| Roukou Wuwei pill,a classic Mongolian prescription,consists of Myristica fragrans Houtt.,Inula helenium,Aucklandia lappa Decne,Choerospondias axillaris fruit and Piper longum L.The pill was contained in the "People’s Republic of China Ministry of Health Drug Standards"(Mongolian Medicine Volume),for the treatment of Heart "He Yi" disease.Modern pharmacological studies have shown that Roukou Wuwei pill is also effective in treatment of depression.The results of the screening of the pharmacological substances showed that the volatile oil of nutmeg,the sesquiterpene of I.helenium and A.lappa Decne,the phenolic acid of C.axillaris fruit and the alkaloid of P.longum L were the main active substances.The existing pharmacokinetic data suggest that the bioavailability of these components is poor except phenolic compounds,due to poor water solubility.In order to meet the needs of the modernization of Traditional Chinese Medicine preparation,the main purpose of this study was to develop new preparation for the clinical under the guidance of the theory of Chinese medicine,including the extraction,separation,purification,new pharmaceutical technologies,quality evaluation and pharmacokinetics.Part 1 Extraction,separation and purification of active ingredients from herbsObjective: To optimize the extraction of active ingredients by ultrasonic-assisted extraction.The separation and purification of sesquiterpene components from I.helenium and A.lappa Decne by silica gel column chromatography.The macroporous resin was used to purify gallic acid and piperine.Methods: The extraction process of alantolactone and isoalantolactone from I.helenium,costundide and dehydrocostus lactone from A.lappa Decne,gallic acid from C.axillaris fruit and piperine from P.longum L.were optimized.Several factors such as extraction solvent,ultrasonic time and liquid/material ratio were evaluated.Purification process of the sesquiterpene from I.helenium and A.lappa Decne were studied by particle size,loading amount,elution flow rate and eluent content.In addition,the suitable macroporous resin was screened and the factors affecting the elution were investigated by dynamic adsorption test.Finally,the purification process of gallic acid and piperine by macroporous resin was established.Results: The optimal extraction conditions and separation conditions for I.helenium were as follows: extraction solvent,methanol;extraction time,40 min;solid–liquid ratio,1:20(g/mL);silica gel column,100-200 mesh;loading amount,1:40;eluted solvent,petroleum ether:acetone(30:1)and flow rate 2.0 BV/h.Under these optimum conditions,the yield was 32.68 g/kg,which contained 14.64 g(44.80%)of isoalantolactone and 9.29 g(29.50%)of alantolactone.The optimal extraction conditions and separation conditions for A.lappa Decne were as follows: extraction solvent,ethanol;solid–liquid ratio,1:30(g/mL);extraction time,20 min;silica gel column,100-200 mesh;loading amount,1:30;eluted solvent,petroleum ether:ethyl acetate(25: 1)and flow rate 2.0 BV/h.Under these optimum conditions,the yield was 37.14 g/kg,which contained 10.52 g(28.33%)of costundide and 12.16 g(32.74%)of dehydrocostus lactone.The optimized extraction conditions for C.axillaris fruit were as follows: extraction solvent,80% ethanol;solid–liquid ratio,1:30(g/mL)and ultrasonic extraction time,30 min.The gallic acid was purified by NKA-9 macroporous resin with sample concentration of 2.94 mg/mL,flow rate of 2 BV/h,and eluent of 80% ethanol.Under these optimum conditions,the yield was 1.54 g/kg,which contained 1.13 g(73.46%)of gallic acid.The optimized extraction conditions for P.longum L.were as follows: extraction solvent,ethanol;solid–liquid ratio,1:30(g/mL)and ultrasonic extraction time,20 min.The piperine was purified by HPD 100 macroporous resin with sample concentration of 2.4 mg/mL,flow rate of 2 BV/h,and eluent of 95% ethanol(p H3).Under these optimum conditions,the yield was 25.72 g/kg,which contained 19.40 g(75.43%)of piperine.Conclusions: The contents of effective parts obtained from I.helenium,A.lappa Decne,C.axillaris fruit and P.longum L.were more than 50%,which was in accordance with the requirements of effective parts.Part 2 Preparation of intermediates and pharmacodynamics experimentObjective: To improve the stability and solubility of the extract and observe the effect of the intermediate on the behavior of depressed rats.Methods: Volatile oil of nutmeg was prepared into cyclodextrin(b-CD)inclusion complex to improve its stability.An orthogonal assay was used to optimize the inclusion process.Differential scanning calorimetry(DSC)and infrared spectroscopy(IR)was applied for the characterization of the inclusion complex.The solid dispersion(SD)was prepared by using poloxamer 188,PEG 6000 and polyvinyl pyrrolidone K30 as carrier materials.The effects of carrier materials and drug/carrier ratio on the dissolution rate were evaluated,and the characterization of the SD was assessed by DSC.To investigate the effect of Roukou Wuwei intermediates on chronic unpredictable mild stress(CUMS)combined with isolated induced depressive rat model.Results: The influence factor to the inclusion rate declined in the sequential order: inclusion time> volatile