本文選題：當(dāng)歸 + 有效組分�。� 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的根據(jù)課題組前期在細(xì)胞模型和動(dòng)物模型上的研究結(jié)果,通過(guò)葡聚糖硫酸鈉(DSS)誘導(dǎo)大鼠產(chǎn)生潰瘍性結(jié)腸炎模型,驗(yàn)證當(dāng)歸超臨界提取物與酚酸類提取物對(duì)大鼠潰瘍性結(jié)腸炎相關(guān)癥狀的改善作用;根據(jù)其生物學(xué)特性,利用擠出滾圓和包衣方法將二者制成結(jié)腸定位微丸,使其更好的發(fā)揮作用;通過(guò)體外溶出和體內(nèi)活體成像技術(shù)對(duì)結(jié)腸定位微丸的性能進(jìn)行評(píng)價(jià),并通過(guò)DSS誘導(dǎo)小鼠產(chǎn)生潰瘍性結(jié)直腸炎癥模型,進(jìn)一步驗(yàn)證當(dāng)歸有效組分結(jié)腸定位微丸的藥效作用。1.采用葡聚糖硫酸鈉誘導(dǎo)大鼠產(chǎn)生潰瘍性結(jié)腸炎模型,驗(yàn)證當(dāng)歸超臨界提取物與酚酸類提取物合用對(duì)大鼠潰瘍性結(jié)腸炎相關(guān)癥狀的改善作用;2.利用擠出滾圓及包衣鍋包衣法制備當(dāng)歸有效組分結(jié)腸定位微丸,使藥物口服后在結(jié)腸釋放,提高藥物在結(jié)腸的濃度,使其發(fā)揮局部和全身的治療作用;3.通過(guò)體內(nèi)、外評(píng)價(jià)方法對(duì)結(jié)腸定位微丸的性能進(jìn)行評(píng)價(jià),并通過(guò)DSS誘導(dǎo)小鼠產(chǎn)生潰瘍性結(jié)直腸炎癥模型,進(jìn)一步驗(yàn)證當(dāng)歸有效組分結(jié)腸定位微丸的藥效作用。為當(dāng)歸有效組分進(jìn)一步的開(kāi)發(fā)利用提供理論依據(jù)。為中藥新型口服結(jié)腸定位制劑的研制和系統(tǒng)評(píng)價(jià)體系的建立進(jìn)行一些有益的研究和嘗試。方法1.當(dāng)歸有效組分合用對(duì)大鼠潰瘍性結(jié)腸炎的保護(hù)作用:采用3%的葡聚糖硫酸鈉溶液誘導(dǎo)大鼠產(chǎn)生潰瘍性結(jié)腸炎模型,從造模第二天開(kāi)始給藥,每天1次。每日同一時(shí)段灌腸,灌腸前先刺激大鼠肛周,使其大小便盡量排空并稱取體重,抽取藥液1mL連接灌胃針,緩慢勻速推入肛門,推后保持原位30s,空白組及模型組給予等量生理鹽水保留灌腸,繼續(xù)喂養(yǎng)6天。觀察當(dāng)歸超臨界提取物與酚酸類提取物合用對(duì)大鼠體質(zhì)量、糞便性狀、便血情況的影響。在第8天給藥后,禁食12小時(shí),過(guò)量注射10%的水合氯醛使大鼠安樂(lè)死,解剖腹腔,取出肛門至盲腸末端的整個(gè)腸段,分別測(cè)量結(jié)腸長(zhǎng)度。然后縱向剖開(kāi)結(jié)腸,用冰生理鹽水沖洗干凈后立即肉眼觀察結(jié)腸組織大體形態(tài)并打分。HE染色分析大鼠結(jié)腸組織病理?yè)p傷和炎癥細(xì)胞浸潤(rùn)程度,驗(yàn)證當(dāng)歸有效組分對(duì)大鼠結(jié)腸的抗炎能力,探討其對(duì)大鼠在結(jié)腸炎癥發(fā)生發(fā)展的過(guò)程中對(duì)結(jié)腸的保護(hù)作用。2.當(dāng)歸有效組分結(jié)腸定位微丸的制備工藝研究:以微晶纖維素(MCC)為微丸賦形劑,以羧甲基纖維素鈉為粘合劑,以水為潤(rùn)濕劑,采用擠出滾圓法制備當(dāng)歸有效組分微丸丸芯。以18-24目收率和平面臨界角為指標(biāo),單因素考察粘合劑用量、粘合劑濃度、潤(rùn)濕劑用量、藥物用量、滾圓時(shí)間和滾圓轉(zhuǎn)速,確定影響微丸成形、18-24目收率和平面臨界角的主要影響因素,采用Box-Behnken試驗(yàn)設(shè)計(jì),對(duì)丸芯處方進(jìn)行優(yōu)化,并根據(jù)優(yōu)化的處方進(jìn)行工藝驗(yàn)證。