复方中药连萸胃滞留渗透泵控释制剂的设计与评价
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摘要
左金方是经典药方,为黄连、吴茱萸(6：1)二药配伍,其丸剂及胶囊剂为2010年版《中国药典》收载品种,具有抑制胃酸分泌、抗溃疡、抗菌、镇痛抗炎等药理作用,临床上常用来治疗胃部炎症及溃疡性疾病。用生物热动力学、抗幽门螺杆菌方法对左金方的药效物质进行了筛选研究证明主要活性物质以黄连、吴茱萸总生物碱类为主。已有的药代动力学研究表明,该类成分的体内吸收很差,其作用基础是在病变局部保持一定浓度才能发挥治疗作用。左金方的普通制剂如丸剂或胶囊,口服后会随胃排空迅速进入肠道,不能在胃局部保持有效的浓度,因此治疗效果不甚理想。
开发连萸胃滞留渗透泵控释给药系统,该制剂可显著减少患者服用剂量,长时间滞留于胃液中,并具有明显的零级释药特征,能够使黄连与吴茱萸的有效部位在胃中持续定量释放,可有效避免药物的突释效引起的刺激性,同时可使有效成分直达患处,可显著提高疗效和患者的依从性,使治疗胃部炎症及溃疡性疾病获得成功。
传统骨架型胃漂浮缓释制剂药物的释放与其溶解度有直接关系,对于中药复方多组分药物,由于溶解度不同,难以保证各组分同步释放。本研究制备连萸胃滞留渗透泵控释片(LIFOPT),由推动层与药物层组成,其中推动层含有轻质多孔微球,使片剂总表观密度小于水,由于浮力作用,片子漂浮于胃液中,水分渗入药物层形成软膏状药物混悬液,推动层吸水膨胀,将药物层推出释药孔。此种释药方式可保证药物层各成分完全同步释放,同时可获得明显的零级释药特征。
第一部分黄连与吴茱萸有效部位的提取分离纯化
目的：采用大孔树脂吸附技术对黄连总生物碱进行有效分离纯化并确定最佳工艺,采用中性氧化铝柱色谱对吴茱萸中生物碱和柠檬苦素进行纯化,并优化其工艺。
方法：称取黄连药材1kg,粉碎成粗粉,加入12倍体积水,回流提取3次,每次2h,合并提取液并浓缩至8L,控制上样生药质量浓度在0.10-0.15g／mL,采用AB-8大孔吸附树脂分离,纯化水洗脱杂质3BV,50%乙醇洗脱生物碱3BV,减压回收乙醇,真空干燥即得。黄连总生物碱的含量测定方法：采用Diamonsil C18色谱柱(250mm×4.6mm,5μ m),乙腈-0.05mol/L磷酸二氢钾溶液(50：50)为流动相,检测波长为345nm。吴茱萸加入8倍量的70%乙醇,提取3次,每次2h,滤过,合并滤液,滤液减压回收乙醇至无醇味,加入24倍量的蒸馏水,然后用盐酸调pH值至3。静止放置2h,在4500r/min离心30min,得下层沉淀。沉淀加入24倍药材量的95%乙醇溶解后,减压回收乙醇,浸膏加入适量中性氧化铝,少量甲醇,置60℃水浴中挥干甲醇,搅拌均匀,装柱,醋酸乙酯-二氯甲烷(70：30)洗脱6BV,洗脱液减压回收溶剂,真空干燥即得。吴茱萸中柠檬苦素、吴茱萸碱、吴茱萸次碱含量测定方法：采用DiamonsilC18色谱柱(250mm X4.6mm,5μ m),乙腈-水-四氢呋喃-冰乙酸(41：59：1：0.2)为流动相,检测波长为225nm。
结果：黄连总生物碱的含量测定方法：巴马汀、黄连碱在5～100μ g/mL,小檗碱在10.07-201.4μg/mL的浓度范围内与峰面积均呈良好的线性关系；平均回收率分别为9947%(RSD=1.29%),99.73%(RSD=1.16%)、99.41(RSD=0.99%)。黄连总生物碱平均转移率91.72%,提取物中总生物碱平均含量81.87%,其中小檗碱、巴马汀、黄连碱、表小檗碱平均含量分别为48.54%、13.25%、12.83%、7.26%。吴茱萸中柠檬苦素、吴茱萸碱、吴茱萸次碱含量测定方法：吴茱萸碱和吴茱萸次碱在10～200μg/mL,柠檬苦素在25.27-505.4μg/mL的浓度范围内与峰面积均呈良好的线性关系；平均回收率分别为99.64%(RSD=1.46%)、98.90%(RSD=1.55%)、99.00%(RSD=1.10%)。吴茱萸中有效部位平均转移率72.04%,提取物中有效部位平均含量56.45%,其中吴茱萸碱、吴茱萸次碱、柠檬苦素平均含量分别为8.73%、17.83%、30.18%。
结论：本实验方法制备的黄连与吴茱萸的提取物中的有效部位含量均大于50%,符合有效部位的要求。
第二部分精制中间体与左金丸的药效学比较
目的：比较精制中间体与左金丸对抗大鼠实验性胃溃疡及胃酸分泌的作用,以此验证提取工艺的合理性。
方法：考察不同剂量精制中间体与左金丸对吲哚美辛胃溃疡的影响,以及对乙醇胃溃疡的影响,并考察高剂量组精制中间体与左金丸对正常大鼠胃液分泌的影响。
结果：对吲哚美辛胃溃疡的影响：精制中间体0.096、0.135g／kg及左金丸1.05g／kg组的溃疡数与对照组比较有显著性差异。对乙醇胃溃疡的影响：精制中间体0.135g／kg组及左金丸1.05g/kg组的溃疡数均较对照组显著为低。对正常大鼠胃液分泌的影响：精制中间体及左金丸均可显著减少胃液分泌量和胃酸分泌。
结论：经过主要药效学指标对比表明,精制中间体与左金丸的药效无显著差异(p>0.05),说明提取精制工艺合理。
第三部分连萸胃滞留渗透泵控释片的制备
目的：采用渗透泵制剂技术,制备连萸胃滞留渗透泵控释片。
方法：连萸胃滞留渗透泵控释片的制备方法：(1)轻质多孔微球的制备：将聚丙烯酸树脂RS溶于适量乙醇-二氯甲烷中,加入0.3%十二烷基硫酸钠水溶液,50-100r/min搅拌,至油珠粒径为0.3-0.8mm时,开始缓慢升温,至40℃时固化4h,过滤,装柱,10倍柱体积热水淋洗后,干燥即得。(2)助推层的制备：将轻质多孔微球、PEO、 HPMC、氯化钠、PVPk30按处方量混合均匀,加入无水乙醇制软材,过20目筛制粒,40℃干燥12h,加入硬脂酸镁混匀。(3)含药层的制备：将精制中间体、PEO按处方量混合均匀,加入无水乙醇制软材,过20目筛制粒,40℃干燥12h,加入硬脂酸镁混匀。(4)压片与包衣：用上述助推层与含药层颗粒压制双层片芯后,用醋酸纤维素丙酮溶液包衣,在片剂的含药层表面打一直径0.8mm的小孔,即得。分别考察含药层促渗剂种类及用量、助推层渗透剂用量、包衣液中致孔剂用量、释药孔直径、片芯硬度对释药行为的影响,含药层助悬剂种类和用量对药物混悬液流变性的影响,膨胀漂浮层膨胀剂材料对膨胀速度的影响,醋酸纤维素薄膜厚度对薄膜弹性模量及断裂强度的影响,包衣液中致孔剂PEG400对醋酸纤维素(CA)膜水渗透速率的影响。在单因素考察的基础上,,以助推层膨胀剂及渗透剂的用量、包衣液致孔剂的用量作为考察因素,进行三因素三水平的正交设计优化处方。测定不同渗透压介质中渗透泵片释放度,验证介质渗透压对药物释放的影响。
