37 > 0,05 < 0,05 Average improvement D0 - 0,40 ± 0,62 0,25 ± 0,44 > 0,05 D30 p (D0 - D30) < 0,001 < 0,001 After treatment, a statistically significant decrease in the average number of swollen joints was found in both groups (p < 0,001) At D30, the reductions in the study group was more pronounced than in the control group (p < 0,05) * Analgesic effects Table 3.4 Improvement in the pain intensity assessed by VAS1 Study group Control group p VAS1 index (point) study-control D0 D30 Average improvement D0 D30 ± SD (n = 60) ± SD (n = 60) 6,90 ± 0,82 2,19 ± 1,10 6,93 ± 0,80 4,07 ± 1,03 > 0,05 < 0,001 4,71 ± 1,26 2,87 ± 1,06 < 0,001 15 p (D0-D30) < 0,001 < 0,001 Tu dieu tan granule improved the average number of painful joints at D30 and improved the pain intensity assessed by VAS higher than the control group and pre-treatment, the difference was statistically significantly (p < 0,05) * Improvement in physical activity function Table 3.5 Improvement in physical activity function according to VAS2, VAS3, HAQ scores Average Study group Control group p study-control improvement D0 ± SD (n = 60) ± SD (n = 60) D30 VAS2 index (point) 4,56 ± 1,53 2,97 ± 1,12 < 0,001 VAS3 index (point) 4,58 ± 1,25 2,68 ± 1,14 < 0,001 HAQ index (point) - 0,96 ± 0,32 - 0,42 ± 0,22 < 0,001 After treatment, both two groups had statistically significant improvement in physical activities fucntion according to VAS2, VAS3, HAQ scores better than pre-treatment (p < 0,001) The improvement in the study group being more pronounced than the control group (p < 0,001) * Hypouricemic effects Table 3.6 SUA levels changed in the two groups pre- and posttreatment SUA levels (μmol/l) Study group ± SD (n = 60) Control group ± SD (n = 60) D0 D30 521,07 ± 69,16 320,65 ± 83,88 498,18 ± 63,26 438,65 ± 95,59 > 0,05 < 0,001 Average improvement D0 200,42 ± 100,14 57,53 ± 101,89 < 0,001 - D30 p (D0 - D30) < 0,001 < 0,001 p study-control After treatment, both two groups had statistically significant decreased in SUA levels more than pre-treatment (p < 0,001); the reduction in the study group being more pronounced than the control group (p < 0,001) 16 The percentage of patients in the study group who had the hypouricemic treatment efficacy was 98,33%, higher than the control group (71,67%), the effectiveness in the study group being more pronounced than in the control group (p < 0,001) 3.2.2.2 Supporting gout treatment in clinical and subclinical of Tu dieu tan granule according to traditional medicine * The effect of Tu dieu tan granule on Tong Feng treatment according to traditional medicine: Patient was assessed the Tong Feng treatment efficacy according to Nimodiping score After treatment, the percentage of patients in the study group had completed and pronounced results (98,33%) higher than the control group (15%), the difference was statistically significantly (p < 0,001) * The effect of Tu dieu tan granule on Tong Feng treatment in two traditional patterns Table 3.7 Changes in treatment evaluation indicators of two traditional patterns in the study group Average improvement D0 D30 Nimodiping index (point) Number of swollen joints (joint) Number of painful joints (joint) VAS1 index (point) VAS2 index (point) VAS3 index (point) HAQ index (point) SUA (μmol/l) Wind damp heat impediment pattern Phlegm blood stagnation pattern P(1-2) (1) ± SD (n = 12) (2) ± SD (n = 48) 10,92 ± 2,31 10,62 ± 2,83 > 0,05 0,42 ± 0,51 0,39 ± 0,64 > 0,05 0,42 ± 0,52 0,44 ± 0,58 > 0,05 > 0,05 2,52 ± 0,97 2,83 ± 0,80 > 0,05 4,00 ± 1,65 4,7 ± 1,48 > 0,05 4,08 ± 1,08 4,71 ± 1,27 > 0,05 0,88 ± 0,22 1,03 ± 0,32 222,26 ± 114,84 194,97 ± 96,69 > 0,05 After treatment, the study indicators were used to evaluate the anti-inflammatory, analgesic, hypouricemic effects, Nimodiping score in both two traditional patterns improved better than pretreatment, the difference between two patterns was not statistically significantly (p > 0,05) 3.3 Adverse effects 17 * In clinical: During treatment, the percentage of patients in control group had unexpected symptoms such as digestive disorders were 5% (3/60), itchy rash was 1,67% (1/60) Patients in the study group did not have unexpected symptoms * In subclinical: There was no significant differences in the number of red blood cells, white blood cells, platelets, hemoglobin, glucose, triglyceride, HDL-C and laboratory tests of liver and kidney functions, such as urea, creatinine, AST, and ALT between pre- and post-treatment, and between