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-infla
Trang 11 The urgency of the study
Gout is a common disease, due to the deposition of monosodiumurate (MSU) crystals in synovial fluid or tissues Between 1 - 4% ofthe adult population worldwide has gout In developed countries, theincidence of gout in men is between 3 - 6% of the population, andbetween 1 - 2% of the population In some developing countries, theincidence of gout may increase to 10% Gout usually develops instages About 25% of gout patients have joint damage, irreversiblejoint deformity The quality of mental and physical life of goutpatients decreased, due to the frequent affects of acute gout attacks,the number of swollen, painful joints increased over time, theprogression and spread of tophus, using colchicin, corticosteroids
To research into new herbal medicine for gout treatment that canlimit unwanted effects is meaningful and necessary Therefore, overthe past decade, scientists have studied a variety of herbal andtraditional medicines for the gout treatment Vietnam traditionalmedicine has many effective formulas in treating gout, Si Miao Yanformula 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 theeffects of Tu dieu tan granule on two objectives:
2 Study objectives
1. Evaluating the effectiveness of Tu dieu tan granule on acute and sub-chronic toxicity, anti-inflammatory, analgesic and lowering serum urate level in experimental
2. Evaluating anti-inflammatory, analgesic and lowering serum urate level and unexpected effects of Tu dieu tan granule in patient with chronic gout.
3 Practical implications and new contributions of the thesis
This scientific study based on the effect evaluation of a newherbal product called Tu dieu tan granule which was used in chronic
Trang 2gout treatment According to the clinical experience, we reduce thetraditional formula Si Miao to suit Vietnamese climate, physiologicalcharacteristics 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 hypouricemiceffects of Tu dieu tan granule in experimental and clinical
The experimental results showed that Tu dieu tan granule has notshown the acute and subchronic toxicity Tu dieu tan granule hasanti-inflammatory, analgesic and hypouricemic effects by excretinguric acid through urinary system in the experimental model After 30days of treatment, the clinical results showed that the combined of Tudieu tan granule with allopurinol reduced serum uric acid (SUA)levels were 200,42 ± 100,14μmol/l; the effectiveness of loweringmol/l; the effectiveness of loweringSUA decreased by 98,33%; decreased the number of swollen, painfuljoints, improved mean pain intensity on VAS1 scale and physicalactivities function according to VAS2, VAS3 Improving medianfunction assessed by HAQ was 0,96 ± 0,32 point; Its efficacy ofimproving traditional symptoms was 98,33% The improvement ofthese indicators in the study group were statistically significantlyhigher 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 decreased222,26 ± 114,84μmol/l; the effectiveness of loweringmol/l; 194,97 ± 96,69μmol/l; the effectiveness of loweringmol/l and Nimodiping scoreimprovement 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 wereobserved These findings are scientific indications of the safety, anti-inflammatory and analgesic, hypouricemic effects of Tu dieu tangranule in treating chronic gout
4.Thesis structure Apart from the Introduction and Conclusions
chapters, the thesis consists of four chapters
Chapter 2: Study subjects and methods 25 pages
Chapter 3: Results 37 pages
And 38 tables, 6 graphs, 5 images, 13 appendixes, and 151references (34 Vietnamese references, 93 English references, 24Chinese references)