结果：根据单因素考察结果,含药层促渗剂为氯化钠20mg,含药层助悬剂为PEO (Mw,200,000),用量为提取物：PEO=4：1助推层膨胀剂为PEO:HPMC=4:1,助推层渗透剂为氯化钠,包衣膜厚度控制在0.18-0.20mm。根据正交试验结果,助推层膨胀剂用量为50mg；助推层促渗剂的用量20mg；包衣液致孔剂的用量10%。以0.1mol/L盐酸溶液(加2mol/L氯化钠)为释放介质,药物释放速率明显下降,表明释放的驱动力为释放介质与制剂内部的渗透压差。
结论：按优化的处方与工艺制备的连萸胃滞留渗透泵片在12h内药物释放大于90%,释放曲线符合零级动力学方程,符合设计要求。
第四部分连萸胃滞留渗透泵控释片体外释放度研究
目的：建立HPLC法同时测定本制剂释放介质中的小檗碱、巴马汀、吴茱萸碱、吴茱萸次碱,考察本制剂的释放度并建立控制标准。
方法：HPLC方法：采用Diamonsil C18色谱柱(250mm X4.6mm,5μm),乙睛-0.025mol/L磷酸二氢钾溶液(含20mmol/L十二烷基磺酸钠,磷酸调至pH3.5)(55：45)为流动相,检测波长为225nm,流速：1mL/mino释放度测定方法：照释放度测定法(附录XD第一法),采用溶出度测定法第二法装置,以100ml0.1mol/L盐酸溶液为释放介质,转速为每min100转,依法操作,经2、4、6、8、10、12h分别取溶液2ml滤过,取续滤液测定即得。分别以0.1mol/L盐酸溶液、水及磷酸盐缓冲液(pH6.8)为释放介质,照释放度测定法进行测定,采用零级动力学方程对释放度数据进行拟合。采用f2因子法评价四种成分的释放曲线相似性。
结果：小檗碱在500.8-25.04μg/ml,巴马汀为1.914-47.85μg/ml,吴茱萸碱与吴茱萸次碱在0.1023-2.046μg/ml的浓度范围内与峰面积均呈良好的线性关系；平均回收率分别为98.84%(RSD=1.51%).99.17%(RSD=1.12%)、97.94%(RSD=1.34%)、99.46%(RSD=1.05%)。在0.1mol/L盐酸溶液、水及磷酸盐缓冲液(pH6.8)释放介质中,小檗碱、巴马汀、吴茱萸碱、吴茱萸次碱的释放度数据用零级动力学方程进行拟合,所得各回归方程r值均大于0.98,同时各成分的释放曲线相比较,f2因子均大于50。
结论：利用HPLC法建立了同时测定本制剂中小檗碱、巴马汀、吴茱萸碱、吴茱萸次碱释放度方法,经方法学验证,精密度、准确性、稳定性均符合要求,本制剂在各释放介质中的释放曲线均符合零级动力学方程,且各成分释放曲线相似,表明本品可实现各成分以恒定速度同步释放。
第五部分连萸胃滞留渗透泵控释片Beagle犬体内药动学研究
目的：建立可同时测定Beagle犬血浆中小檗碱、巴马汀的检测方法,研究连萸胃滞留渗透泵控释片中小檗碱与巴马汀在Beagle犬体内药代动力学。
方法：Beagle犬随机分成2组,采用双周期交叉试验,一组服用受试制剂连萸渗透泵片,另一组服用参比制剂连萸片,剂量均为300mg。2周后进行交叉试验。于口服给药前(0h)和给药后1、2、3、4、6、8、、10、12、24、36、48、72、96、120h h取后肢静脉血5ml,并立即移入肝素化离心管中,10000r/min离心10min,分离血浆,于-20℃冰箱中保存待测。色谱柱为Kromasil C18(250min×4.6mm,5μm),流动相：A为0.1%甲酸水溶液, B为0.1%甲酸乙腈溶液,梯度洗脱,检测波长为345nm,流速：1mL/min.采用DAS软件计算药代动力学参数。
结果：小檗碱的线性范围0.1～5.0μg/ml,日内与日间RSD<15%,回收率84.16%-96.30%；巴马汀线性范围0.01～0.5μg/ml,日内与日间RSD<15%,回收率81.05％～93.29％.Beagle犬口服参比制剂和受试制剂后的主要药动学参数：小檗碱：tmax分别为(3.890±1.524)h和(6.761±2.496)h,Cmax分别为(1.4073±0.3804)u g/ml和(0.8773±0.2395)μg/ml,t1/2分别为(14.26±4.85)h和(18.33±6.39)h,AUC0～96分别为(35.94±8.86)μg.ml-1·h和(39.27±7.94)μg.ml-1·h,相对生物利用度为109.3±22.1%。巴马汀：tmax分别为(4.191±1.527)h和(8.835±2.906)h,cmax分别为(0.2515±0.0719)μ g/ml和(0.1773±0.0525)μg/ml,t1/2分别为(11.47±5.63h和(16.64±5.34)h,AUC0～96分别为(7.48±2.61)μg·m-1·h和(8.54±2.39)μg·ml-1·h,相对生物利用度为114.2±31.9%。
结论：小檗碱、巴马汀体内血浆样品测定灵敏度、精密度、准确度良好,线性范围内能满足样品的分析。以小檗碱、巴马汀AUCo-96计算,受试制剂与参比制剂相比生物利用度无显著性差异,tmax明显延长,Cmax显著降低,两种制剂在Beagle犬体内生物作用等效且具有一定的缓释作用。
Zuojin (ZJ) prescription is a classic prescription with Coptis and Evodia (6:1), the pills and capsules was collected in the2010edition of "Chinese Pharmacopoeia". Pharmacological effects of ZJ prescription are as follows: theinhibition of gastric acid secretion, anti-ulcer, antimicrobial, analgesic and anti-inflammatory. ZJ preseription is used clinically to treat stomach inflammation and ulcer disease.
