摘要
六茜素为中草药的抗菌有效成分,具有抗菌谱广、抗菌作用强、低毒、无残留、不易产生耐药性等优点,临床主要用于治疗奶牛乳房炎、猪水肿病、猪丹毒以及多种病原菌引起的畜禽消化道疾病。由于在六茜素工业化生产工艺流程中的重结晶所用试剂对合成者的身体健康有严重危害,而且六茜素存在极易氧化变质降低药效鲁问题。因此,本文进行了对六茜素的生产工艺流程的改进、新制剂的研制、质量控制及稳定性等方面研究,为六茜素在兽医临床上的正式生产、使用提供科学依据。
1.六茜素原料药的工业化生产的工艺流程改进:先将邻甲基苯胺与二氧化锰在硫酸的催化下于室温中经氧化得到邻甲基苯醌混合物,再将其通过水蒸气蒸馏得到邻甲基苯醌水溶液,再被保险粉还原后得到六茜素混合物,最后经乙醚萃取、浓缩、1%的亚硫酸氢钠重结晶后得到六茜素原料药。结果表明此法与其它方法相比具有操作简单、安全、成本低等优点。
2.六茜素制剂的制备:六茜素原料药通过干燥、称重、分装、紫外灯灭菌及封口得到注射用六茜素1号;通过在六茜素原料药中加入亚硫酸氢钠及依地酸二钠制成注射用六茜素2号;通过在六茜素原料药水溶液中按一定比例加入亚硫酸氢钠及依地酸二钠等过程制成六茜素注射液。
3.建立六茜素的含量测定方法:采用反相高效液相色谱法,内标物为对苯二酚,色谱柱为Symmetry(?)C_(18)(4.6mm×150mm,5μm),检测波长为302nm,流动相为甲醇:水(20:80),流速为0.8ml/min。结果表明六茜素在60μg/ml～140μg/ml范围内呈良好线性关系Y=0.0086X+0.00416(r=0.9999)。此法具有操作简便快速、结果准确的特点,可用于注射用六茜素1号、2号及六茜素注射液含量的测定。
4.六茜素制剂的质量标准考察:注射剂质量标准的考察项目主要包括澄明度测定、无菌检查、渗透压测定、溶血性试验、酸度测定、局部刺激性试验、热原检查、过敏反应试验、含量测定。结果显示,注射用六茜素1号、2号为白色晶体,在灭菌注射用水中澄清、无颗粒、无毒、无菌、渗透压适中、不溶血、pH值为6.1～6.2、无刺激、无热原、无过敏、稳定。六茜素注射液的质量也符合注射剂的一般要求。
5.六茜素制剂的稳定性研究及其有效期的计算:稳定性试验包括影响因素试验、加速试验和长期试验。(1)六茜素原料药在高温(60℃)、干燥、避光条件下放置10d后,其含量变化分别为:第5d为98.15%,第10d为98.23%,外观色泽、pH、澄明度等均无明显变化。说明六茜素原料药在高温试验条件下很稳定。(2)六茜素原料药在高湿度(相对湿度90%±5%)、室温、避光条件下放置10d后,其含量变化分别为:第5d为94.81%,第10d为89.03%,颜色由无色变为粉红色,含量明显下降,有吸湿增重性。说明六茜素原料药在高湿度试验条件下很不稳定。(3)六茜素原料药在强光(照度为45001x±5001x)、室温、干燥条件下10d后,其含量变化分别为:第5d为98.15%,第10d为97.83%,外观色泽、pH、澄明度等均无明显变化。说明六茜素原料药在强光照射试验条件下很稳定。(4)将六茜素原料的水溶液在室温、干燥、避光条件下放置10d,其含量变化分别为:第5d为79.15%,第10d为60.83%,颜色由无色先变为澄红色后变为棕红色或黑色,且随着放置时间越长溶液越浑浊。说明六茜素原料药在水中极不稳定。(5)将注射用六茜素1号、2号和六茜素注射液在超常条件(温度40±2℃,相对湿度75%)下放置6个月,其含量、外观色泽、pH、澄明度等均未见明显变化。说明注射用六茜素1号、2号和六茜素注射液在超常条件下都较稳定。(6)注射用六茜素l号、2号和六茜素注射液的各3个批号在实际贮存条件(温度25±2℃,相对湿度60±5%)下长期放置1.5年,经统计分析计算得出注射用六茜素1号有效期为29.5个月;注射用六茜素2号有效期为36个月;六茜素注射液有效期为24.2个月。说明注射用六茜素1号、2号和六茜素注射液在实际贮存条件下都很稳定。
Pyroline is a new veterinary drugs with high efficiency, broad spectrum, low toxicity and no residual for treatment of bovine mastitis, bowel oedema disease, bovine endometritis, pullorum disease, swine yellow dysentery, swine white dysentery and swine comprehensiveness diarrhea. The drug is white crystal like lamellar or needle, easily dissolved in water,ethylether and other organic solvent. Because the measure of recrystallization used in the industrialized manufacture of pyroline is harmful to those who synthesis the drug and the praeparatum of pyroline used at present is easy to be oxidated to decrease its potency. So we improve its technology flowsheet in order to fit industrialized manufacture and prepare pyroline's new praeparatum, and study on its quality control & stability ,all of these above will supply the scientific bases to its formal use in veterinary clinic.