Trang 3Chapter 1 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 SUAlevel rises over saturation, uric acid deposits in the form of MSU injoint or tissues, triggering inflammatory reactions that relating acutegout attacks Between gout flares, MSU are deposited and formedtophus The more uric acid is deposited the more inflammatory reactionbecomes 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 mostimportant target is reducing SUA, which allows patients to preventcomplications of chronic renal failure, organ damage Treatment Principle:Patients maintain a diet of chronic gout, combined with hypouricemicdrug Start at low doses, gradually increasing to therapeutic doses andmaintain continuously, uninterrupted, which can be combined withNSAIDs or colchicin to prevent acute gout attacks Target is loweringSUA levels less than 6mg/dl (360μmol/l; the effectiveness of loweringmol/l) in chronic gout or 5mg/dl(300μmol/l; the effectiveness of loweringmol/l) in chronic gout with tophus Common drug are xanthinoxidase inhibitors (allopurinol, febuxostat ) which inhibit xanthinoxidase According to American College of Rheumatology, startingdose of allopurinol should be no greater than 100mg/day and start at50mg/day in stage 4 or severe CKD Dosage can be raised above300mg daily, even with renal impairment Febuxostat is indicated forpatients with allopurinol-resistant or allergic allopurinol Febuxostatinhibits vasoactive agents and prevents inflammation In addition,other urate lowering therapy such as uricosuric therapy (probenecid,benzbromarone), uricolytic drugs (Uricozym, Pegloticase), urinealkalinization (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, notcleared) The causes of “Tong bi” include six external climatic factors,seven emotional factors, and other pathogenic factors
Trang 41.2.2 Treatment of gout
Depending on the clinical types, there are appropriate treatmentsprinciples and specific formulas According to Hao Xian Gou and JieTian Ping – China (2008), Tong Feng is divided into five patterns:Wind cold damp impediment pattern, wind damp heat impedimentpattern, phlegm blood stagnation pattern, spleen and kidney yangdeficiency pattern, liver and kidney yin deficiency pattern For thewind cold damp impediment pattern, the principles include ofdispelling wind, scattering cold, eliminating dampness, circulatingmeridians; the appropriate herbal formula are Tigao Tai Tang orFangjian Fengge Tang For the wind damp heat impediment pattern,the principles include of dispelling wind, clearing heat, eliminatingdampness; the appropriate herbal formula are Er Miao San combinedwith Bai Hu Gui Zhi Tang; for the damp heat pattern includenumbness, the principles include of clearing heat, eliminatingdampness, circulating meridians, analgesic, the appropriate herbalformula are Si Miao San combined with Long Dan Da Gan Tang Forthe phlegm blood stagnation pattern, the principles include ofcirculating blood, dispelling stasis, transforming phlegm; theappropriate herbal formula are Fu Yuan Huo Xue Tang combinedwith Er Chen Tang For the spleen and kidney yang deficiencypattern, the principles include of tonifying spleen qi, supplementingkidney yang; the appropriate herbal formula is Wu Tou Gui ZhiTang For the liver and kidney yin deficiency pattern, the principlesinclude of nourishing yin, supplementing kidney and liver yin; theappropriate 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 SiMiao Although the formula composition does not change, but due tothe reduction of each dose in the formula compared to the old andconverted formulation, Tu dieu tan granule should be determinedtoxicity to ensure safety for testing the next phase Tu dieu tan granulewas evaluated acute and sub-chronic toxicity It was evaluated anti-inflammatory, analgesic and hypouricemic effects in experimentalmodels such as the carrageenin-induced rat paw oedema, rat peritonitisinflammatory, rat arthritis after intrasynovial injection of sodium urate;hot plate, hyperalgesia to thermal stimulation in rat by the ThermalPlantar Test Instrument; potassium oxonat induced hyperuricemia in
Trang 5rat, 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 andmaintaining SUA levels below 6mg/dL (<360μmol/l; the effectiveness of loweringmol/L) Next-generation drugs were being clinical used: febuxostat; 3,4-dihydroxy-5-nitrobenzaldehyde (DHNB)… In addition, based on the gout flaremechanism, researchers was still testing IL-1β inhibitors (canakinumab,anakinra) on anti-inflammatory and analgesic effects in gout flares InVietnam, there has been no research on new medicine or gout treatmentmedication in the last decade Most studies of Western medicine focused
on the medicine abuse (corticosteroid) Some studies assessed theknowledge of patients in preventing and using gout medicines
