当归有效组分结肠定位微丸的制备及体内外评价

发布时间：2018-04-24 00:21

本文选题：当归 + 有效组分　；参考：《北京中医药大学》2017年硕士论文

【摘要】：目的根据课题组前期在细胞模型和动物模型上的研究结果,通过葡聚糖硫酸钠(DSS)诱导大鼠产生溃疡性结肠炎模型,验证当归超临界提取物与酚酸类提取物对大鼠溃疡性结肠炎相关症状的改善作用;根据其生物学特性,利用挤出滚圆和包衣方法将二者制成结肠定位微丸,使其更好的发挥作用;通过体外溶出和体内活体成像技术对结肠定位微丸的性能进行评价,并通过DSS诱导小鼠产生溃疡性结直肠炎症模型,进一步验证当归有效组分结肠定位微丸的药效作用。1.采用葡聚糖硫酸钠诱导大鼠产生溃疡性结肠炎模型,验证当归超临界提取物与酚酸类提取物合用对大鼠溃疡性结肠炎相关症状的改善作用;2.利用挤出滚圆及包衣锅包衣法制备当归有效组分结肠定位微丸,使药物口服后在结肠释放,提高药物在结肠的浓度,使其发挥局部和全身的治疗作用;3.通过体内、外评价方法对结肠定位微丸的性能进行评价,并通过DSS诱导小鼠产生溃疡性结直肠炎症模型,进一步验证当归有效组分结肠定位微丸的药效作用。为当归有效组分进一步的开发利用提供理论依据。为中药新型口服结肠定位制剂的研制和系统评价体系的建立进行一些有益的研究和尝试。方法1.当归有效组分合用对大鼠溃疡性结肠炎的保护作用:采用3%的葡聚糖硫酸钠溶液诱导大鼠产生溃疡性结肠炎模型,从造模第二天开始给药,每天1次。每日同一时段灌肠,灌肠前先刺激大鼠肛周,使其大小便尽量排空并称取体重,抽取药液1mL连接灌胃针,缓慢匀速推入肛门,推后保持原位30s,空白组及模型组给予等量生理盐水保留灌肠,继续喂养6天。观察当归超临界提取物与酚酸类提取物合用对大鼠体质量、粪便性状、便血情况的影响。在第8天给药后,禁食12小时,过量注射10%的水合氯醛使大鼠安乐死,解剖腹腔,取出肛门至盲肠末端的整个肠段,分别测量结肠长度。然后纵向剖开结肠,用冰生理盐水冲洗干净后立即肉眼观察结肠组织大体形态并打分。HE染色分析大鼠结肠组织病理损伤和炎症细胞浸润程度,验证当归有效组分对大鼠结肠的抗炎能力,探讨其对大鼠在结肠炎症发生发展的过程中对结肠的保护作用。2.当归有效组分结肠定位微丸的制备工艺研究:以微晶纤维素(MCC)为微丸赋形剂,以羧甲基纤维素钠为粘合剂,以水为润湿剂,采用挤出滚圆法制备当归有效组分微丸丸芯。以18-24目收率和平面临界角为指标,单因素考察粘合剂用量、粘合剂浓度、润湿剂用量、药物用量、滚圆时间和滚圆转速,确定影响微丸成形、18-24目收率和平面临界角的主要影响因素,采用Box-Behnken试验设计,对丸芯处方进行优化,并根据优化的处方进行工艺验证。利用包衣锅对优化的丸芯进行包衣,以体外释放度为指标,考察不同包衣材料、不同处方和不同包衣厚度对结肠靶向性的影响,确定最优的包衣工艺,并进行工艺验证实验,采用f2相似因子法对释放过程中的一致性进行评价。3.当归有效组分结肠定位微丸的质量及药效学评价:以包衣微丸表面形态和圆整度、堆密度、脆碎度为指标,对包衣微丸进行质量考察。同时将丸芯和包衣微丸置密封洁净容器中,分别在60℃和相对湿度(75±5)%条件下放置10天,于0、5、10天取样检测,考察其色泽、性状、含量等稳定性。借助小动物活体成像技术,将荧光显色和X-射线显影技术结合,判定微丸在给药后的不同时间在动物体内的位置及释放情况,评价微丸的结肠靶向性。采用3%DSS制备小鼠溃疡性结肠炎模型,观察小鼠给药后的体质量、粪便性状、便血情况、结肠长度、组织病理损伤和炎症细胞浸润程度,验证当归有效组分结肠定位微丸对小鼠结肠的保护作用。结果1.当归有效组分合用对大鼠溃疡性结肠炎的保护作用:实验中各组均出现了不同程度的炎症细胞浸润,但各给药组在给予药物干预后,无论是从动物的日常行为观察还是结肠大体评分还是病理切片分析,均不同程度的表现出对溃疡性结肠炎发生发展的延缓和改善作用。其中SFE:PAE(纯化)=1:2组表现出一定的抗炎作用,验证了细胞模型上的抗炎结果;同时SFE:PAE=1:2组在大鼠身上也表现出较好的药理作用,提示可能未富集的酚酸类提取物中除了阿魏酸以外的其他共存成分也有较好的抗炎能力,共同产生协同作用。2.当归有效组分结肠定位微丸的制备工艺研究:采用挤出滚圆法制备了当归有效组分微丸丸芯,建立了丸芯中指标性成分藁本内酯和阿魏酸含量测定的方法学。以微丸18-24目收率和平面临界角为指标,采用单因素实验考察了制备过程中和处方中影响微丸成形、18-24目收率和平面临界角的主要影响因素。然后采用Box-Behnken实验设计对丸芯的处方进行优化,通过模型拟合确定最佳处方为:MCC 20 g,药物9 g,润湿剂水12g,粘合剂为0.8%的CMC-Na7.4g,挤出速度40Hz,滚圆速度30Hz,滚圆时间120 s,烘干。并进行3批验证实验,预测值与实测值较为接近,说明建立的预测模型的预测能力较好。通过该方法获得了收率较高、平面临界角较小的当归有效组分微丸丸芯。体外释放研究发现丸芯释放较好,在20min累计释放达到80%,2 h达到95%左右,同时可以看出指标性成分藁本内酯与阿魏酸二者能够保持同步释放。包衣过程中考察了:内层包酸溶衣、外层包肠溶衣,增重30%以上,微丸在人工胃液中也溶出较多;内层包时滞型包衣液RSPO、外层包肠溶衣,也不能满足释放要求,后期优化不再考虑该处方。按照前期研究的包衣处方增重15%时,基本能达到结肠定位的目的,但在人工胃液中累计释放超过20%,需要进行优化。在优化处方时内层包裹时滞型包衣液RSPO后会延缓药物的溶出,特别是在结肠中6个小时较难达到80%,不能达到结肠定位迅速释药的目的。按照原包衣处方增重16%时,能较好达到结肠定位的目的。故最终选用尤特奇FS30D包衣材料,微丸增重16%,能较好的实现结肠靶向性。3.当归有效组分结肠定位微丸的质量及药效学评价:根据包衣微丸的圆整度、堆密度、脆碎度等考察结果,说明结肠定位微丸性质稳定均一。通过扫描电镜观察微丸表面形态,微丸表面包衣膜完整,光滑,大小均一。在温度25℃,相对湿度75%±5%条件下10天后微丸的形态和含量未发生显著变化;在温度60℃条件下,10天后包衣微丸发生部分粘连,影响其释放,因而微丸需放置常温下更利于保存。通过荧光成像系统和X-射线显影结果,可见丸芯在小鼠体内释放和代谢正常,在体内无蓄积;包衣微丸随着时间的推移,出现在小鼠体内不同部位,在正常小鼠和炎症小鼠体内表现行为基本一致,可基本实现结肠定位目的,且释放效果较好。在药效学实验中,模型组小鼠的整体状况较差,造模期间有小鼠死亡,病理切片看出结肠存在大面积深层炎性细胞浸润。给药组能改善小鼠日常状态和便血便稀情况,并延缓结肠炎症的发生和发展,根据结果可以看出微丸组对小鼠的效果略优于阳性药组,可能由于微丸到达结肠后在病变部位迅速释药,起到改善局部症状的作用。结论当归超临界提取物与酚酸类提取物联合使用对大鼠溃疡性结肠炎的发生发展有延缓和抑制的作用,表明二者合用具有较好的抗炎效果,同时验证了课题组前期在细胞模型上的实验。并成功制备了含超临界提取物与酚酸类提取物的结肠定位载药微丸,制备工艺合理稳定、简单可行,制得的微丸释放性好,通过体外释放实验和体内动物活体成像结果显示微丸在体内外结肠靶向性强,药效学研究表明微丸能改善小鼠整体状态,对小鼠溃疡性结肠炎的发生发展有延缓和抑制作用。研究结果达到预期目的。
