β-欖香烯—姜黃素復(fù)方脂質(zhì)體的制備與聯(lián)用多西他賽抗肺癌作用的研究
發(fā)布時(shí)間：2018-08-04 13:02
【摘要】：肺癌是目前患病率、死亡率均較高的一種癌癥,肺癌的治療手段多為傳統(tǒng)的放化療以及手術(shù)切除,這些方式往往在殺傷腫瘤細(xì)胞的同時(shí)也損傷了正常組織,對(duì)患者身體造成極大的傷害。中藥作為我國(guó)臨床治療肺癌的主要手段之一,在臨床上與化療藥物合用以證明可減毒增效、延長(zhǎng)生存期和生存質(zhì)量。中藥莪術(shù)是姜科植物蓬莪術(shù)、廣西莪術(shù)和溫郁金的干燥根莖。味辛、苦、溫,歸肝、脾經(jīng),具有行氣破血、消積止痛之功。β-欖香烯和姜黃素分別是莪術(shù)中揮發(fā)油類成分和姜黃素類成分的代表,這兩種藥物單獨(dú)對(duì)癌癥的研究很多,本課題將β-欖香烯和姜黃素配伍應(yīng)用,探究?jī)伤幰种品伟┘?xì)胞生長(zhǎng)的協(xié)同作用。進(jìn)而與臨床抗癌一線藥物多西他賽聯(lián)用,共同探究中西藥對(duì)肺癌細(xì)胞的協(xié)同抑制作用。脂質(zhì)體由磷脂組成,在水中磷脂分子親水頭部插入到水中,疏水尾部則伸向空氣,攪動(dòng)后形成雙層磷脂分子的球形囊泡,可將藥物包封于雙分子層內(nèi)提高藥物的穩(wěn)定性同時(shí)降低藥物的毒性。脂質(zhì)體在體內(nèi)會(huì)被巨噬細(xì)胞吞噬,集中在肺部等器官,具有靶向性、細(xì)胞親和性和組織相容性。由于藥物包封在脂質(zhì)體內(nèi)部,在靶組織釋放緩慢,具有一定的緩釋性,可以彌補(bǔ)β-欖香烯和姜黃素在體內(nèi)代謝過(guò)快的情況。因此將β-欖香烯和姜黃素制成復(fù)方脂質(zhì)體,以達(dá)到靶向、緩釋等效用。霧化吸入給藥對(duì)于治療肺部疾病有很好的療效,可以將藥物直接遞送到靶器官,可進(jìn)一步提高靶向性,同時(shí)避免肝臟首過(guò)效應(yīng)。中醫(yī)理論中提到"肺為嬌臟",是對(duì)肺的生理病理特征的概括。肺部具有大量肺泡,肺泡是由單層上皮細(xì)胞構(gòu)成的半球狀囊泡,周圍還有許多毛細(xì)血管,非常利于藥物的吸收,但同時(shí)也容易受到刺激。因此霧化吸入給藥的安全性需要保證,本文對(duì)脂質(zhì)體的霧化吸入刺激性做了初步評(píng)價(jià)。1.β-欖香烯和姜黃素最佳配伍比例的確定首先建立了穩(wěn)定的人肺癌細(xì)胞A549細(xì)胞和鼠肺腺癌細(xì)胞LLC細(xì)胞的體外傳代和培養(yǎng)方法。MTT法的原理為活細(xì)胞線粒體中的琥珀酸脫氫酶能使外源性MTT還原為不溶性的藍(lán)紫色結(jié)晶—甲佨(Formazan)并沉積在細(xì)胞中,而死細(xì)胞無(wú)此功能。DMSO能溶解細(xì)胞中的甲佨,并通過(guò)酶聯(lián)免疫檢測(cè)儀測(cè)定其吸光值,即可間接反映活細(xì)胞數(shù)量。通過(guò)MTT法研究不同濃度β-欖香烯和姜黃素單獨(dú)及配伍對(duì)兩種細(xì)胞的生長(zhǎng)抑制作用。采用金氏公式(q=(EA+B)/(EA+EB-EA × EB))來(lái)評(píng)價(jià)藥物聯(lián)用的協(xié)同作用,q值越大,協(xié)同作用越明顯。最終通過(guò)兩藥與多西他賽聯(lián)用后協(xié)同作用的大小確定β-欖香烯和姜黃素的最佳濃度配伍比例為1:4。當(dāng)β-欖香烯濃度為10 mg·L-1、姜黃素濃度為40 mg·L-1、多西他賽濃度為800 mg.L-1時(shí),三種藥物聯(lián)用對(duì)LLC細(xì)胞的抑制率可達(dá)87.44%。2.β-欖香烯-姜黃素復(fù)方脂質(zhì)體的制備與評(píng)價(jià)以確定的最佳配伍比例將β-欖香烯和姜黃素制成復(fù)方脂質(zhì)體。首先建立復(fù)方脂質(zhì)體中β-欖香烯和姜黃素的含量測(cè)定方法。通過(guò)單因素考察篩選出三因素三水平,以β-欖香烯和姜黃素的包封率和載藥量為指標(biāo),利用效應(yīng)面法進(jìn)行優(yōu)化處方。采用Design-Expert.8.05b軟件,利用Box-Behnken法優(yōu)選出最佳處方為姜黃素20 mg、β-欖香烯5 mg、卵磷脂666.7 mg、膽固醇133.3 mg,最終采用pH7.0的磷酸緩沖鹽10 mL旋轉(zhuǎn)水化。驗(yàn)證實(shí)驗(yàn)結(jié)果顯示β-欖香烯包封率為(97.86±1.53)%、姜黃素包封率為(97.71±1.53)%,總載藥量為(0.1925±0.006)%。制得的脂質(zhì)體外觀呈黃色,分散均勻無(wú)沉淀,微觀形態(tài)呈球形,大小均勻。復(fù)方脂質(zhì)體粒徑為(232.0±6.4)nm,電位為(0.71±0.1)mV。通過(guò)細(xì)胞實(shí)驗(yàn)將復(fù)方脂質(zhì)體和溶液劑分別與多西他賽聯(lián)用,對(duì)比結(jié)果顯示復(fù)方脂質(zhì)體在較高濃度多西他賽時(shí)對(duì)LLC細(xì)胞的生長(zhǎng)抑制率顯著高于溶液劑(P0.05)。3.脂質(zhì)體霧化吸入刺激性的研究由于制得的β-欖香烯-姜黃素復(fù)方脂質(zhì)體載藥量較小,考慮采用霧化吸入給藥方式。為考察脂質(zhì)體霧化吸入后對(duì)肺部的刺激性,設(shè)計(jì)大鼠霧化吸入實(shí)驗(yàn),采用自主研發(fā)的霧化吸入裝置(專利號(hào):ZL201520442646.5)按臨床折算劑量對(duì)大鼠進(jìn)行霧化吸入給藥。以生理鹽水為對(duì)照,比較β-欖香烯和姜黃素脂質(zhì)體對(duì)大鼠的肺部刺激性,通過(guò)肺部組織HE染色切片和肺泡灌洗液進(jìn)行BCA總蛋白的含量和乳酸脫氫酶活力進(jìn)行評(píng)價(jià)。結(jié)果顯示,無(wú)論是病理切片的觀察還是肺泡灌洗液的分析結(jié)果均顯示β-欖香烯和姜黃素脂質(zhì)體組與生理鹽水組無(wú)顯著性差異(P0.05)。初步表明兩藥脂質(zhì)體對(duì)大鼠的肺部無(wú)顯著刺激性,為后期研究奠定了基礎(chǔ)。