The dynamics of bio-heat, anti-Helicobacter pylori method on the active ingredients of ZJ prescription screening studies were used to prove that the main active substance is Coptis and Evodia alkaloids. The existing pharmacokinetic studies have shown that such ingredients in vivo absorption is poor. Alkaloids only to maintain a certain concentration can play a therapeutic role in the local lesion. The ordinary preparations of ZJ prescription are pills and capsules. Because these agents rapidly into the intestine after oral administration with gastric emptying, so the effective concentration can not be maintained in the stomach. This treatment effect is not ideal.
The researcher developed Lianyu intragastric floating osmotic pump controlled release formulations (LIFOPT). This preparation can significantly reduce the dosage of patients, remain in the gastric juice long time, and has a zero-order release characteristics. It enables the effective parts of Coptis and Evodia continuously and quantitatively release in the stomach, and can effectively avoid irritating due to the sudden release of the drug. The active ingredient was directly released to the lesion, so the efficacy and patient compliance can be significantlyimproved. This treatment can successfully cure stomach inflammation and ulcer disease.
The traditional framework gastric floating sustained-release formulations has a direct relationship between drug release and solubility. For multi-component drugs, the various componentsis difficult to synchronize the release because of their different solubility. The LIFOPT contains promoting layer and drug layer. Promoting layer contains light porous microspheres, the tablets float in the gastric juice because apparent density of tablets less than water. Ointment-like drug suspension was formed due to water infiltrating into the drug layer, promoting layer absorbed water and expanded, thedrug suspension was introduced from the release hole. The release style can guarantee that each component is synchronously released, The release curve has a zero-order release characteristics.
PART1The extraction and purification of Coptis and Evodia Effective parts
Objective:To separate and purify the Coptis total alkaloids with macroporous adsorption resin technology and determine the best technology, purify Evodia alkaloids and limonoids with neutral alumina column chromatography and optimize the processes.
Methods:Coptis herbs1kg was crushed into coarse powder and reflux extracted3times(each for2h) with12times volume of water. The combined extractand was concentrated to8L and controlled to0.10to0.15g/mL of crude drug mass concentration. AB-8macroporous resin was used to separate alkaloids. The purification process:the impurities were eluted with3BV purified water, alkaloids were eluted with3BV50%ethanol, the extract were derived through vacuum drying after recovery of ethanol.
The contents of Coptis alkaloids were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-0.05mol/L potassium dihydrogen phosphate solution (50:50) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at345nm..
Evodia herbs was extracted3times(each for2h) with8times volume of70%ethanol. The combined extractand was filtered and the filtrate was evaporated to alcohol-free taste for recycling ethanol. The Concentrate was added24times volume of water and adjusted pH to3with hydrochloric acid. The precipitation was derived thro μ gh4500r/min centrifμgation after Stationary for2h and redissolved with24times volume of95%ethanol. The solution was added into neutral alumina with little methanol after decompression recycling ethanol. The mixture was stired and packed into chromatography column after methanol was evaporated at60℃. The purification process:the effective parts were eluted with6BV ethyl acetate-dichloromethane(70:30), the extract were derived throμgh vacuum drying after recovery of solvent.
The contents of Evodia effective parts were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-water-tetrahydrofuran-acetic acid (41:59:1:0.2) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at225nm.
Results:For the method of Coptis alkaloids determination, there were good linear relationships for coptisine and palmatine within the range of5～100μg/mL and berberine within the range of10.07～201.4μ g/mL. The average recoveries were9947%(RSD=1.29%)、99.73%(RSD=1.16%)、99.41%(RSD=0.99%)
The Coptis alkaloids transfer rate were91.72%, the average content of total alkaloids in the extract were81.87%, the average concentration of berberine, palmatine, coptisine, epiberberine was48.54%,13.25%,12.83%,7.26%.
For the method of Evodia effective ingredients determination, there were good linear relationships for evodiamine and rutacarpine within the range of10～200μ g/mL and limonin within the range of25.27-505.4μ g/mL. The average recoveries were99.64%(RSD=1.46%)、98.90%(RSD=1.55%).99.00%(RSD=1.10%)
The average transfer rate of effective parts of the Evodia was72.04%, the average content of effective parts in the extract were56.45%, the average concentration of evodiamine,rutaecarpine, limonin was8.73%,17.83%,30.18%.
Conclusions:The average content of effective parts of the prepared extracts of Coptis and Evodia were more r than50%in line with the requirements of the effective parts.
Part2The pharmacodynamics comparison between the refined intermediate and Zuojin pills
Objective:To verify the rationality of the extraction process with the comparison of effect against the experimental gastric ulcer in rats and the gastric acid secretion between refining intermediates and Zuojin pills.
Methods:The impact of different dosages of refined intermediate and Zuojin pills on indomethacin gastric ulcer was investigated, as well as ethanol gastric ulcer, the impact of the high dosage group refined intermediate and Zuojin pills on gastric secretion of normal rats was studied.
Results:The influence on indomethacin gastric ulcer was as follows:the number of ulcers of the refined intermediate0.096,0.135g/kg groups and Zuojin pills1.05g/kg group had significant difference compared with control group. The influence on ethanol gastric ulcer show that the number of ulcers of the refined intermediate0.135g/kg groups and Zuojin pills1.05g/kg group were significantly less than control group. The influence on gastric secretion of normal rats show that the refined intermediate group and Zuojin pills group can significantly reduce the secretion of gastric juice and gastric acid secretion.
Conclusions:After major pharmacodynamic comparison, the efficacy of refining intermediates and Zuojin pills had no significant differences, indicating that the extraction and purification process were reasonable.
Part3The preparation of Lianyu intragastric floating osmotic pump
controlled release tablets
Objective:To prepare Lianyu intragastric floating osmotic pump controlled release tablets with osmotic pump formulations technology.