1. The improvement of the technology flowsheet of pyroline' industrialized manufacture: Firstly put the o-toluidine and manganese dioxide together at room temperature catalysed by sulphuric acid and then get the admixture of orth-methyl benzoquinone, Secondly aqueous solution of orth-methyl benzoquinone is got through wet distillation, Thirdly the admixture of pyroline is got after reducted by sodium dithionite, then pharmaceutical product of pyroline is got through extracted and concentrated by ethylether and recrystalled by 1% sodium bisulfite.The results showed that this synthesis procedure is easily operation, safe and low cost compared with other methods.
2. The preparation of praeparatum for pyroline: Pyroline for injection No.1 is got through drying, Weighing, subpackage, ultraviolet lamp sterilizing and sealing of the pharmaceutical product of pyroline; Put sodium bisulfite and edathamil disodium to the pharmaceutical product of pyroline get pyroline for injection No.2; Put sodium bisulfite and edathamil disodium to the aqueous solution of pharmaceutical product of pyroline get pyroline injection.
3. Establishment of a method for determining pyroline content:RP-HPLC was carried out with the internal standard 1, 4-dihydroxybenzene. Symmetry (?)C_(18) column( 4.6mm×150mm, 5μm) ,the detection wavelength atλ_(mas)= 302nm,The mobile phase was composed of CH_3OH:H_2O(20:80), The flow rate was 0.8ml/min. The results showed the liner relationship was presented over the range of 60μg/ml～140μg/ml with equation Y= 0.0086X+0.00416( r = 0.9999). The proposed method is simple,accurate and precise which can be applied in the determination of the content for pyroline for injection No. 1, pyroline for injection No.2 and pyroline injection.
4. The quality standard investigation of praeparatum for pyroline: The quality standard investigation for injection includes clarity,asepsis,osmotic pressure, haemolyticus, acidity,localized stimulus, pyrogen, anaphylactic response and assay. The results showed that pyroline for injection No.1 and pyroline for injection No.2 were white crystal, It is clear,no particle, innoxious, asepsis, moderate osmotic pressure, no haemolysis,PH6.1, no stimulus, nonpyrogenic,no allergy and stable. The quality of pyroline injection was also fit the common requirement of injection.
5. The study on the stability of praeparatum for pyroline and its expiration date calculation: The stability test includes influential factor test, accelerated test and long-term test.(1)The results showed that the content of pharmaceutical product of pyroline were 98.15 % after 5 days and 98.23% after 10 days respectively under the high temperature(60℃) with no significant difference on aspects of color, pH and clarity. (2) The content of pharmaceutical product of pyroline were 94.81 % after 5 days and 89.03 % after 10 days respectively under the high humidity(relative humidity 90%±5%), room temperature away from light, with color changed from achromatism to pink, the content decreased obviously, moisture regain weight gain.(3) The content of pharmaceutical product of pyroline were 98.15 % after 5 days and 97.83% after 10 days respectively under the strong light-strucking (the illuminance is 45001x±5001x), room temperature ,dry condition ,with no significant difference on aspects of color, pH and clarity.The results showed that the pharmaceutical product of pyroline is stable.(4)The content of aqueous solution of pharmaceutical product of pyroline were 79.15% after 5 days and 60.83 % after 10 days respectively under the high humidity(relative humidity 90%±5%), room temperature away from light, with color changed from achromatism to pink and then to brown or black, the longer time it was placed the more muddy it was. The results showed that the pharmaceutical product of pyroline is instability at the aqueous solution. (5) Put the pyroline for injection No.1 , pyroline for injection No.2 and pyroline injection under the accelerated condition(temperature 40±2℃, relative humidity 75%),six months later the content ,aspects of color, pH and clarity have no significant change. the results showed that the pyroline for injection No.1 , pyroline for injection No.2 and pyroline injection is stable under the accelerated condition. (6) Put the three batches of pyroline for injection No.1 , pyroline for injection No.2 and pyroline injection for 1.5 years under storage condition( temperature 25±2℃, relative humidity 60±5%),after statistical analysis and calculation we get the conclusion that the term of validity of pyroline for injection No.l, pyroline for injection No.2 and pyroline injection is 29.5,36,24.2 monthes respectively, the results showed that the pyroline for injection No. 1 , pyroline for injection No.2 and pyroline injection is stable under the storage condition.
引文
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