纳米脂质体槲皮素对肝损伤大鼠保肝作用的研究
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摘要
目的肝脏易发生各种代谢性、感染性疾病以及原发性和继发性肿瘤。我国是肝病高发区,有十分之一多的人口感染肝炎,而乙型肝炎表面抗原阳性患者比阴性者显著增高。肝炎导致的原发性肝癌是在我国肿瘤死因中占第二位的常见病。因此研究肝靶向性治疗药物具有重要意义。
使用特殊的载体是目前实现肝靶向药物运输的主要方式,而纳米载体可以改变药物的体内分布,显示出体内分布的靶向性。载药纳米颗粒进入人体内迅速被网状内皮系统(RES)摄取,而RES主要分布于肝,因而可借此种被动靶向作用达到肝靶向给药的目的。脂质体(liposome)是磷脂依靠疏水缔合作用在水中自发形成的一种分子有序组合体,为多层囊泡结构,每层均为类脂双分子膜,层间和脂质体内核为水相,双分子膜为油相。载药脂质体纳米粒具有与细胞结构相似,有生物膜的特性和功能,进入体内被生物降解,免疫原性小、毒性极低；已广泛用于运载水溶性和脂溶性药物。
槲皮素(Quercetin)及其衍生物是自然界分布最广的一种黄酮类化合物,其具有抗癌、抗炎症、抗纤维化、抗氧化和清除自由基、抗病毒、逆转某些肿瘤的耐药性、扩张血管、降低血压的药理活性,并对机体免疫有增强作用；它可对肝癌细胞显出较强的细胞毒作用,能有效抑制肝癌细胞的生长；对肝损伤具有良好的保护作用及一定的抗肝纤维化的作用,在治疗肝脏疾病中有着广阔的应用前景。但由于其普通制剂不溶于水,肝靶向性差,直接应用可影响凝血功能,限制了其在肝病治疗中的应用。根据上述思想,以纳米脂质体为药物载体包裹中药成分槲皮素制成纳米脂质体槲皮素,改善给药系统水溶性,实现肝靶向给药,使纳米药物在肝脏中更加聚集。
本课题目的是首先制备出水溶性较好的纳米脂质体槲皮素,验证其肝脏靶向性及体内代谢的药效成分,然后应用于肝损伤模型动物考察其对肝脏的保护作用。
方法采用高压均质-喷雾干燥法制备纳米脂质体槲皮素,使用透射电镜、粒度仪观察其形态及测量其粒径,HPLC法测量药物包封率。使用CCL4加猪血清配合乙醇饮食建立复合大鼠肝损伤模型,同时建立测定模型大鼠血浆及各组织中槲皮素的HPLC法,肝损伤大鼠分组后分别给予槲皮素和纳米脂质体槲皮素,观察各组模型大鼠体内槲皮素的时间空间分布,并验证其靶向性。给予正常大鼠纳米脂质体槲皮素,使用高效液相色谱-质谱法(LCMS)分析大鼠血浆中的药物代谢产物是否存在药效成分,为药效学研究提供依据。将肝损伤模型大鼠按体重随机分为正常对照组、损伤模型对照组、纳米脂质体槲皮素组、槲皮素药物组,空白纳米脂质体组后按不同剂量给药,比较各组大鼠血清ALT、AST、TBiLi、TBA、TP;各组大鼠肝脏系数；各组大鼠肝脏的病理改变；各组大鼠肝脏MDA、GSH-Px、SOD、LN、HA、PCⅢ、Ⅳ-C。同时通过各组大鼠肝脏Bcl-2和Bax蛋白表达的比较,探讨纳米脂质体槲皮素保护肝脏的机制。
结果采用本实验方法制得的纳米脂质体槲皮素粒径为123±31nm,纳米粒子呈类圆形,表面有厚度均匀的包衣包裹,且粒度分布均匀。为测定槲皮素纳米药物所建立的HPLC法色谱分离良好,精密度、重现性、方法回收率良好。纳米脂质体槲皮素的包封率为91.18±0.78%,载药量为0.61±0.08mg/mg。本实验所建立的复合肝损伤模型大鼠达到实验要求。用于测定血浆及各组织中槲皮素的HPLC法色谱分离及专属性良好,精密度、重现性、方法回收率良好。槲皮素在肝损伤大鼠体内的药物半衰期为10min,而纳米脂质体槲皮素在大鼠体内的药物半衰期达到2h,脂质体延长了槲皮素在大鼠体内的循环时间,纳米脂质体槲皮素具有一定的缓释性。