利用包衣鍋對(duì)優(yōu)化的丸芯進(jìn)行包衣,以體外釋放度為指標(biāo),考察不同包衣材料、不同處方和不同包衣厚度對(duì)結(jié)腸靶向性的影響,確定最優(yōu)的包衣工藝,并進(jìn)行工藝驗(yàn)證實(shí)驗(yàn),采用f2相似因子法對(duì)釋放過(guò)程中的一致性進(jìn)行評(píng)價(jià)。3.當(dāng)歸有效組分結(jié)腸定位微丸的質(zhì)量及藥效學(xué)評(píng)價(jià):以包衣微丸表面形態(tài)和圓整度、堆密度、脆碎度為指標(biāo),對(duì)包衣微丸進(jìn)行質(zhì)量考察。同時(shí)將丸芯和包衣微丸置密封潔凈容器中,分別在60℃和相對(duì)濕度(75±5)%條件下放置10天,于0、5、10天取樣檢測(cè),考察其色澤、性狀、含量等穩(wěn)定性。借助小動(dòng)物活體成像技術(shù),將熒光顯色和X-射線顯影技術(shù)結(jié)合,判定微丸在給藥后的不同時(shí)間在動(dòng)物體內(nèi)的位置及釋放情況,評(píng)價(jià)微丸的結(jié)腸靶向性。采用3%DSS制備小鼠潰瘍性結(jié)腸炎模型,觀察小鼠給藥后的體質(zhì)量、糞便性狀、便血情況、結(jié)腸長(zhǎng)度、組織病理?yè)p傷和炎癥細(xì)胞浸潤(rùn)程度,驗(yàn)證當(dāng)歸有效組分結(jié)腸定位微丸對(duì)小鼠結(jié)腸的保護(hù)作用。結(jié)果1.當(dāng)歸有效組分合用對(duì)大鼠潰瘍性結(jié)腸炎的保護(hù)作用:實(shí)驗(yàn)中各組均出現(xiàn)了不同程度的炎癥細(xì)胞浸潤(rùn),但各給藥組在給予藥物干預(yù)后,無(wú)論是從動(dòng)物的日常行為觀察還是結(jié)腸大體評(píng)分還是病理切片分析,均不同程度的表現(xiàn)出對(duì)潰瘍性結(jié)腸炎發(fā)生發(fā)展的延緩和改善作用。其中SFE:PAE(純化)=1:2組表現(xiàn)出一定的抗炎作用,驗(yàn)證了細(xì)胞模型上的抗炎結(jié)果;同時(shí)SFE:PAE=1:2組在大鼠身上也表現(xiàn)出較好的藥理作用,提示可能未富集的酚酸類提取物中除了阿魏酸以外的其他共存成分也有較好的抗炎能力,共同產(chǎn)生協(xié)同作用。2.當(dāng)歸有效組分結(jié)腸定位微丸的制備工藝研究:采用擠出滾圓法制備了當(dāng)歸有效組分微丸丸芯,建立了丸芯中指標(biāo)性成分藁本內(nèi)酯和阿魏酸含量測(cè)定的方法學(xué)。以微丸18-24目收率和平面臨界角為指標(biāo),采用單因素實(shí)驗(yàn)考察了制備過(guò)程中和處方中影響微丸成形、18-24目收率和平面臨界角的主要影響因素。然后采用Box-Behnken實(shí)驗(yàn)設(shè)計(jì)對(duì)丸芯的處方進(jìn)行優(yōu)化,通過(guò)模型擬合確定最佳處方為:MCC 20 g,藥物9 g,潤(rùn)濕劑水12g,粘合劑為0.8%的CMC-Na7.4g,擠出速度40Hz,滾圓速度30Hz,滾圓時(shí)間120 s,烘干。并進(jìn)行3批驗(yàn)證實(shí)驗(yàn),預(yù)測(cè)值與實(shí)測(cè)值較為接近,說(shuō)明建立的預(yù)測(cè)模型的預(yù)測(cè)能力較好。通過(guò)該方法獲得了收率較高、平面臨界角較小的當(dāng)歸有效組分微丸丸芯。體外釋放研究發(fā)現(xiàn)丸芯釋放較好,在20min累計(jì)釋放達(dá)到80%,2 h達(dá)到95%左右,同時(shí)可以看出指標(biāo)性成分藁本內(nèi)酯與阿魏酸二者能夠保持同步釋放。