1.2.2 Review on gout treatment studies according
to traditional medicine
In experimental, many models were built up to evaluate theeffectiveness of gout medication on anti-inflammatory, analgesic, andlowering SUA level such as: Xanthin oxidase inhibatory in vitro and in
vivo models, rat arthritis after intrasynovial injection of sodium urate,evaluating hypouricemic effects in potassium oxonat inducedhyperuricemia in rat Shen Wei Zeng, Pang Xue Feng used rat arthritisafter intrasynovial injection of sodium urate in evaluating anti-inflammatory and analgesic effects of Dang Gui Nian Tong Tang, ZhiTong Qu Feng Tang In clinical, many scientists have built up andstudied the effectiveness of new herbal formulas such as: HA1 herbalformula, GLP hypouricaemia Other authors have investigated theassociation between Western medicine and traditional medicine in order
to have higher therapeutic efficacy than either Western medicine ortraditional medicine All researchs have the same procedure is controlledtrial, comparison study (Studying on the effectiveness of Tong FengWan, 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 Si Miao
in Fang Ji Xue book, with changing doses and replacing Phellodendron
sp Rutaceae and Achyranthes bidenta with Cortex Oroxyli and Radix Achyranthes asperae The increased dosage of Tu dieu tan granule was
Trang 6intended to enhance the effectiveness on excreting phlegm, eliminatingdampness, clearing heat, circulating blood
1.3.2 Components and efficacy of Tu dieu tan granule
- Tu dieu tan granule 7,5g with the composition of 1,13g dried extract
Radix Achyranthes asperae; 0,56g dried extract Cortex Oroxyli; 1,5g dried extract Semen Coicis; 0,56g dried extract Rhizoma Atractylodis;
excipients enough for 7,5g
- Semen Coicis has effective on tonifying spleen qi, diuretic Chemical composition: Semen Coicis contains starch, protein, amino
acid Substances isolated from the semen include coixenolid,policosanol, phytosterols, lactams, phenol compounds P-coumaric
acid in Semen Coicis granule inhibits the nucleation, growth and
aggregation of stone crystal, inhibits the binding of crystals on therenal tubular epithelium, inhibitits the formation of calcium oxalatecrystals which depends on acid p-coumaric concentration
- Radix Achyranthes asperae has effective on circulating blood,
stimulating meridians, diuretic, nourishing liver and kidney,dispelling wind dampness Chemical composition: Betaine,
achyrathine, β-ecdysone, stigmasterol Radix Achyranthes asperae
contains saponin, polysaccharide, ketosteroid and β-sitosterol
Saponin in Radix Achyranthes asperae has an inhibitory effect on
cytokines, which reduces swelling and arthritis Achyranthine hasdiuretic effect in white mice and does not change the pH in urine
- Cortex Oroxyli has effective on clearing heat, detoxifying,
eliminating dampness in lower burner Chemical composition:Baicalein, oroxylin A, chrysin, tetuin, β-sitosterol, tannic acid
Cortex Oroxyli contains p-coumaric acid Baicalein, oroxylin A in Cortex Oroxyli has acute anti-inflammatory effects by inhibiting NF-
QB, thereby inhibiting the release of inflammatory response factorsPGE2, IL6, IL-1β
- Rhizoma Atractylodis has effective on scattering cold and
circulating cuticle, tonifying spleen qi, detoxifying, eliminatingphlegm Chemical composition: Mostly are hinesol or β-eudesmol,atractylon Polysaccharide has effective on regulating the intestineimmune system against candida albicans β-eudesmol and atractylonprotect the liver cells
Trang 7Chapter 2 STUDY MATERIALS, SUBJECTS AND METHODS 2.1 Study materials
2.1.1 Baseline medicines
- Hypouricemic medicine: Allopurinol oral tablet contains 300mgallopurinol, excipients just enough This product is distributed byDomesco Medical Import Export Joint Stock Corporation
- NSAIDs: Mofen oral tablet contains 400mg ibuprofen, excipientsjust enough This product is distributed by Medopharm – India
2.1.2 Studied medicines
- Tu dieu tan granule 7,5g with the composition of 1,13g dried extract
Radix Achyranthes asperae; 0,56g dried extract Cortex Oroxyli; 1,5g dried extract Semen Coicis; 0,56g dried extract Rhizoma Atractylodis;