模型大鼠体内槲皮素由注射纳米药物后5min起即可较浓集于肝,以10min后更为明显,且明显高于对照组(P<0.05),槲皮素被制成纳米药物后提高了其对肝脏、脾脏的趋向性,降低了其对心脏、肾脏的趋向性；纳米脂质体槲皮素较槲皮素有很好的肝靶向性。当纳米脂质体槲皮素药物剂量增加到100mg/kg时,大鼠出现死亡。大鼠血浆中槲皮素代谢产物测定的色谱及质谱条件可满足实验需要,使用液相色谱-质谱联用法可确认大鼠血浆代谢产物中含有槲皮素、柽柳素、异鼠李素等药效成分。纳米脂质体槲皮素组及槲皮素组大鼠的一般状况明显优于损伤模型对照组和空白纳米脂质体组,但较正常对照组稍差,同剂量纳米脂质体槲皮素组在血清AST、ALT、TBiLi、TBA、TP、ALB及肝脏MDA、GSH-Px、SOD、LN、HA、PCⅢ、Ⅳ-C等指标总体较损伤对照组、槲皮素组及空白脂质体纳米药物组有明显的提高。纳米脂质体槲皮素、槲皮素及空白脂质体纳米药物对肝损伤大鼠肝功能保护在AST、ALT、TBiLi、TBA上呈现出由高到低的剂量-效应依赖关系。各组大鼠肝组织光镜观察可见相同剂量的槲皮素纳米脂质体药物组的肝组织病变情况好于槲皮素组和空白脂质体纳米粒组及损伤模型组,特别是高剂量的纳米脂质体槲皮素组大鼠的肝组织病理情况接近正常对照组；同时,空白脂质体纳米粒对肝损伤大鼠的肝脏病理情况亦有所改善。损伤模型对照组、纳米脂质体槲皮素高剂量组、槲皮素药物高剂量组,空白纳米脂质体高剂量组及正常对照组的大鼠肝脏Bcl-2蛋白的表达依次升高,而Bax蛋白表达则是依次降低。
结论所制备的纳米脂质体槲皮素粒度分布均匀,载药量、包封率高。本实验制备的纳米脂质体槲皮素与肝损伤模型大鼠肝脏有较强的亲和力,具有良好的肝靶向性,是十分理想的靶向性给药系统。本实验制备的纳米脂质体槲皮素同时具有较好的脾靶向性,有一定的药物缓释性,可较长时间维持血药浓度。纳米脂质体槲皮素在正常大鼠体内的血浆代谢产物中存在药效成分槲皮素、柽柳素及异鼠李素。纳米脂质体槲皮素对模型大鼠的肝脏保护优于未纳米化的槲皮素及空白脂质体纳米药物,且呈正相量效关系,100mg/kg以上的纳米脂质体槲皮素会产生致命的毒性反应。纳米脂质体槲皮素保护肝脏的机制是降低肝脏MDA水平,增强肝脏SOD和GSH-Px的活性及增强Bcl-2蛋白的表达和减弱Bax蛋白的表达。空白脂质体纳米药物亦有保护肝脏的功效,亦呈现正相量效关系。
Objective Currently metabolic diseases and infectious diseases,as well as primary and secondary tumors, are easily found in liver. China is hepatopathy high risk area and more than one-tenth of the population has HBV infection, with a significantly higher occurrence of positive hepatitis B than negative HBsAg. Primary liver cancer ranking the second leading cause among all tumor deaths in China, it is of great significance to study therapeutic drug of hepatic targeting property.
