【作者】 孟祥东；

【导师】 祖元刚；

【作者基本信息】 东北林业大学 ， 植物学， 2011， 博士

【摘要】 辅酶Q10是一种在高等动植物中广泛存在的抗氧化剂,它在治疗心血管疾病和神经退化性疾病方面具有显著的疗效。人体内的辅酶Q10在20岁左右达到巅峰后呈现下降趋势,老年时体内的辅酶Q10会比20岁时减少约57%,所以外源补充辅酶Q10对维持人体正常的新陈代谢具有积极的意义。然而由于辅酶Q10不能溶于水,导致它在人体内的吸收较差,生物利用度较低(2-3%),达不到预期的治疗作用与营养保健功效。为了提高辅酶Q10的生物利用度,国内外研究者做了大量的工作,但依然存在作用不明显、安全性不高、稳定性差等问题。微粉化技术可以直接将原料药物加工成微/纳米粒,通过粒径的减少,增加了药物颗粒的表面积,从而达到提高其水溶性的目的。这种方法无需使用任何有机溶剂和载体材料,所以安全性更高。本课题的主要目的是采用微粉化技术制备一种新型的辅酶Q10纳米粒,并对其物理化学表征、生物利用度、抗氧化活性以及心肌保护功能进行研究和评价,期望能够得到一种功效显著的水溶性辅酶Q10微粉剂型。本课题在对超临界快速膨胀(Rapid expansion of supercritical solution,RESS)设备进行改良的基础上,通过对正交实验和影响纳米粒平均粒径主要因素的分析,得出RESS技术生产辅酶Q10纳米粒的最优工艺条件:溶解压力是25Mpa;溶解温度是40℃;喷嘴直径是200μm;结晶温度足25℃。在此条件下,制备得到了平均直径为147.9±27.3nm的辅酶Q10纳米粒。同时,通过LC-MS/MS、X射线衍射、DSC差热、激光粒度以及红外光谱分析,对辅酶Q10纳米粒的理化表征、溶解度及稳定性进行考察发现,与辅酶Q10原粉相比较,辅酶Q10纳米粒的分子量和化学结构没有发生改变,但结晶度和熔点显著下降,证实RESS过程引起了辅酶Q10晶型的改变,较小的晶体结构和纳米尺寸的粒径导致辅酶Q10纳米粒水溶性和溶出速率的提高,并且辅酶Q10纳米粒在室温避光条件下具有良好的物理化学稳定性。本课题对辅酶Q10纳米粒的生物利用度进行了研究,通过反相HPLC法测定大鼠口服辅酶Q10纳米粒后的体内血药浓度,以辅酶Q10原粉为参比,求算出它们的药物动力学参数。结果表明,辅酶Q10纳米粒在大鼠体内的生物利用度是辅酶Q10原粉的1.96倍。本课题采用自然衰老模型,以辅酶Q10原粉和维生素E为参比,考查了辅酶Q10纳米粒对大鼠血浆中辅酶Q10含量以及抗氧化能力的影响。结果发现:外源补充辅酶Q10纳米粒,对衰老大鼠血浆中辅酶Q10含量、超氧化物歧化酶(SOD)活力、谷胱甘肽过氧化物酶(GSH-Px)活力有明显的提高,并且使衰老大鼠血浆中丙二醛(MDA)的含量显著降低,衰老大鼠的抗氧化能力超过正常青年大鼠(对照组)的水平;辅酶Q10纳米粒提高机体抗氧化活性的能力好于原粉,但不及维生素E。我们制备的水溶性辅酶Q10纳米粒,为在细胞水平上研究辅酶Q10的心肌保护作用提供了平台。本课题利用体外分离培养的乳鼠原代心肌细胞,建立缺氧/复氧模型,探讨了辅酶Q10对心肌细胞保护作用的部分机制。通过对细胞存活率、活性氧水平、乳酸脱氢酶(LDH)漏出量、SOD活力、MDA含量、线粒体膜电位以及细胞凋亡率的检测,结果发现:在缺氧/复氧的条件下,辅酶Q10能够提高心肌细胞的存活率,降低心肌细胞中LDH的漏出量和MDA的含量,抑制线粒体膜电位的下降,减少了心肌细胞的凋亡比率,但辅酶Q10在降低心肌细胞内活性氧水平和提高SOD活力方面的作用,并不非常显著。通过上述结果可以得出结论:本研究提供了一种新型的水溶性辅酶Q10微粉剂型,在改善辅酶Q10口服生物利用度以及提高其体内抗氧化活性方面,具有显著的功效。更多还原