[Abstract]:Objective to induce ulcerative colitis in rats by sodium sulfate sodium (DSS), and to verify the effect of the extract of Angelica sinensis and phenolic acids on the symptoms of ulcerative colitis in rats, and to use the extrusion circle according to its biological characteristics. The two groups were made into colonic positioning pellets to make them play a better role. The properties of colon specific pellets were evaluated by in vitro dissolution and in vivo imaging techniques, and the mice were induced by DSS to produce ulcerative colitis model, and the effect of the effective component of Angelica sinensis on colon location micro pills was further verified by.1. extraction. The rat model of ulcerative colitis was induced with sodium sulfate sodium sulfate, and the effect of the combination of the supercritical extract of Angelica and the extract of phenolic acid on the related symptoms of ulcerative colitis in rats was verified. 2. the effective component of the colon of Angelica sinensis was prepared by extrusion circle and coating method, and the drug was released in the colon after oral administration. The concentration of high drug in the colon makes it play the role of local and whole body; 3. the performance of colon specific pellets was evaluated by the external evaluation method in vivo, and the mice were induced by DSS to produce ulcerative colitis model, and the effective component of Angelica sinensis was further verified by the effective component of Angelica sinensis. It provides a theoretical basis for the development and utilization of the step. Some beneficial studies and attempts have been made for the development of a new oral colon specific preparation and the system of systematic evaluation of Chinese medicine. Method 1. the protective effect of the effective component of Angelica sinensis on ulcerative colitis in rats: using 3% sodium dextran sulfate solution to induce ulcerative colitis in rats The model was given 1 times a day from second days. Before the same time, the enema was irrigated at the same time. Before the enema, the rats were irrigated and the body was emptied and weighed as far as possible. The 1mL connected the anus with the medicine liquid, then slowly and uniformly pushed into the anus, and then kept in situ 30s. The blank group and the model group were given the same amount of saline retention enema and continued to feed 6. The effects of the combination of the supercritical extract of the angelica and the extract of the phenolic acid on the body mass, stool character and blood condition in rats. After eighth days of administration, the rats were fasted for 12 hours, and 10% of chloral hydrate was overdosed to euthanasia, dissecting the abdominal cavity, taking out the anus to the end of the cecum and measuring the length of the colon respectively. Then the length of the colon was measured and the length was opened longitudinally. After rinsing the colon with ice physiological saline, the gross morphology of the colon tissue was observed by the naked eye and the.HE staining was used to analyze the pathological damage of the colon tissue and the degree of infiltration of inflammatory cells in the rats. The anti-inflammatory ability of the effective components of Angelica sinensis on the colon of rats was verified, and the protection of the colon in the process of the development of colitis in rats was discussed. Study on the preparation process of colon specific pellets with.2. Angelica effective components: using microcrystalline cellulose (MCC) as a pellet excipient, sodium carboxymethyl cellulose as adhesive and water as wetting agent, the effective component of Angelica pellet pellet core was prepared by extrusion round method. 