[Abstract]:Lung cancer is a kind of cancer with high morbidity and mortality, and the treatment of lung cancer is mostly traditional radiotherapy and chemotherapy and surgical excision. These methods often damage the normal tissue while killing the tumor cells, and cause great harm to the patient's body. It is used in bed with chemotherapeutic drugs to prove that it can reduce toxicity and increase efficiency and prolong life and quality of life. The rhizome of zedoary zedoary turmeric, Guangxi zedoary turmeric and Wen Yujin are the dried rhizomes of zedoary turmeric and zedoary. It is a kind of volatile oil component and curcumin in zedoary. The two drugs, represented by these drugs, have a lot of research on cancer alone. This subject applies the combination of beta elemene and curcumin to explore the synergistic effect of two drugs to inhibit the growth of lung cancer cells. Then, the synergistic inhibition effect of Chinese and Western drugs on lung cancer cells is explored together with the clinical anticancer drug, docetaxel. Liposomes are from the phospholipid group. In the water, the phospholipid molecules are inserted into the water and the hydrophobic tail is extended to the air and stirs into the spherical vesicles of the bilayer phospholipid molecules. The drug can be encapsulated in the double molecular layer to improve the stability of the drug and reduce the toxicity of the drug. The liposomes will be phagocytic in the body of the giant macrophage in the body and are concentrated in the lungs and other organs. The release of beta elemene and curcumin in the body is slow, which can make up for the rapid metabolism of beta elemene and curcumin in the body. Therefore, beta elemene and curcumin are made into compound liposomes to achieve target and slow release. Aerosol inhalation is used. It has a good effect on the treatment of lung disease. It can be delivered directly to the target organ, which can further improve the targeting and avoid the first effect of the liver. The theory of Chinese medicine refers to "the lungs are dirty", which is a summary of the physiological and pathological characteristics of the lung. The lungs have a large number of alveoli, and the alveoli are hemispherical vesicles consisting of single layer epithelial cells. There are many capillaries around it, which are very beneficial to the absorption of drugs, but also easy to be stimulated. Therefore, the safety of inhalation inhalation needs to be ensured. In this paper, a preliminary evaluation of the optimum compatibility of.1. beta elemene and curcumin by atomization inhalation of liposomes is first established to establish a stable human lung cancer cell A549 fine. The principle of.MTT method for the generation and culture of LLC cells in cell and rat lung adenocarcinoma cells is that succinic dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue purple crystals - Formazan and deposit in cells. Dead cells do not have the function.DMSO to dissolve the thyroid gland in the cells and be immunized by enzyme linked immunosorbent assay. The number of living cells could be indirectly reflected by the measuring instrument, and the inhibitory effects of the two cells on the growth of two kinds of cells were studied by the method of q= (EA+B) / (EA+EB-EA x EB). The