Methods:The method to prepare LIFOPT:(1) the preparation of light and porous micro spheres:polyacrylate resin RS was dissolved in appropriate amount of ethanol-dichloromethane; mix the solution obtained with0.3%dodecyl sulfate aqueous solution; stir at50-100r/min until the oil droplet diameter increase to0.3-0.8mm; heat the mixture to40℃slowly and solidify for4hours; filter the mixture and pack the microspheres into column, wash with10BV hot water, the light and porous microspheres were derived after drying.(2) the preparation of promoing layer:mix light and porous microspheres, PEO, HPMC, sodium chloride, PVPk30according the prescription; prepare wet mixture with ethanol, prepare granules throμ gh20mesh sieve, dry for12hour at40℃, add magnesium stearate into the granules.(3) the preparation of drug layer: mix the refined intermediate and PEO according the prescription; prepare wet mixture with ethanol, prepare granules through20mesh sieve, dry for12hour at40℃, add magnesium stearate into the granules.4) Pressing tablets and coating:bilayer tablet core was prepared with the above promoing layer and drug layer granules; coat tablets core with the acetone solution of cellulose acetate; an orifice of0.8mm diameter was drilled by micro drilling machine on surface of the drug layer.
The main investigated factors were as follows:the influence of different osmotic promoting agent and dosage in promoing layer and drug layer on drug release, the influence of different amount of PEG400in coating solution, the diameter of release hole and hardness of tablet core on drug release, the influence of the suspending agent on flow propertyof suspension including API, the influence of the swelling agent on swelling rate, the elastic modulus and fracture strength of acetyl cellulose film with defferent thickness, the influence of the porous agent on water permeation rate of CA membrane.
On the basis of single factor investigation, the orthogonal experiment was designed to optimize formula in which the amount of osmotic agents, swelling agent, PEG400were taken as three influential factors and three different levels were selected to part, each of them was selected refer to the orthogonal design table. The accumulative release percentage of LIFOPT in different osmotic media was determined to verify the influence of the medium osmotic pressure on drug release.
Results:According to the results of single factor investigation, the osmotic promoting agent in drug layer was sodium chloride20mg; suspending agents in drug layer was PEO(Mw200,000)(the refined intermediate:PEO=4:1); swelling agent was PEO:HPMC=4:1; the osmotic promoting agent in promoting layer was sodium chloride; the thickness of coating film was0.18-0.20mm. According to the results of orthogonal test, the amount of osmotic agents was20mg; swelling agent was50mg; PEG400in coating solution was10%. The drug release rate decreased significantly in0.1mol/L hydrochloric acid solution (including2mol/L sodium chloride). The phenomenon showed that the driving force of drug release was the osmotic pressure diffefence between the release media and internal preparation.
Conclusions:The accumulative ralease percentage within12hours of the osmotic pump tablets prepared according to optimal prescription and process is more than90%, the release curse was in line with the zero-order kinetics equation to meet the design requirements.
Part4Studies on dissolution test in vitro of Lianyu intragastric floating osmotic pump controlled release tablets
Objective:To establish an HPLC method for simultaneous determination of berberine, palmatine, evodiamine and rutaecarpine in release medium, investigate the release character of the formulation and set up dissolution test standards.
Methods:The contents of berberine, palmatine, evodiamine and rutaecarpine in release medium were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-0.025mol/L potassium dihydrogen phosphate solution (including20mmol/L sodium dodecyl sulfate, adjust pH to3.5with phosphate)(55:45) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at225nm.
Dissolution test:Medium:0.1mol/L hydrochloric acid solution1000ml; apparatus2:100rpm; times:2、4、6、8、10and12hours; test solution:pass a portion of the solution under test throμgh a suitable0.45-μm filter. The zero-order kinetic equation was taken to fit the release data, the similarity of release curve of the four components were evaluated acording to f2factor.
Results:For the method of determination of berberine, palmatine, evodiamine and rutaecarpine in release medium, there were good linear relationships for berberine within the range of500.8-25.04μg/ml, palmatine within the range of1.914-47.85μg/ml, evodiamine and rutacarpine within the range of0.1023-2.046μg/ml. The average recoveries were98.84%(RSD=1.51%),99.17%(RSD=1.12%),97.94%(RSD=1.34%),99.46%(RSD=1.05%). The zero-order kinetic equation was taken to fit the release data in water,0.1mol/L hydrochloric acid solution and phosphate buffer (pH6.8), the r values of regression equation were greater than0.98. The comparison results of release curve of each component showed that the f2factor were greater than50.
Conclusions:The HPLC method wasestablished for simultaneous determination of berberine, palmatine, evodiamine and rutaecarpine in release medium. By the method validation, precision, accuracy, and stability meet the requirements of pharmacopoeia. In various release media the release curves of the preparation are in line with the zero-order kinetics equation, the release curve of different composition were similar, indicating that the product can achieve synchronous release of each component at a constant speed. Part5Studies on pharmacokinetics in beagle dogs of Lianyu
intragastric floating osmotic pump controlled release tablets
Objective:To establish an HPLC method for simultaneous determination of berberine and palmatine in plasma of beagle dogs, study the pharmacokinetics in beagle dogs of Lianyu intragastric floating osmotic pump controlled release tablets.
Methods:Beagle dogs were randomly divided into twogroups, two-period crossover trial were employed to arrange experiment, the test preparation(LIFOPT) and the reference preparation(normal tablets) were taken respectively, the dosage was300mg, crossover study was carried out after two weeks. Sampling time:before administration and1、2、3、4、6、8、10、12、24、36、48、72、96、120hours after administration. Plasma processing: take hindlimb blood5ml, and immediately transfer into heparinized tube, centrif μ ge at10000r/min for10min, separate plasma and store at-20℃
The contents of berberine and palmatine in plasma were determined by HPLC. The separation was carried out on a C18column with a mixture of0.1%formic acid and0.1%formic acid in acetonitrile(gradient elution) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at345nm.. The pharmacokinetic parameters were calculated by DAS software.
Results:For the method of determination of berberine and palmatine in plasma, there were good linear relationships for berberine within the range of0.1-5.0μ g/ml, palmatine within the range of0.01-0.5μ g/ml, within-day and between-day RSD were less than15%, the recoveries were84.16%-96.30%and81.05%～93.29%. The main pharmacokinetics parameters were respectively as follows:berberine:tmax (3.890±1.524) and (6.761±2.496) h;cmax (1.4073±0.3804) and (0.8773±0.2395)μ g/ml; t1/2(14.26+4.85) h and (18.33±6.39) h; AUC0～96(35.94±8.86) μg·ml-1·h and (39.27±7.94) μ g·ml-1·h. The relative bioavailability was109.3±22.1%. Palmatine:tmax(4.191±1.527and (8.835±2.906)h; cmax (0.2515±0.0719) and (0.1773±0.0525) μ g/ml; t1/2(11.47±5.63) h and (16.64±5.34) h; AUCo-96(7.48±2.61) μg·ml-1·h and (8.54±2.39) μ g·ml-1·h. The relative bioavailability was114.2±31.9%.