the two groups In the study group, cholesterol and LDL-C decreased statistically significantly compared to pre-treatment (p < 0,001) Chapter DISCUSSION 4.1 Discussion on the findings of experimental studies 4.1.1 Investigating the acute and sub-chronic toxicity effects * Acute toxicity: The dose of 78,1g/kg was the highest dose possible used for mices but no mices died within 72 hours, thus LD50 of Tu dieu tan granule have not been determined by oral in mices According to WHO guideline, Tu dieu tan granule is safety in clinical In the two groups which had diarrhea, mices were used Tu dieu tan granule at the dose of 17 times clinical dose (62,5g/kg) and 21 times clinical dose (78,1g/kg) This symptom may be due to the effect of gum in Radix Achyranthes asperae Gum is a herbal carbohydrate, it is not absorbed through the gastrointestinal membranes then causes laxation, which can cause diarrhea if being used at high dose * Subchronic toxicity: According to results, Tu dieu tan granule at the dose of 1,8g/kg after weeks of continuous administration and 5,4g/kg after weeks of continuous administration showed no haematopoietic toxicity and the treatment did not change the rats’ liver and kidney function indicated by biochemiscal as well as histopathological tests Tu dieu tan granule at the dose of 5,4g/kg after weeks of continuous administration showed the changes in hematological indicators but still in normal standards The bias of reducing number of red blood cells, hemoglobin levels, and hematocrit may be due to the action of saponin in Radix Achyranthes 18 asperae Saponin used in high dose for long time can rupture the red blood cells, thus affects hemoglobin level and hematocrit This indicator needs to be monitored in clinical studies 4.1.2 Acute anti-inflammatory effects * The carrageenin-induced rat paw oedema model: The results showed that Tu dieu tan granule at the dose of 1,8g/kg and 5,4g/kg had no anti-inflammatory effect at all times after causing inflammation This finding is not consistent with the previous studies on the anti-inflammatory effects of Rhizoma Atractylodis and Radix Achyranthes asperae on the carrageenin-induced rat paw oedema model Thus, we continue to conduct the rat peritonitis inflammatory model to evaluate the mechanism of Tu dieu tan granule * The rat peritonitis inflammatory model: The results on table 3.1 showed that Tu dieu tan granule at both two doses reduced the volume and protein content of rat peritonitis inflammatory statistically significantly compared to the biological control group (p < 0,001), reduced the number of white blood cells (p < 0,01) This result is consistent with some studies on the anti-inflammatory effect of Radix Achyranthes asperae, Rhizoma Atractylodis, Cortex Oroxyli, which were found in Tu dieu tan granule component Nguyen Huong Giang (2014) found that the hydroxyl at position of benzoxazinone in Semen Coicis extract has anti-inflammatory effect which inhibits the cell proliferation of nitric oxide and PGE2 by reducing the production of nitric oxide synthetase and cyclooxygenase * The rat arthritis after intrasynovial injection of sodium urate: This model was built up by Faires and McCarty which is specialize for evaluating the anti-inflammatory effects on gouty arthritis The results showed that Tu dieu tan granule at the dose of 2,1g/kg had effect on reducing knee oedema, synovial fluid, lowering the temperature of the inflammatory sites, reducing the infiltration of inflammatory cells after hrs and 24 hours The difference was statistically significantly compared to the biological group (p < 0,05) This result is consistent with the studies of Chen Guang Liang, Yang Yan Hua which evaluated the effectiveness of Tu dieu tan formula in the rat arthritis after intrasynovial injection of sodium urate Observing the images of knee pathology in rats treated Tu dieu tan granule (image 3.1) showed that: The synovial fluid has a 19 sloughed area, however it is easy to observe The synovial fluid had less oedema fluid, urate crystals and infiltration of inflammed cells than the biological control group In particular, the urate crystals in the synovium were rarely seen These images showed that Tu dieu tan granule excreted and reduced urate crystals, thereby inhibiting the spread of the inflammatory factors We suppose that saponin in Radix Achyranthes asperae, polysaccharide in Rhizoma Atractylodis and baicalein, oroxylin A in Cortex Oroxyli