greater the Q value, the more obvious the synergism was. Finally, through two. The synergistic effect of drug and docetaxel combined to determine the optimum concentration ratio of beta elemene and curcumin was 1:4. when the concentration of beta elemene was 10 mg L-1, the concentration of curcumin was 40 mg. L-1, and the concentration of docetaxel was 800 mg.L-1, and the inhibition rate of three drugs on LLC cells could reach 87.44%.2. beta elemene curcumin compound fat. The preparation and evaluation of plastids to determine the optimum compatibility ratio of beta elemene and curcumin into compound liposomes. First, the content determination method of beta elemene and curcumin in compound liposomes was established. Three factors and three levels were screened out by single factor investigation, and the encapsulation efficiency and drug loading of beta elemene and Jiang Huang were used as indexes. Design-Expert.8.05b software was used to optimize the prescription. The best formula was selected by Design-Expert.8.05b software. The best prescription was curcumin 20 mg, beta elemene 5 mg, lecithin 666.7 mg, cholesterol 133.3 mg, and finally using pH7.0 phosphate buffer salt 10 mL to rotate hydration. The experimental results showed that the encapsulation efficiency of beta elemene was (97.86 + 1.53)%, curcumin. The encapsulation rate was (97.71 + 1.53)% and the total load was (0.1925 + 0.006)%. The liposomes were yellow, dispersed evenly and without precipitation. The micromorphology was spherical and uniform. The particle size of the compound liposome was (232 + 6.4) nm, and the potential was (0.71 + 0.1) mV. combined with docetaxel and the compound liposome and solution agent respectively. The results showed that the growth inhibition rate of compound liposomes at high concentration of docetaxel was significantly higher than that of solution agent (P0.05).3. liposome atomization inhalation. The effect of the prepared beta elemene curcumin compound liposome was smaller and the aerosol inhalation method was considered. The effect of liposome inhalation on the lung after liposome inhalation was investigated. The irritation of the rats was designed by atomization inhalation experiment. The atomization inhalation of rats was carried out by the self developed atomization inhalation device (patent number: ZL201520442646.5). The rats were compared with the physiological saline. The lung irritation of rats was compared with the liposomes of beta elemene and curcumin liposomes, and the lung tissue was stained with HE staining and lung tissue. The total protein content of BCA and the activity of lactate dehydrogenase were evaluated. The results showed that there was no significant difference between the liposomes group and the saline group (P0.05), no matter the observation of pathological section or the analysis of the alveolar lavage fluid (P0.05). The initial step showed that the two drug liposomes were not significant to the lungs of the rats. Irritation has laid a foundation for later research.