oil/ b-CD ratio> inclusion temperature.The results demonstrated that the optimal conditions were 2 h of inclusion time,1:10 of volatile oil/ b-CD ratio and 50°C of inclusion temperature.Under these optimal conditions,the inclusion rate was 86.7%.It was found that the solid dispersion prepared with P188 as the carrier was the most appropriate,and the optimal drug/P188 ratio was 1:8 for A.Radix sesquiterpenoids composition,1:6 for I.helenium sesquiterpenoids composition and piperine,respectively.The results of DSC showed that the endothermic peak of the drug disappeared in the solid dispersion,showing only the endothermic peak of P188,which indicated that the solid dispersion was successfully prepared.Roukou Wuwei intermediates(200,100,50 mg/kg)can improve the immobility time of CUMS rats and the incubation period of novel inhibition of feeding.It has anti-anxiety and anti-depressant effect.Conclusions: Pharmacokinetic test confirmed that Roukou Wuwei intermediates were effective for CUMS combined with isolated rat model,indicating that extraction,purification and intermediate preparation process were reasonable and feasible.Part 3 Preparation of Roukou Wuwei sustained release pelletsObjective: The aim of this study was to develop Roukou Wuwei sustained-release pellets using extrusion roll and pot coating,respectively and establish a quality control method for the sustained-release pellets.Methods: The effects of excipients,adhesive agents and drug loading on pellet formation were investigated with roundness as an index.On the basis of this,the orthogonal experiment was designed to optimize the preparation process with the extrusion speed,the roll speed and the spheronization time as the influencing factors.The effect of the coating pan speed,spray pressure,flow rate,temperature and coating weight gain on the drug release were also evaluated with Eudragit RS100 as the coating material.In order to improve the quality control of the drug,a UPLC method for simultaneous determination of gallic acid,piperine,costundide,dehydrocostus lactone,isoalantolactone and alantolactone was established.The release profiles of Roukou Wuwei sustained-release pellets were compared in different dissolution medium.And the mechanism of drug release was clarified.Results: The single factor experiment results suggested that the appropriate prescription is drug/MCC ratio of 3:7 and adhesive agents of 0.5% hydroxypropylmethylcellulose.Orthogonal test shows that the spheronization speed has the greatest effect on the pellet forming,and then the spheronization time and the extrusion speed.The final preparation process was as follows: M.fragrans Hott.essential oil/b-CD inclusion complexes 40 g,I.helenium sesquiterpenoids SD 18.3 g,A.Radix sesquiterpenoids SD 26.7 g,C.axillaris fruit extracts 0.058 g,P.longum L.SD 1.8 g,excipient MCC 202.6 g,crushed to sieve 80 mesh and mixed with 0.5% HPMC as adhesive agents,with extrusion speed of 40 rpm,roll speed of 800 rpm and the spheronization time of 3.5 min.Coating conditions were inlet temperature of 35°C,coating pot speed of 40 rpm,jet flow rate of 4 mL/min,and coating weight gain of 4%.The mobile phase consisted of acetonitrile(A)-water(B),gradient elution program of 0~0.8 min(8:92),0.8~1.0 min(8:92~52:48),1.0~6.5 min(52:48),6.5~7.0 min(52:48~8:92).The detection wavelength of gallic acid and piperine was 270 nm and 343 nm,respectively.Costundide,dehydrocostus lactone,isoalantolactone and alantolactone was detected at 225 nm.The inject volume was 0.3 mL/min,and the injection volume was 4 μL.The 6 components determined in Roukou Wuwei sustained-release pellets showed good linear relationship.Dissolution results expressed that Fick diffusion was the main drug release mechanism.It is a membrane controlled sustained-release pellets.Conclusions: The dissolution rate of all the components were more than 85% for 12 h,and the release was in accordance with Fick’s diffusion.The UPLC method is simple,rapid and sensitive,and it can be used for the determination of the content of Roukou Wuwei sustained-release pellets,which provides a theoretical basis for the quality evaluation.Part 4 Study on pharmacokinetics of Roukou Wuwei sustained releasepelletsObjective: To establish a UPLC method for simultaneous determination of costundide,dehydrocostus lactone,isoalantolactone and alantolactone in plasma of beagle dogs.And the pharmacokinetics parametes of Roukou Wuwei sustained-release pellets were also determined.Methods: Beagle dogs were randomly divided into two groups.A single dose crossover experiment was performed to evaluate the pharmacokinetic parameters in Beagle dogs between Roukou Wuwei sustained-release pellets and Roukou Wuwei pills.Results: The standard curve of each component has good linearity,and the results of recovery and precision were in accordance with the requirements.Compared with the Roukou Wuwei pills,Tmax was prolonged and the bioavailability was improved in Roukou Wuwei sustained-release pellets.Conclusions: The Roukou Wuwei sustained-release pellets prepared by modern technology can improve the bioavailability of drugs and have sustained-release effect. |
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