18-24 order yield and boundary angle as index, single factor investigation of adhesive dosage, adhesive The concentration of mixture, the amount of wetting agent, the dosage of drug, the time of rolling circle and the rotational speed of the ball, determine the main influencing factors of the pellet forming, the 18-24 mesh yield and the plane critical angle. The Box-Behnken test design is used to optimize the prescription of the pellet core and to verify the process according to the optimized prescription. In vitro release degree, the effects of different coating materials, different prescriptions and different coating thickness on the colon targeting were investigated, the optimal coating technology was determined, and the process validation experiment was carried out. The consistency of the release process was evaluated by F2 similarity factor method to evaluate the quality and pharmacodynamic evaluation of.3. Angelica colonic positioning pellets. The quality of coating pellets was investigated with the surface morphology and roundness of the coated pellets, the density and the brittleness of the coated pellets. At the same time, the pellets and coated pellets were placed in the sealed and clean containers for 10 days at 60 degrees and relative humidity (75 + 5)% respectively. The stability of the color, character and content of the pellets was examined by 0,5,10 days, and the small animals were examined with the aid of small animals. In vivo imaging technique, the location and release of the pellets at different time in the animals were determined by combining the fluorescence coloration with the X- ray imaging technique to evaluate the colon targeting of the pellets. The mice ulcerative colitis model was prepared by 3%DSS, and the body mass, fecal character, blood condition, the colon length and the length of the colon were observed. The protective effect of the effective component of Angelica on the colon of mice was verified by the pathological damage and the infiltration of inflammatory cells. Results 1. the effective components of Angelica sinensis were used to protect the ulcerative colitis in rats. SFE:PAE (purified) =1:2 group showed a certain anti-inflammatory effect and demonstrated the anti-inflammatory results on the cell model, and the SFE:PAE=1:2 group was in the rat body. It also showed good pharmacological effects, suggesting that the other coexisting components other than ferulic acid in the unenriched phenolic acid extract also had better anti-inflammatory ability, and CO produced the preparation process of the effective component of.2. Angelica colonic positioning pellets. A methodology for determining the content of ligustilide and ferulic acid in the pellet core was established. The factors affecting the pellet forming, the 18-24 order yield and the plane critical angle in the preparation process and the prescription were investigated by the single factor experiment, and the Box-Behnken experimental design was adopted. The Box-Behnken experimental design was used. The prescription of the pellet core was optimized. Through the model fitting, the best prescription was determined as: MCC 20 g, drug 9 g, wetting agent water 12g, the adhesive of 0.8%, the extrusion speed 40Hz, the rolling velocity 30Hz, the round time 120 s, and drying. And the 3 batch test was carried out, the prediction value was close to the measured value, indicating the predictive ability of the predicted model. In vitro release study found that the release of pellets was better, the cumulative release of 20min reached 80%, and 2 h reached about 95%. At the same time, it could be seen that the indexes of ligustilide and ferulic acid two were able to keep synchronized release. The internal layer of acid soluble clothing and outer coated enteric coated clothing, with more than 30% weight gain, and more pellets dissolved in the artificial