involved in inhibiting gouty arthritis with different mechanisms 4.1.3 Analgesic effects The results in the hot plate model and the hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test Instrument showed that Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had effects on prolonging reaction time for temperature, increasing the endurance for painful force, increasing the response time for painful stimulation, the difference was statistically significantly compared to the biological control group and pre-treatment (p < 0,05) The response time for painful stimulation was prolonged shows the analgesic effects of Tu dieu tan granule This effect may be due to β-eudesmol in Rhizoma Atractylodis and other substances in Radix Achyranthes asperae β-eudesmol has analgesic effect in the hot plate model and the acetic acid writhing and colorectal distention models 4.1.4 Hypouricemic effects * The potassium oxonat induced hyperuricemia in rat The results on table 3.2 showed that Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had hypouricemic effects, the difference was statistically significantly compared to the induction control group (p < 0,001) The SUA levels reduction at the dose 10,8g/kg higher than the dose 3,6g/kg Thus, Tu dieu tan granule is effective in lowering SUA levels in experimental According to Liu Ku, Rhizoma Atractylodis has effective treatment on mice with kidney failure caused by hyperuricemia According to Xu Jia Xin, Semen Coicis promoted the excretion of uric acid through the urinary system, thereby reducing SUA levels * The xanthin oxidase inhibatory activity in vitro and in vivo models: The results in both two models showed that Tu dieu tan 20 granule had no effects on inhibiting xanthin oxidase in vivo and in vitro These results suggested us to find out the other hypouricemic mechanism of Tu dieu tan granule * The diuretic and uricosuric activity in mice: The results on chart 3.1 showed that Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg increased uric acid excretion in mice, the difference was statistically significantly compared to the biological control group (p < 0,05) Thus, Tu dieu tan granule had hypouricemic effects by excreting uric acid through the urinary system This mechanism is related to the effects of achyranthine in Radix Achyranthes asperae and stigmasterol, p-coumaric acid in Semen Coicis Achyranthine has diuretic effect in mice This effect was demonstrated in the Lipschitz diuretic model Stigmasterol and p-coumaric acid eliminate uric acid which inhibit the proliferation, aggregation of calcium oxalate crystals, thereby inhibiting the binding of urate crystals on epithelial cells According to traditional medicine, Semen Coicis has effective on tonifying spleen qi which has been used on treating the damp stasis pattern with numbness, difficulty in movement Radix Achyranthes asperae has effective on circulating blood, stimulating meridians, diuretic, nourishing liver and kidney, dispelling wind dampness Radix Achyranthes asperae combined with Cortex Oroxyli and Rhizoma Atractylodis improved the effective of Semen Coicis on eliminating dampness, dỉuretic, analgesic The four herbal ingredients interacted with each other on lowering SUA levels in experimental 4.2 Discussion on clinical study results 4.2.1 The comparability in the characteristics of two groups of patients The age, gender, disease severity indicated (number of swollen and painful joints, average duration of chronic gout, VAS1, VAS2, VAS3, HAQ scores) were comparable between the study group and control group This is a major criterion in controlled experimental studies to ensure the study objectivity 4.2.2 Results of supporting gout treatment in clinical and subclinical 21 4.2.2.1 Supporting gout treatment in clinical and subclinical of Tu dieu tan granule according to Western medicine * Acute anti-inflammatory effects: The results on table 3.3 showed that all patients had the reductions of swollen joints At D30, the reductions in the study group was more pronounced than in the control group (p < 0,05) Thus, Tu dieu tan granule had antiinflammatory effect in clinical These effects were demonstrated in experimental models Capably, saponin in Radix Achyranthes asperae, polysaccharide in Rhizoma Atractylodis and baicalein, oroxylin A in Cortex Oroxyli involved in inhibiting gouty arthritis with different mechanisms * Analgesic effects: The results showed that Tu dieu tan