Conclusions:The method of determination of berberine and palmatine in plasma had good sensitivity, precision and accuracy, and the sample concentration was within the linear range. On the basis of AUC0～96of berberine and palmatine, the bioavailability had no significant difference between test formulation and reference formulation, tmax significantly extended and Cmax significantly reduced. It could be concluded that the LIFOPT and the normal tablets were bioequivalent, LIFOPT had good effect of sustained-release in vivo.
开发连萸胃滞留渗透泵控释给药系统,该制剂可显著减少患者服用剂量,长时间滞留于胃液中,并具有明显的零级释药特征,能够使黄连与吴茱萸的有效部位在胃中持续定量释放,可有效避免药物的突释效引起的刺激性,同时可使有效成分直达患处,可显著提高疗效和患者的依从性,使治疗胃部炎症及溃疡性疾病获得成功。
传统骨架型胃漂浮缓释制剂药物的释放与其溶解度有直接关系,对于中药复方多组分药物,由于溶解度不同,难以保证各组分同步释放。本研究制备连萸胃滞留渗透泵控释片(LIFOPT),由推动层与药物层组成,其中推动层含有轻质多孔微球,使片剂总表观密度小于水,由于浮力作用,片子漂浮于胃液中,水分渗入药物层形成软膏状药物混悬液,推动层吸水膨胀,将药物层推出释药孔。此种释药方式可保证药物层各成分完全同步释放,同时可获得明显的零级释药特征。
第一部分黄连与吴茱萸有效部位的提取分离纯化
目的：采用大孔树脂吸附技术对黄连总生物碱进行有效分离纯化并确定最佳工艺,采用中性氧化铝柱色谱对吴茱萸中生物碱和柠檬苦素进行纯化,并优化其工艺。
方法：称取黄连药材1kg,粉碎成粗粉,加入12倍体积水,回流提取3次,每次2h,合并提取液并浓缩至8L,控制上样生药质量浓度在0.10-0.15g／mL,采用AB-8大孔吸附树脂分离,纯化水洗脱杂质3BV,50%乙醇洗脱生物碱3BV,减压回收乙醇,真空干燥即得。黄连总生物碱的含量测定方法：采用Diamonsil C18色谱柱(250mm×4.6mm,5μ m),乙腈-0.05mol/L磷酸二氢钾溶液(50：50)为流动相,检测波长为345nm。吴茱萸加入8倍量的70%乙醇,提取3次,每次2h,滤过,合并滤液,滤液减压回收乙醇至无醇味,加入24倍量的蒸馏水,然后用盐酸调pH值至3。静止放置2h,在4500r/min离心30min,得下层沉淀。沉淀加入24倍药材量的95%乙醇溶解后,减压回收乙醇,浸膏加入适量中性氧化铝,少量甲醇,置60℃水浴中挥干甲醇,搅拌均匀,装柱,醋酸乙酯-二氯甲烷(70：30)洗脱6BV,洗脱液减压回收溶剂,真空干燥即得。吴茱萸中柠檬苦素、吴茱萸碱、吴茱萸次碱含量测定方法：采用DiamonsilC18色谱柱(250mm X4.6mm,5μ m),乙腈-水-四氢呋喃-冰乙酸(41：59：1：0.2)为流动相,检测波长为225nm。
结果：黄连总生物碱的含量测定方法：巴马汀、黄连碱在5～100μ g/mL,小檗碱在10.07-201.4μg/mL的浓度范围内与峰面积均呈良好的线性关系；平均回收率分别为9947%(RSD=1.29%),99.73%(RSD=1.16%)、99.41(RSD=0.99%)。黄连总生物碱平均转移率91.72%,提取物中总生物碱平均含量81.87%,其中小檗碱、巴马汀、黄连碱、表小檗碱平均含量分别为48.54%、13.25%、12.83%、7.26%。吴茱萸中柠檬苦素、吴茱萸碱、吴茱萸次碱含量测定方法：吴茱萸碱和吴茱萸次碱在10～200μg/mL,柠檬苦素在25.27-505.4μg/mL的浓度范围内与峰面积均呈良好的线性关系；平均回收率分别为99.64%(RSD=1.46%)、98.90%(RSD=1.55%)、99.00%(RSD=1.10%)。吴茱萸中有效部位平均转移率72.04%,提取物中有效部位平均含量56.45%,其中吴茱萸碱、吴茱萸次碱、柠檬苦素平均含量分别为8.73%、17.83%、30.18%。
结论：本实验方法制备的黄连与吴茱萸的提取物中的有效部位含量均大于50%,符合有效部位的要求。
第二部分精制中间体与左金丸的药效学比较
目的：比较精制中间体与左金丸对抗大鼠实验性胃溃疡及胃酸分泌的作用,以此验证提取工艺的合理性。
方法：考察不同剂量精制中间体与左金丸对吲哚美辛胃溃疡的影响,以及对乙醇胃溃疡的影响,并考察高剂量组精制中间体与左金丸对正常大鼠胃液分泌的影响。
结果：对吲哚美辛胃溃疡的影响：精制中间体0.096、0.135g／kg及左金丸1.05g／kg组的溃疡数与对照组比较有显著性差异。对乙醇胃溃疡的影响：精制中间体0.135g／kg组及左金丸1.05g/kg组的溃疡数均较对照组显著为低。对正常大鼠胃液分泌的影响：精制中间体及左金丸均可显著减少胃液分泌量和胃酸分泌。
结论：经过主要药效学指标对比表明,精制中间体与左金丸的药效无显著差异(p>0.05),说明提取精制工艺合理。
第三部分连萸胃滞留渗透泵控释片的制备
目的：采用渗透泵制剂技术,制备连萸胃滞留渗透泵控释片。
方法：连萸胃滞留渗透泵控释片的制备方法：(1)轻质多孔微球的制备：将聚丙烯酸树脂RS溶于适量乙醇-二氯甲烷中,加入0.3%十二烷基硫酸钠水溶液,50-100r/min搅拌,至油珠粒径为0.3-0.8mm时,开始缓慢升温,至40℃时固化4h,过滤,装柱,10倍柱体积热水淋洗后,干燥即得。(2)助推层的制备：将轻质多孔微球、PEO、 HPMC、氯化钠、PVPk30按处方量混合均匀,加入无水乙醇制软材,过20目筛制粒,40℃干燥12h,加入硬脂酸镁混匀。(3)含药层的制备：将精制中间体、PEO按处方量混合均匀,加入无水乙醇制软材,过20目筛制粒,40℃干燥12h,加入硬脂酸镁混匀。(4)压片与包衣：用上述助推层与含药层颗粒压制双层片芯后,用醋酸纤维素丙酮溶液包衣,在片剂的含药层表面打一直径0.8mm的小孔,即得。分别考察含药层促渗剂种类及用量、助推层渗透剂用量、包衣液中致孔剂用量、释药孔直径、片芯硬度对释药行为的影响,含药层助悬剂种类和用量对药物混悬液流变性的影响,膨胀漂浮层膨胀剂材料对膨胀速度的影响,醋酸纤维素薄膜厚度对薄膜弹性模量及断裂强度的影响,包衣液中致孔剂PEG400对醋酸纤维素(CA)膜水渗透速率的影响。在单因素考察的基础上,,以助推层膨胀剂及渗透剂的用量、包衣液致孔剂的用量作为考察因素,进行三因素三水平的正交设计优化处方。测定不同渗透压介质中渗透泵片释放度,验证介质渗透压对药物释放的影响。