excipients enough for 7,5g Tu dieu tan granule is produced byDepartment of Pharmacy/Military Institute of Traditional Medicine(MITM), has been granted certification basic-level standard
- Placebo: Presenting format like Tu dieu tan granule One packagewith the composition of 7,5g lactose, excipients enough Placebo isproduced by Department of Pharmacy/Military Institute of TraditionalMedicine
2.2 Study subjects
The studies were conducted from March 2015 to May 2017
2.2.1 Subjects in experimental studies: Swiss mice, Wistar rats, both
species are healthy, qualify for participation in experimental models.Location of the experimental studies: Department of Pharmacology/HanoiMedical University, Experimental Laboratory/MITM, Department ofPharmacology/Hanoi University of Pharmacy
2.2.2 Subjects in clinical studies: The sample size was calculated
according to formulas for clinical studies established by World
Health Organisation The study was performed on 120 inpatients at MITM, qualify the clinical inclusion and exclusion criterias:
* Inclusion criteria: Selecting patients regardless of age, gender,
occupation, residence, consent to participate in the study, based on thecriteria of Western medicine and traditional medicine According toWestern medicine: Patient with chronic gout (include gout flare) wasdiagnosed according to Bennett and Wood criteria (1968), with SUAlevels > 420μmol/l; the effectiveness of loweringmol/l for male and > 360μmol/l; the effectiveness of loweringmol/l for female According totraditional medicine: Patient was diagnosed wind damp heatimpediment pattern or phlegm blood stagnation pattern
Trang 8* Exclusion criteria: Subjects with hepatic failure, renal failure,
myocardial infarction, cerebral hemorrhage; Long-term gastrointestinaldisturbances affect the process of drug absorption and metabolism; mentalillness, malnutrition, infectious diseases; contraindications for allopurinol;Tong Feng symptoms do not belong to the wind damp heat impedimentpattern 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 micereceived the medication under investigation with increasing dosesthrough oral use The aim was to identify the non-lethal highest dose(0%), the lethal lowest dose (100%) and the intermediate doses Themice were observed for 72 hours to monitor their mortality rate andoverall health condition; and were observed for 7 days sincemedication administration to monitor mortality rate
* Sub-chronic toxicity: According to WHO guideline, Wistar rat
were devided into 3 groups: Controll group (distilled water);Treatment group 1 (Tu dieu tan granule 1,8g/kg/day - corresponding
to the clinical dose); Treatment group 2 (Tu dieu tan granule 5,4g/kg/day - three times higher than the clinical dose) Rats were drunkwater and Tu dieu tan granule for 8 weeks continuously The ratswere observed to monitor their weight, eating, sleeping, activity,digestion, hematology, biochemistry, liver and kidney function, liverand kidney histopathology Comparisons were made between pre-and 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 thecarrageenin-induced rat paw oedema, rat peritonitis inflammatory, ratarthritis after intrasynovial injection of sodium urate The carrageenin-induced rat paw oedema model was conducted according to Wintermethod The rat peritonitis inflammatory model was conducted according
to Patel method The rat arthritis after intrasynovial injection of sodiumurate model was conducted according to Faires and McCarty method
2.3.1.3 Analgesic effect
Trang 9Evaluating analgesic effect in experimental models such as thehot plate model (evaluation the central analgesic effect), thehyperalgesia to thermal stimulation in rat by the Thermal Plantar TestInstrument (evaluation the peripheral analgesic effect) The hot platemodel was conducted according to Vogel method The hyperalgesia
to thermal stimulation in rat by the Thermal Plantar Test Instrumentwas conducted according to Randall-Selitto method
2.3.1.4 Hypouricemic effect
Evaluating hypouricemic effect in experimental models such as thepotassium oxonat induced hyperuricemia in rat (evaluating hypouricemiceffect), 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) Thepotassium 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 toStarvic method
presented in the same format