gastric juice; the internal coating time delay coating liquid RSPO, the outer coated enteric coated clothing, can not meet the release requirements, and the later optimization no longer consider the prescription. It can basically reach the goal of colon location according to the weight gain of 15% of the previous study package. However, the cumulative release of more than 20% in the artificial gastric juice needs to be optimized. In the optimization of the prescription, the internal layer of the time delay coating liquid RSPO will delay the dissolution of the drug, especially in the colon, which is difficult to reach 80% in the 6 hour of the colon. It can not reach the purpose of rapid colon location. When the weight gain of the original coat prescription is 16%, it can be better to reach the colon location. Objective. Therefore, the quality and pharmacodynamics evaluation of the colon targeted.3. Angelica effective component colon specific pellets can be achieved by using the FS30D coating material and the weight increase of 16%. The results show that the properties of the colon specific pellets are stable and homogeneous according to the roundness, density and fragility of the coated pellets. The scanning electron microscope is observed by scanning electron microscope. The surface morphology of the pellets, the coating film of the surface of the pellets is complete, smooth and uniform. The morphology and content of the pellets have not changed significantly at temperature 25 C and relative humidity of 75% + 5% conditions. At the temperature of 60 degrees, the coated pellets have partial adhesion after 10 days, affecting the release of the pellets, so the pellets need to be stored at room temperature. The results of optical imaging system and X- ray development show that the release and metabolism of the pellets are normal in the mice and no accumulation in the body. The coated pellets appear in different parts of the mice in the body of the mice in different parts of the body, and the behavior of the mice in the normal mice and the inflammatory mice is basically the same. In the experiment, the whole condition of the model group was poor, the mice died during the model, and the pathological section showed that there was a large area of inflammatory cell infiltration in the colon. The drug group could improve the daily state of mice and the dilute condition of the stool, and postpone the occurrence and development of the colitis, and the effect of the pellet group on the mice could be seen according to the results. The effect of the combination of the supercritical extract of Angelica and the extract of phenolic acids on the occurrence and development of ulcerative colitis in rats was delayed and inhibited by the combination of the extract of Angelica and the extract of phenolic acids, which showed that the combination of the two groups had better anti-inflammatory effects. The experiment on the cell model of the group was verified. The colon specific pill containing the extracts of supercritical and phenolic acids was successfully prepared. The preparation process was reasonable, simple and feasible. The release of pellets was good. The pellets were targeted in vitro and in vivo by in vitro release experiments and in vivo animal living imaging results. The study of pharmacodynamics showed that the pellets could improve the overall state of mice and postpone and inhibit the development of ulcerative colitis in mice. The results reached the expected goal.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R283.6;R285.5

【参考文献】