结果：根据单因素考察结果,含药层促渗剂为氯化钠20mg,含药层助悬剂为PEO (Mw,200,000),用量为提取物：PEO=4：1助推层膨胀剂为PEO:HPMC=4:1,助推层渗透剂为氯化钠,包衣膜厚度控制在0.18-0.20mm。根据正交试验结果,助推层膨胀剂用量为50mg；助推层促渗剂的用量20mg；包衣液致孔剂的用量10%。以0.1mol/L盐酸溶液(加2mol/L氯化钠)为释放介质,药物释放速率明显下降,表明释放的驱动力为释放介质与制剂内部的渗透压差。
结论：按优化的处方与工艺制备的连萸胃滞留渗透泵片在12h内药物释放大于90%,释放曲线符合零级动力学方程,符合设计要求。
第四部分连萸胃滞留渗透泵控释片体外释放度研究
目的：建立HPLC法同时测定本制剂释放介质中的小檗碱、巴马汀、吴茱萸碱、吴茱萸次碱,考察本制剂的释放度并建立控制标准。
方法：HPLC方法：采用Diamonsil C18色谱柱(250mm X4.6mm,5μm),乙睛-0.025mol/L磷酸二氢钾溶液(含20mmol/L十二烷基磺酸钠,磷酸调至pH3.5)(55：45)为流动相,检测波长为225nm,流速：1mL/mino释放度测定方法：照释放度测定法(附录XD第一法),采用溶出度测定法第二法装置,以100ml0.1mol/L盐酸溶液为释放介质,转速为每min100转,依法操作,经2、4、6、8、10、12h分别取溶液2ml滤过,取续滤液测定即得。分别以0.1mol/L盐酸溶液、水及磷酸盐缓冲液(pH6.8)为释放介质,照释放度测定法进行测定,采用零级动力学方程对释放度数据进行拟合。采用f2因子法评价四种成分的释放曲线相似性。
结果：小檗碱在500.8-25.04μg/ml,巴马汀为1.914-47.85μg/ml,吴茱萸碱与吴茱萸次碱在0.1023-2.046μg/ml的浓度范围内与峰面积均呈良好的线性关系；平均回收率分别为98.84%(RSD=1.51%).99.17%(RSD=1.12%)、97.94%(RSD=1.34%)、99.46%(RSD=1.05%)。在0.1mol/L盐酸溶液、水及磷酸盐缓冲液(pH6.8)释放介质中,小檗碱、巴马汀、吴茱萸碱、吴茱萸次碱的释放度数据用零级动力学方程进行拟合,所得各回归方程r值均大于0.98,同时各成分的释放曲线相比较,f2因子均大于50。
结论：利用HPLC法建立了同时测定本制剂中小檗碱、巴马汀、吴茱萸碱、吴茱萸次碱释放度方法,经方法学验证,精密度、准确性、稳定性均符合要求,本制剂在各释放介质中的释放曲线均符合零级动力学方程,且各成分释放曲线相似,表明本品可实现各成分以恒定速度同步释放。
第五部分连萸胃滞留渗透泵控释片Beagle犬体内药动学研究
目的：建立可同时测定Beagle犬血浆中小檗碱、巴马汀的检测方法,研究连萸胃滞留渗透泵控释片中小檗碱与巴马汀在Beagle犬体内药代动力学。
方法：Beagle犬随机分成2组,采用双周期交叉试验,一组服用受试制剂连萸渗透泵片,另一组服用参比制剂连萸片,剂量均为300mg。2周后进行交叉试验。于口服给药前(0h)和给药后1、2、3、4、6、8、、10、12、24、36、48、72、96、120h h取后肢静脉血5ml,并立即移入肝素化离心管中,10000r/min离心10min,分离血浆,于-20℃冰箱中保存待测。色谱柱为Kromasil C18(250min×4.6mm,5μm),流动相：A为0.1%甲酸水溶液, B为0.1%甲酸乙腈溶液,梯度洗脱,检测波长为345nm,流速：1mL/min.采用DAS软件计算药代动力学参数。
结果：小檗碱的线性范围0.1～5.0μg/ml,日内与日间RSD<15%,回收率84.16%-96.30%；巴马汀线性范围0.01～0.5μg/ml,日内与日间RSD<15%,回收率81.05％～93.29％.Beagle犬口服参比制剂和受试制剂后的主要药动学参数：小檗碱：tmax分别为(3.890±1.524)h和(6.761±2.496)h,Cmax分别为(1.4073±0.3804)u g/ml和(0.8773±0.2395)μg/ml,t1/2分别为(14.26±4.85)h和(18.33±6.39)h,AUC0～96分别为(35.94±8.86)μg.ml-1·h和(39.27±7.94)μg.ml-1·h,相对生物利用度为109.3±22.1%。巴马汀：tmax分别为(4.191±1.527)h和(8.835±2.906)h,cmax分别为(0.2515±0.0719)μ g/ml和(0.1773±0.0525)μg/ml,t1/2分别为(11.47±5.63h和(16.64±5.34)h,AUC0～96分别为(7.48±2.61)μg·m-1·h和(8.54±2.39)μg·ml-1·h,相对生物利用度为114.2±31.9%。
结论：小檗碱、巴马汀体内血浆样品测定灵敏度、精密度、准确度良好,线性范围内能满足样品的分析。以小檗碱、巴马汀AUCo-96计算,受试制剂与参比制剂相比生物利用度无显著性差异,tmax明显延长,Cmax显著降低,两种制剂在Beagle犬体内生物作用等效且具有一定的缓释作用。
Zuojin (ZJ) prescription is a classic prescription with Coptis and Evodia (6:1), the pills and capsules was collected in the2010edition of "Chinese Pharmacopoeia". Pharmacological effects of ZJ prescription are as follows: theinhibition of gastric acid secretion, anti-ulcer, antimicrobial, analgesic and anti-inflammatory. ZJ preseription is used clinically to treat stomach inflammation and ulcer disease.