During 30 treatment days, patients in the study group usedallopurinol and Tu dieu tan granule, patients in the control groupused allopurinol and placebo Patient was treated on the first day ofthe study (D0): Allopurinol (01 tablet taken once per day, after meal);
Tu dieu tan granule (1 package per time taken twice per day, 1 hourbefore meal); placebo (1 package per time taken twice per day, 1hour before meal) During the study, patients did not use othermedicines In case of patient with gout flare was painful and ask for
Trang 10analgesic medicine, he/she would be allowed to use Mofen 400mgin
5 days maximum (01 tablet per time, taken twice or three times perday) The patients’ identities were only revealed after the course oftreatment was completed, then data collection, analysis and reportwere 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 theeffectiveness of Tu dieu tan granule in clinical and subclinical andcompare the effectiveness between the control and study group, pre-and post-treatment These indicators were assessed in the first day(D0) and the 30th day (D30) of the treatment course: Anti-inflammatoyeffect (reduced the number of swollen joints); analgesic effect(reduced the number of painful joints, improved VAS1 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); evaluatingSUA levels reduction in the study and control groups, in twotraditional patterns
- Unexpected effects were monitored during the whole course of
treatment: The unexpected symptoms in clinical; hematologicalindicators 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 thestudy to be conducted at MITM was retrieved from the hospital’s
General Sciences Council
Chapter 3 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 4 to 6 hours, all rats showed normal function, agility, smoothfur, normal eating activities and dry stool Rats drank Tu dieu tangranule at the dose 62,5g and 78,1g were diarrhea on the last 2 days.There were no adverse symptoms, no rats died within 72 hours ofadministration and for 7 days Acute toxicity and LD50 of Tu dieu tangranule have not been determined
Trang 11* Subchronic toxicity: During the experiments, the rats in all 3
groups showed normal function There were no difference in overallconditions and weight Tu dieu tan granule at the dose of 1,8g/kgafter 8 weeks of continuous administration and 5,4g/kg after 4 weeks
of continuous administration showed no haematopoietic toxicity andthe treatment did not change the rats’ liver and kidney functionindicated by biochemiscal as well as histopathological tests Tu dieutan granule at the dose of 5,4g/kg after 8 weeks of continuousadministration showed the changes in hematological indicators butstill 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-inflammatoryeffect 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
Indicators
Biologicalcontrolgroup(n = 10)
Aspirin200mg/kg(n = 10)
Tu dieu tan
granule 1,8g/
kg(n = 10)
Tu dieu tan
granule 5,4g/kg(n = 10)Volume of
** p ≤ 0,01 and *** p ≤ 0,001 compared to control group
The results showed that Tu dieu tan granule at the two dosesreduced the volume and protein content of rat peritonitisinflammatory statistically significantly compared to the biological
Trang 12control 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, sizeand temperature of the knee joint: Tu dieu tan granule at the dose of2,1g/kg had effect on reducing knee oedema, synovial fluid, loweringthe temperature of the inflammatory site, reducing the infiltration ofinflammatory cells after 6 hrs and 24 hours The difference wasstatistically significantly compared to the biological group(p < 0,05)
- Knee pathology in rats treated Tu dieu tan granule: The synovialfluid has a sloughed area which is easy to observe The synovial fluidhad less oedema fluid, urate crystals and infiltration of inflammedcells than the biological control group
Picture 3.1 Knee pathology in rats treated Tu dieu tan granule
(Rat number 25, treated Tu dieu tan granule)
1 The synovial fluid had less edema fluid, urate crystals
2 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 thebiological 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 differencewas statistically significantly compared to the biological controlgroup and to pre-treatment (p < 0,05)
2