包衣過(guò)程中考察了:內(nèi)層包酸溶衣、外層包腸溶衣,增重30%以上,微丸在人工胃液中也溶出較多;內(nèi)層包時(shí)滯型包衣液RSPO、外層包腸溶衣,也不能滿足釋放要求,后期優(yōu)化不再考慮該處方。按照前期研究的包衣處方增重15%時(shí),基本能達(dá)到結(jié)腸定位的目的,但在人工胃液中累計(jì)釋放超過(guò)20%,需要進(jìn)行優(yōu)化。在優(yōu)化處方時(shí)內(nèi)層包裹時(shí)滯型包衣液RSPO后會(huì)延緩藥物的溶出,特別是在結(jié)腸中6個(gè)小時(shí)較難達(dá)到80%,不能達(dá)到結(jié)腸定位迅速釋藥的目的。按照原包衣處方增重16%時(shí),能較好達(dá)到結(jié)腸定位的目的。故最終選用尤特奇FS30D包衣材料,微丸增重16%,能較好的實(shí)現(xiàn)結(jié)腸靶向性。3.當(dāng)歸有效組分結(jié)腸定位微丸的質(zhì)量及藥效學(xué)評(píng)價(jià):根據(jù)包衣微丸的圓整度、堆密度、脆碎度等考察結(jié)果,說(shuō)明結(jié)腸定位微丸性質(zhì)穩(wěn)定均一。通過(guò)掃描電鏡觀察微丸表面形態(tài),微丸表面包衣膜完整,光滑,大小均一。在溫度25℃,相對(duì)濕度75%±5%條件下10天后微丸的形態(tài)和含量未發(fā)生顯著變化;在溫度60℃條件下,10天后包衣微丸發(fā)生部分粘連,影響其釋放,因而微丸需放置常溫下更利于保存。通過(guò)熒光成像系統(tǒng)和X-射線顯影結(jié)果,可見(jiàn)丸芯在小鼠體內(nèi)釋放和代謝正常,在體內(nèi)無(wú)蓄積;包衣微丸隨著時(shí)間的推移,出現(xiàn)在小鼠體內(nèi)不同部位,在正常小鼠和炎癥小鼠體內(nèi)表現(xiàn)行為基本一致,可基本實(shí)現(xiàn)結(jié)腸定位目的,且釋放效果較好。在藥效學(xué)實(shí)驗(yàn)中,模型組小鼠的整體狀況較差,造模期間有小鼠死亡,病理切片看出結(jié)腸存在大面積深層炎性細(xì)胞浸潤(rùn)。給藥組能改善小鼠日常狀態(tài)和便血便稀情況,并延緩結(jié)腸炎癥的發(fā)生和發(fā)展,根據(jù)結(jié)果可以看出微丸組對(duì)小鼠的效果略優(yōu)于陽(yáng)性藥組,可能由于微丸到達(dá)結(jié)腸后在病變部位迅速釋藥,起到改善局部癥狀的作用。結(jié)論當(dāng)歸超臨界提取物與酚酸類提取物聯(lián)合使用對(duì)大鼠潰瘍性結(jié)腸炎的發(fā)生發(fā)展有延緩和抑制的作用,表明二者合用具有較好的抗炎效果,同時(shí)驗(yàn)證了課題組前期在細(xì)胞模型上的實(shí)驗(yàn)。并成功制備了含超臨界提取物與酚酸類提取物的結(jié)腸定位載藥微丸,制備工藝合理穩(wěn)定、簡(jiǎn)單可行,制得的微丸釋放性好,通過(guò)體外釋放實(shí)驗(yàn)和體內(nèi)動(dòng)物活體成像結(jié)果顯示微丸在體內(nèi)外結(jié)腸靶向性強(qiáng),藥效學(xué)研究表明微丸能改善小鼠整體狀態(tài),對(duì)小鼠潰瘍性結(jié)腸炎的發(fā)生發(fā)展有延緩和抑制作用。研究結(jié)果達(dá)到預(yù)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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R283.6;R285.5

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本文編號(hào)：1794302