The dynamics of bio-heat, anti-Helicobacter pylori method on the active ingredients of ZJ prescription screening studies were used to prove that the main active substance is Coptis and Evodia alkaloids. The existing pharmacokinetic studies have shown that such ingredients in vivo absorption is poor. Alkaloids only to maintain a certain concentration can play a therapeutic role in the local lesion. The ordinary preparations of ZJ prescription are pills and capsules. Because these agents rapidly into the intestine after oral administration with gastric emptying, so the effective concentration can not be maintained in the stomach. This treatment effect is not ideal.
The researcher developed Lianyu intragastric floating osmotic pump controlled release formulations (LIFOPT). This preparation can significantly reduce the dosage of patients, remain in the gastric juice long time, and has a zero-order release characteristics. It enables the effective parts of Coptis and Evodia continuously and quantitatively release in the stomach, and can effectively avoid irritating due to the sudden release of the drug. The active ingredient was directly released to the lesion, so the efficacy and patient compliance can be significantlyimproved. This treatment can successfully cure stomach inflammation and ulcer disease.
The traditional framework gastric floating sustained-release formulations has a direct relationship between drug release and solubility. For multi-component drugs, the various componentsis difficult to synchronize the release because of their different solubility. The LIFOPT contains promoting layer and drug layer. Promoting layer contains light porous microspheres, the tablets float in the gastric juice because apparent density of tablets less than water. Ointment-like drug suspension was formed due to water infiltrating into the drug layer, promoting layer absorbed water and expanded, thedrug suspension was introduced from the release hole. The release style can guarantee that each component is synchronously released, The release curve has a zero-order release characteristics.
PART1The extraction and purification of Coptis and Evodia Effective parts
Objective:To separate and purify the Coptis total alkaloids with macroporous adsorption resin technology and determine the best technology, purify Evodia alkaloids and limonoids with neutral alumina column chromatography and optimize the processes.
Methods:Coptis herbs1kg was crushed into coarse powder and reflux extracted3times(each for2h) with12times volume of water. The combined extractand was concentrated to8L and controlled to0.10to0.15g/mL of crude drug mass concentration. AB-8macroporous resin was used to separate alkaloids. The purification process:the impurities were eluted with3BV purified water, alkaloids were eluted with3BV50%ethanol, the extract were derived through vacuum drying after recovery of ethanol.
The contents of Coptis alkaloids were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-0.05mol/L potassium dihydrogen phosphate solution (50:50) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at345nm..
Evodia herbs was extracted3times(each for2h) with8times volume of70%ethanol. The combined extractand was filtered and the filtrate was evaporated to alcohol-free taste for recycling ethanol. The Concentrate was added24times volume of water and adjusted pH to3with hydrochloric acid. The precipitation was derived thro μ gh4500r/min centrifμgation after Stationary for2h and redissolved with24times volume of95%ethanol. The solution was added into neutral alumina with little methanol after decompression recycling ethanol. The mixture was stired and packed into chromatography column after methanol was evaporated at60℃. The purification process:the effective parts were eluted with6BV ethyl acetate-dichloromethane(70:30), the extract were derived throμgh vacuum drying after recovery of solvent.
The contents of Evodia effective parts were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-water-tetrahydrofuran-acetic acid (41:59:1:0.2) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at225nm.
Results:For the method of Coptis alkaloids determination, there were good linear relationships for coptisine and palmatine within the range of5～100μg/mL and berberine within the range of10.07～201.4μ g/mL. The average recoveries were9947%(RSD=1.29%)、99.73%(RSD=1.16%)、99.41%(RSD=0.99%)
The Coptis alkaloids transfer rate were91.72%, the average content of total alkaloids in the extract were81.87%, the average concentration of berberine, palmatine, coptisine, epiberberine was48.54%,13.25%,12.83%,7.26%.
For the method of Evodia effective ingredients determination, there were good linear relationships for evodiamine and rutacarpine within the range of10～200μ g/mL and limonin within the range of25.27-505.4μ g/mL. The average recoveries were99.64%(RSD=1.46%)、98.90%(RSD=1.55%).99.00%(RSD=1.10%)
The average transfer rate of effective parts of the Evodia was72.04%, the average content of effective parts in the extract were56.45%, the average concentration of evodiamine,rutaecarpine, limonin was8.73%,17.83%,30.18%.
Conclusions:The average content of effective parts of the prepared extracts of Coptis and Evodia were more r than50%in line with the requirements of the effective parts.
Part2The pharmacodynamics comparison between the refined intermediate and Zuojin pills
Objective:To verify the rationality of the extraction process with the comparison of effect against the experimental gastric ulcer in rats and the gastric acid secretion between refining intermediates and Zuojin pills.
Methods:The impact of different dosages of refined intermediate and Zuojin pills on indomethacin gastric ulcer was investigated, as well as ethanol gastric ulcer, the impact of the high dosage group refined intermediate and Zuojin pills on gastric secretion of normal rats was studied.
Results:The influence on indomethacin gastric ulcer was as follows:the number of ulcers of the refined intermediate0.096,0.135g/kg groups and Zuojin pills1.05g/kg group had significant difference compared with control group. The influence on ethanol gastric ulcer show that the number of ulcers of the refined intermediate0.135g/kg groups and Zuojin pills1.05g/kg group were significantly less than control group. The influence on gastric secretion of normal rats show that the refined intermediate group and Zuojin pills group can significantly reduce the secretion of gastric juice and gastric acid secretion.
Conclusions:After major pharmacodynamic comparison, the efficacy of refining intermediates and Zuojin pills had no significant differences, indicating that the extraction and purification process were reasonable.
Part3The preparation of Lianyu intragastric floating osmotic pump
controlled release tablets
Objective:To prepare Lianyu intragastric floating osmotic pump controlled release tablets with osmotic pump formulations technology.
Methods:The method to prepare LIFOPT:(1) the preparation of light and porous micro spheres:polyacrylate resin RS was dissolved in appropriate amount of ethanol-dichloromethane; mix the solution obtained with0.3%dodecyl sulfate aqueous solution; stir at50-100r/min until the oil droplet diameter increase to0.3-0.8mm; heat the mixture to40℃slowly and solidify for4hours; filter the mixture and pack the microspheres into column, wash with10BV hot water, the light and porous microspheres were derived after drying.(2) the preparation of promoing layer:mix light and porous microspheres, PEO, HPMC, sodium chloride, PVPk30according the prescription; prepare wet mixture with ethanol, prepare granules throμ gh20mesh sieve, dry for12hour at40℃, add magnesium stearate into the granules.(3) the preparation of drug layer: mix the refined intermediate and PEO according the prescription; prepare wet mixture with ethanol, prepare granules through20mesh sieve, dry for12hour at40℃, add magnesium stearate into the granules.4) Pressing tablets and coating:bilayer tablet core was prepared with the above promoing layer and drug layer granules; coat tablets core with the acetone solution of cellulose acetate; an orifice of0.8mm diameter was drilled by micro drilling machine on surface of the drug layer.