【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
本文編號(hào)：2163965
[Abstract]:Lung cancer is a kind of cancer with high morbidity and mortality, and the treatment of lung cancer is mostly traditional radiotherapy and chemotherapy and surgical excision. These methods often damage the normal tissue while killing the tumor cells, and cause great harm to the patient's body. It is used in bed with chemotherapeutic drugs to prove that it can reduce toxicity and increase efficiency and prolong life and quality of life. The rhizome of zedoary zedoary turmeric, Guangxi zedoary turmeric and Wen Yujin are the dried rhizomes of zedoary turmeric and zedoary. It is a kind of volatile oil component and curcumin in zedoary. The two drugs, represented by these drugs, have a lot of research on cancer alone. This subject applies the combination of beta elemene and curcumin to explore the synergistic effect of two drugs to inhibit the growth of lung cancer cells. Then, the synergistic inhibition effect of Chinese and Western drugs on lung cancer cells is explored together with the clinical anticancer drug, docetaxel. Liposomes are from the phospholipid group. In the water, the phospholipid molecules are inserted into the water and the hydrophobic tail is extended to the air and stirs into the spherical vesicles of the bilayer phospholipid molecules. The drug can be encapsulated in the double molecular layer to improve the stability of the drug and reduce the toxicity of the drug. The liposomes will be phagocytic in the body of the giant macrophage in the body and are concentrated in the lungs and other organs. The release of beta elemene and curcumin in the body is slow, which can make up for the rapid metabolism of beta elemene and curcumin in the body. Therefore, beta elemene and curcumin are made into compound liposomes to achieve target and slow release. Aerosol inhalation is used. It has a good effect on the treatment of lung disease. It can be delivered directly to the target organ, which can further improve the targeting and avoid the first effect of the liver. The theory of Chinese medicine refers to "the lungs are dirty", which is a summary of the physiological and pathological characteristics of the lung. The lungs have a large number of alveoli, and the alveoli are hemispherical vesicles consisting of single layer epithelial cells. There are many capillaries around it, which are very beneficial to the absorption of drugs, but also easy to be stimulated. Therefore, the safety of inhalation inhalation needs to be ensured. In this paper, a preliminary evaluation of the optimum compatibility of.1. beta elemene and curcumin by atomization inhalation of liposomes is first established to establish a stable human lung cancer cell A549 fine. The principle of.MTT method for the generation and culture of LLC cells in cell and rat lung adenocarcinoma cells is that succinic dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue purple crystals - Formazan and deposit in cells. Dead cells do not have the function.DMSO to dissolve the thyroid gland in the cells and be immunized by enzyme linked immunosorbent assay. The number of living cells could be indirectly reflected by the measuring instrument, and the inhibitory effects of the two cells on the growth of two kinds of cells were studied by the method of q= (EA+B) / (EA+EB-EA x EB). The greater the Q value, the more obvious the synergism was. Finally, through two. The synergistic effect of drug and docetaxel combined to determine the optimum concentration ratio of beta elemene and curcumin was 1:4. when the concentration of beta elemene was 10 mg L-1, the concentration of curcumin was 40 mg. L-1, and the concentration of docetaxel was 800 mg.L-1, and the inhibition rate of three drugs on LLC cells could reach 87.44%.2. beta elemene curcumin compound fat. The preparation and evaluation of plastids to determine the optimum compatibility ratio of beta elemene and curcumin into compound liposomes. First, the content determination method of beta elemene and curcumin in compound liposomes was established. Three factors and three levels were screened out by single factor investigation, and the encapsulation efficiency and drug loading of beta elemene and Jiang Huang were used as indexes. Design-Expert.8.05b software was used to optimize the prescription. The best formula was selected by Design-Expert.8.05b software. The best prescription was curcumin 20 mg, beta elemene 5 mg, lecithin 666.7 mg, cholesterol 133.3 mg, and finally using pH7.0 phosphate buffer salt 10 mL to rotate hydration. The experimental results showed that the encapsulation efficiency of beta elemene was (97.86 + 1.53)%, curcumin. The encapsulation rate was (97.71 + 1.53)% and the total load was (0.1925 + 0.006)%. The liposomes were yellow, dispersed evenly and without precipitation. The micromorphology was spherical and uniform. The particle size of the compound liposome was (232 + 6.4) nm, and the potential was (0.71 + 0.1) mV. combined with docetaxel and the compound liposome and solution agent respectively. The results showed that the growth inhibition rate of compound liposomes at high concentration of docetaxel was significantly higher than that of solution agent (P0.05).3. liposome atomization inhalation. The effect of the prepared beta elemene curcumin compound liposome was smaller and the aerosol inhalation method was considered. The effect of liposome inhalation on the lung after liposome inhalation was investigated. The irritation of the rats was designed by atomization inhalation experiment. The atomization inhalation of rats was carried out by the self developed atomization inhalation device (patent number: ZL201520442646.5). The rats were compared with the physiological saline. The lung irritation of rats was compared with the liposomes of beta elemene and curcumin liposomes, and the lung tissue was stained with HE staining and lung tissue. The total protein content of BCA and the activity of lactate dehydrogenase were evaluated. The results showed that there was no significant difference between the liposomes group and the saline group (P0.05), no matter the observation of pathological section or the analysis of the alveolar lavage fluid (P0.05). The initial step showed that the two drug liposomes were not significant to the lungs of the rats. Irritation has laid a foundation for later research.
【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
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