granule improved the average number of painful joints at D30 and improved the pain intensity assessed by VAS higher than the control group and pre-treatment, the difference was statistically significantly (p < 0,05) According to traditional theory, the painful on the lower limb occurs when the climate changes, which is related to the wind damp stagnation factors Semen Coicis has effect on tonifying spleen qi, diuretic, clearing heat, analgesic which is the king ingredients of Tu dieu tan granule Radix Achyranthes asperae supports Semen Coicis on diuretic, eliminating dampness, clearing heat, detoxifying that treats the numbness pattern with painful joints, difficulty in movement Rhizoma Atractylodis has effect on tonifying spleen qi, eliminating phlegm that improves the effectiveness of Semen Coicis on eliminating phlegm Therefore, Tu dieu tan granule is suitable for the gouty arthritis treatment on swollen, hot, red, painful symptoms * Improvement in physical activity function: After treatment, both two groups had statistically significant improvement in physical activity fucntion according to VAS2, VAS3, HAQ scores better than pre-treatment (p < 0,001) The improvement in the study group being more pronounced than in the control group (p < 0,001) The effect of improving physical activity function of Tu dieu tan granule may be due to the anti-inflammatory and analgesic effects in clinical By reducing the swelling, heat, redness, painful of joints, patients were capable to the normal activities easier * Hypouricemic effect: After treatment, both two groups had statistically significant decreased in SUA levels more than pre- 22 treatment (p < 0,001); the reduction in the study group being more pronounced than the control group (p < 0,001) The percentage of patients in the study group who had the hypouricemic treatment efficacy was 98,33%, higher than the control group (71,67%), the effectiveness in the study group being more pronounced than in the control group (p < 0,001) The results obtain from experimental model showed that Tu dieu tan granule had hypouricemic effects by excreting uric acid through the urinary system This mechanism is related to the effects of achyranthine in Radix Achyranthes asperae and stigmasterol, p-coumaric acid in Semen Coicis 4.2.2.2 Supporting gout treatment in clinical and subclinical of Tu dieu tan granule according to traditional medicine Our study was the first national study to apply the Nimodiping score, with the aim to qualitative quantification of traditional symptoms in order to diagnose the pattern exactly and improve the clinical efficacy After treatment, the percentage of patients in the study group had completed and pronounced results (98,33%) higher than control group (15%), the difference was statistically significantly (p < 0,001) The symptoms made patient tired were the feeling of thirsty, heat inside heart, anorexia, full stomach, difficulty in sleeping, heavy in the lower limbs, body aches Improving the symptoms of Tong Feng will improve the quality of life, increase the quality and effectiveness on treatment, while minimizing the unexpected effects The results on table 3.7 showed that after treatment, the study indicators were used to evaluate the anti-inflammatoy, analgesic, hypouricemic effects, Nimodiping score in both two traditional patterns improved better than pre-treatment, the difference between two patterns was not statistically significantly (p > 0,05) Tu dieu tan granule has the same effects on the wind damp heat impediment pattern and phlegm blood stagnation pattern 4.2.3 Adverse effects * In clinical: During treatment, the percentage of patients in the control group had unexpected symptoms such as digestive disorders were 5% (3/60), itchy rash was 1,67% (1/60) Patients with gastrointestinal disorders were treated with Berberin, an itchy rash was treated with anti-histamin Patients were stability after taking the 23 medications and continued to participate in the study Patients in the study group did not have unexpected symptoms Thus, the combination of Tu dieu tan granule and allopurinol did not cause adverse effects In our view, the unexpected effects in the control group may be due to inappropriate diet or/and activities, or patient had allergy to allopurinol and/or placebo, or due to interactions between hypertension, diabetes medicines with allopurinol leading to gastrointestinal disorders or rashes The nutrition of patients