【Abstract】 Coenzyme Q10(CoQ10)is an antioxidant which is widespresd in higher plants and animals,it has been found to be effective in cardiovascular diseases and neurodegenerative diseases.COQ10 shows a downward trend after it reaches the peak in the body at 20-year-old,and the content of CoQ10 in old age is decreased by 57%than that of 20 years of age.So the outside supplement of CoQ10 has a positive meaning for maintaining normal metabolism of the human body.However,CoQ10 is not soluble in water,leading to poor absorption and very low bioavailability(2-3%)in the human body,which cannot achieve the desired therapeutic effect and nutritional health benefits.In order to improve the bioavailability of CoQ10,domestic and foreign researchers have done a lot of works.However,there are some problems such as obvious enhancement,poor stability,and uncertain security.Micronized technology can process the raw material into nanoparticles directly.By reducing the particle size,the surface areas of drug particles are increased,resulting in achieve the purpose of improving its water solubility.It dose not employ any organic solvents and carrier material,so security is very high.The main purpose of this project is the use of micronized technology to prepare a new type of CoQ10 nanoparticles,and its physical and chemical characterization,bioavailability,antioxidant activity,and myocardial protection were researched and evaluated,a significant effect water-soluble powder formulation of CoQ10 was expected.In this study,based on the improved rapid expansion of supercritical equipment,analyzing the main factors affecting on mean particle size by the orthogonal experiments,the optimal process conditions to product CoQ10 nanoparticles were developed as follow:dissolved pressure at 25Mpa;solution temperature at 40℃;nozzle diameter at 200μm;crystallization temperature at 25℃.COQ10 nanoparticles were prepared by a rapid expansion of supercritical process(RESS)with an average diameter of 147.9±27.3nm.At the same time,by LC-MS/MS,X-ray diffraction,DSC differential thermal analysis,laser diffraction and infrared spectroscopy,physicochemical properties of CoQ10 nanoparticles were characterized.solubility and stability study showed that,in comparison with the unprocessed CoQ10 powder,CoQ10 nanoparticles has not changed in molecular weight and chemical structure,but its crystallinity and melting point decreased significantly,which indicates a change in crystalline caused by the rapid expansion of supercritical process.The smaller nanoparticle size and the less crystal structure led to improved solubility and dissolution rate of CoQ10 nanoparticles,and coenzyme Q10 nanoparticles process good physical and chemical stability under room temperature and dark conditions.Bioavailability of CoQ10 nanoparticles was studied by RP-HPLC detection of CoQ10 concentration in rats’plasma after a single oral dose in this issue.Pharmacokinetic parameters of CoQ10 nanoparticles were calculated with unprocessed CoQ10 powder as a reference.The results showed that the bioavailability of CoQ10 nanoparticles is 1.96 times than that of unprocessed CoQ10 powder in rats.Using natural aging model the effect of CoQ10 nanoparticles on rat plasma levels of CoQ10 and antioxidant activity was examined in vivo,with unprocessed CoQ10 and vitamin E powder as a reference.The results showed that with exogenous supplement of CoQ10 nanoparticles,the plasma CoQ10 levels,superoxide dismutase activity,glutathione peroxidase activity were significantly increased,and MDA content was significantly reduced in the plasma of old rats,resulting anti-oxidation ability of the old rats was supper than the normal young rats.The antioxidant activity of CoQ10 nanoparticles is better than the unprocessed CoQ10 powder,but less than vitamin E.The CoQ10 nanoparticles prepared by RESS provided a water-soluble dosage form to research the role of CoQ10 on myocardial protection at the cellular level.The primary neonatal rat myocardial cells were isolated and cultured in vitro,and the hypoxia/reoxygenation model was established to study the mechanism of CoQ10 on myocardial protection.Through the detection of the cell survival,reactive oxygen species,LDH leakage volume,SOD activity,MDA content,mitochondrial membrane potential and cell apoptosis rate,the results showed that in the hypoxia/reoxygenation conditions,CoQ10 can improve the myocardial cells survival,reduce the amount of LDH leakage and the content of MDA in myocardial cells,inhibit the decline of mitochondrial membrane potential,leading to reduce the apoptotic rate of myocardial cells.Howerve,CoQ10 has no significant effect on intracellular ROS levels and SOD activity.From the above results,it can be concluded that the CoQ10 nanoparticles provide a new water-soluble micronized powder dosage form of CoQ10,which can enhance the oral bioavailability and antioxidant capacity of CoQ10.更多还原