The main investigated factors were as follows:the influence of different osmotic promoting agent and dosage in promoing layer and drug layer on drug release, the influence of different amount of PEG400in coating solution, the diameter of release hole and hardness of tablet core on drug release, the influence of the suspending agent on flow propertyof suspension including API, the influence of the swelling agent on swelling rate, the elastic modulus and fracture strength of acetyl cellulose film with defferent thickness, the influence of the porous agent on water permeation rate of CA membrane.
On the basis of single factor investigation, the orthogonal experiment was designed to optimize formula in which the amount of osmotic agents, swelling agent, PEG400were taken as three influential factors and three different levels were selected to part, each of them was selected refer to the orthogonal design table. The accumulative release percentage of LIFOPT in different osmotic media was determined to verify the influence of the medium osmotic pressure on drug release.
Results:According to the results of single factor investigation, the osmotic promoting agent in drug layer was sodium chloride20mg; suspending agents in drug layer was PEO(Mw200,000)(the refined intermediate:PEO=4:1); swelling agent was PEO:HPMC=4:1; the osmotic promoting agent in promoting layer was sodium chloride; the thickness of coating film was0.18-0.20mm. According to the results of orthogonal test, the amount of osmotic agents was20mg; swelling agent was50mg; PEG400in coating solution was10%. The drug release rate decreased significantly in0.1mol/L hydrochloric acid solution (including2mol/L sodium chloride). The phenomenon showed that the driving force of drug release was the osmotic pressure diffefence between the release media and internal preparation.
Conclusions:The accumulative ralease percentage within12hours of the osmotic pump tablets prepared according to optimal prescription and process is more than90%, the release curse was in line with the zero-order kinetics equation to meet the design requirements.
Part4Studies on dissolution test in vitro of Lianyu intragastric floating osmotic pump controlled release tablets
Objective:To establish an HPLC method for simultaneous determination of berberine, palmatine, evodiamine and rutaecarpine in release medium, investigate the release character of the formulation and set up dissolution test standards.
Methods:The contents of berberine, palmatine, evodiamine and rutaecarpine in release medium were determined by HPLC. The separation was carried out on a C18column with a mixture of acetonitrile-0.025mol/L potassium dihydrogen phosphate solution (including20mmol/L sodium dodecyl sulfate, adjust pH to3.5with phosphate)(55:45) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at225nm.
Dissolution test:Medium:0.1mol/L hydrochloric acid solution1000ml; apparatus2:100rpm; times:2、4、6、8、10and12hours; test solution:pass a portion of the solution under test throμgh a suitable0.45-μm filter. The zero-order kinetic equation was taken to fit the release data, the similarity of release curve of the four components were evaluated acording to f2factor.
Results:For the method of determination of berberine, palmatine, evodiamine and rutaecarpine in release medium, there were good linear relationships for berberine within the range of500.8-25.04μg/ml, palmatine within the range of1.914-47.85μg/ml, evodiamine and rutacarpine within the range of0.1023-2.046μg/ml. The average recoveries were98.84%(RSD=1.51%),99.17%(RSD=1.12%),97.94%(RSD=1.34%),99.46%(RSD=1.05%). The zero-order kinetic equation was taken to fit the release data in water,0.1mol/L hydrochloric acid solution and phosphate buffer (pH6.8), the r values of regression equation were greater than0.98. The comparison results of release curve of each component showed that the f2factor were greater than50.
Conclusions:The HPLC method wasestablished for simultaneous determination of berberine, palmatine, evodiamine and rutaecarpine in release medium. By the method validation, precision, accuracy, and stability meet the requirements of pharmacopoeia. In various release media the release curves of the preparation are in line with the zero-order kinetics equation, the release curve of different composition were similar, indicating that the product can achieve synchronous release of each component at a constant speed. Part5Studies on pharmacokinetics in beagle dogs of Lianyu
intragastric floating osmotic pump controlled release tablets
Objective:To establish an HPLC method for simultaneous determination of berberine and palmatine in plasma of beagle dogs, study the pharmacokinetics in beagle dogs of Lianyu intragastric floating osmotic pump controlled release tablets.
Methods:Beagle dogs were randomly divided into twogroups, two-period crossover trial were employed to arrange experiment, the test preparation(LIFOPT) and the reference preparation(normal tablets) were taken respectively, the dosage was300mg, crossover study was carried out after two weeks. Sampling time:before administration and1、2、3、4、6、8、10、12、24、36、48、72、96、120hours after administration. Plasma processing: take hindlimb blood5ml, and immediately transfer into heparinized tube, centrif μ ge at10000r/min for10min, separate plasma and store at-20℃
The contents of berberine and palmatine in plasma were determined by HPLC. The separation was carried out on a C18column with a mixture of0.1%formic acid and0.1%formic acid in acetonitrile(gradient elution) as a mobile phase at the flow rate of1.0mL/min and detection wave length was set at345nm.. The pharmacokinetic parameters were calculated by DAS software.
Results:For the method of determination of berberine and palmatine in plasma, there were good linear relationships for berberine within the range of0.1-5.0μ g/ml, palmatine within the range of0.01-0.5μ g/ml, within-day and between-day RSD were less than15%, the recoveries were84.16%-96.30%and81.05%～93.29%. The main pharmacokinetics parameters were respectively as follows:berberine:tmax (3.890±1.524) and (6.761±2.496) h;cmax (1.4073±0.3804) and (0.8773±0.2395)μ g/ml; t1/2(14.26+4.85) h and (18.33±6.39) h; AUC0～96(35.94±8.86) μg·ml-1·h and (39.27±7.94) μ g·ml-1·h. The relative bioavailability was109.3±22.1%. Palmatine:tmax(4.191±1.527and (8.835±2.906)h; cmax (0.2515±0.0719) and (0.1773±0.0525) μ g/ml; t1/2(11.47±5.63) h and (16.64±5.34) h; AUCo-96(7.48±2.61) μg·ml-1·h and (8.54±2.39) μ g·ml-1·h. The relative bioavailability was114.2±31.9%.
Conclusions:The method of determination of berberine and palmatine in plasma had good sensitivity, precision and accuracy, and the sample concentration was within the linear range. On the basis of AUC0～96of berberine and palmatine, the bioavailability had no significant difference between test formulation and reference formulation, tmax significantly extended and Cmax significantly reduced. It could be concluded that the LIFOPT and the normal tablets were bioequivalent, LIFOPT had good effect of sustained-release in vivo.
引文
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