were nursing care with careful monitoring, so the possibility of food poinoning is very low According to Hatoum Hind (2015), long-term use of allopurinol in patients with chronic gout may increase susceptibility, causing itching, drug allergies, reduced the effective of low dose allopurinol * In subclinical: The results showed that there was no significant differences in the number of red blood cells, white blood cells, platelets, hemoglobin, glucose, triglyceride, HDL-C and laboratory tests of liver and kidney functions, such as urea, creatinine, AST, and ALT between pre- and post-treatment, between the two groups Thus, Tu dieu tan granule did not affect the subclinical indexes, it is safe for long-term clinical use CONCLUSIONS Tu dieu tan granule has not shown the acute and subchronic toxicity Tu dieu tan granule has effects on anti-inflammatory, analgesic, hypouricemic by excreting uric acid through the urinary system 1.1 Acute and subchronic toxicity - Tu dieu tan granule at the dose of 78,1g/kg (21 times clinical dose) has not been determined Lethal dose (LD50) - Tu dieu tan granule at the dose of 1,8g/kg (corresponding to the clinical dose) and 5,4g/kg (3 times higher than clinical dose) after weeks of continuous administration showed no haematopoietic toxicity and the treatment did not change the rats’ liver and kidney function indicated by biochemiscal as well as histopathological tests 1.2 Pharmacological effects - Tu dieu tan granule had anti-inflammatory effects in the rat peritonitis inflammatory, the rat arthritis after intrasynovial injection of sodium urate models 24 - Tu dieu tan granule had analgesic effects in the hot plate model, the hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test Instrument - Tu dieu tan granule had hypouricemic effect in the potassium oxonat induced hyperuricemia in rat Tu dieu tan granule had hypouricemic effect by excreting uric acid through the urinary system Tu dieu tan granule had supported the chronic gout treatment, had effect in treating two patterns of Tong Feng Initially, no adverse effects of Tu dieu tan granule on clinical were observed 2.1 Supporting the chronic gout treatment - The hypouricemic effects: In the study group (the combined treatment of Tu dieu tan granule with allopurinol): SUA levels decreased 200,42 ± 100,14μmol/l after 30 days of treatment The effectiveness of lowering urate decreased by 98,33% - The anti-inflammatory and analgesic effects: In the study group, the number of swollen, painful joints decreased after treatment Mean pain intensity on VAS1 scale, physical activities function according to VAS2, VAS3 scale improved higher than pre-treatment Median function assessed by HAQ score was improved 0,96 ± 0,32 point - The improvement of traditional symptoms: Its efficacy of improving traditional symptoms was 98,33% - The improvement of these indicators in the study group were statistically significantly higher compared to the control group (the combined treatment of placebo with allopurinol) 2.2 Treating effect on two traditional patterns - Tu dieu tan granule had effect in treating both the wind damp heat impediment pattern and the phlegm blood stagnation pattern with the SUA levels decreased 222,26 ± 114,84μmol/l; 194,97 ± 96,69μmol/l and Nimodiping score improvement was 10,92 ± 2,31 points; 10,62 ± 2,83 points; the difference between two patterns was not statistically significantly 2.3.Adverse effects - Initially, no adverse effects of Tu dieu tan granule on clinical or subclinical were observed after 30 days of treatment RECOMMENDATIONS 25 The efficacy of Tu dieu tan granule should be investigated on a larger number of patients with a longer duration of study The efficacy of Tu dieu tan granule in the treatment of other arthritis diseases such as rheumatoid arthritis, osteoarthritis… should be investigated  ... group Tu dieu tan 3,6g/kg Tu dieu tan 10,8g/kg Chart 3.1 The effects of Tu dieu tan granule on urinary serum uric acid level in mice Lô nghiên cứu 14 Tu dieu tan granule at the dose of 3,6g/kg... Tong Feng Wan, or the combined of Narcaricin and Si Wu Tang) 1.3 Review of Tu dieu tan granule 1.3.1 Origin and traditional efficacy Tu dieu tan granule is derived from the traditional medicine... Table 3.1 Acute anti-inflammatory effects of Tu dieu tan granule on the volume and components of rat peritonitis inflammatory Tu dieu tan Tu dieu tan Biological Aspirin control granule granule Indicators