The major mechanism of achieving its drug delivery is to use special carriers, of which nano-carrier is able to change the distribution of drugs inside,thus showing the targeting property of drug distribution.
Drug-carried nano-particles will be instantly absorbed by RES. With RES mainly spreading in liver, it is possible to achieve hepatic targeted drug delivery based on the effect of passive targeting. Liposome, with multilamellar vesicle, every layer being membrane, interlayer&liposome kernel in water phase, double molecule membrane being oil phase, is a kind of molecular organized assembly formed spontaneously in water,depending on the hydrophobic association of phospholipid.
Drug-carried nano-particles, which can be degraded with low immunogenicity and low toxicity when taken in, are of the qualities and functions of biofilm structure like cell structure,. Therefore, they have been extensively applied in water-soluble and lipophilic drugs.
Quercetin and its derivatives are the most diverse and widespread flavonoids in nature, with the pharmacological activity of anti cancer, anti inflammation, free radical scavenging, antivirus, drug resistance against some tumors, vessels dilation, decreasing blood pressure, and intensifying immunity. Quercetin shows significant cytotoxicity on hepatoma cells, being able to inhibit effectively the growth of hepatoma cells, with good protective effect of liver injury and certain anti-hepatic fibrosis effects.
Since its common preparation with poor quality of targeting is undissolved, the direct application of it has an effect on blood coagulative system,which limits the application of liver disease therapy. On the above basis, nano-granulated quercetin liposome made of Quercetin encapsulated in nano-Liposome as Carrier is able to improve the water solubility of drug delivery system,achieving hepatic targeted drug delivery and aggregating nano-drug in liver.
This paper is first to prepare nano-granulated quercetin liposome, to verify its targeting property and its efficiency in vivo metabolism,and then to apply it to the evaluation of model animal with liver injuries and the protective effect.
Methods High pressure homogeneous and pressure evaporation technology was adopted in preparing nano-granulated quercetin liposome; transmission electron microscope and particle size analyzer were used to observe its morphological feature and measure its particle size; the drug content and entrapment efficiency were measured by HPLC. The hepatectomined mouse modal was made by CCL4plus swain serum and alcoholic diet; HPLC method was established to measure plasma of the modal mouse and quercetin in various tissues. Given quercetin and quercetin nanoliposomes, the grouped hepatectomined mice were observed to detect the time and space distribution of quercetin in their bodies and to testify its targeting index. In order to provide evidence to pharmacodynamics, some normal rats were given nano-granulated quercetin liposome and their metabolites in serum were analyzed to see if there were pharmaceutical components by liquid chromatography masss pectrometry (LCMS). The hepatectomined rats were grouped by weight as normal group, hepatectomined modal group, nano-granulated quercetin liposome group, quercetin group and empty nanoliposomes group, and given different doses of drug. After that, ALT, AST, TBiLi, TBA, TP in the serum, liver coefficient, pathological change in liver and MDA, GSAH-px, SOD, LN, HA, PCⅢ, IV-C of the five groups were compared. At the same time, by comparing Bcl-2of liver and Bax protein expression of the five groups, the liver-protecting mechanism of quercetin nanoliposomes was explored.
Results The size of nano-granulated quercetin liposome obtained from the experiment is123±31nm, the Nanoparticles are kind of circular which covered by coating of uniform thickness and with a uniform size distribution. The HPLC system built for testing nano-granulated quercetin liposome separated well, has a good precision, reproducibility and method recovery as well.The entrapment rate was91.18±0.78%drugloading was0.61±0.08mg/mg.The hepatectomined rats in this experiment can meet the requirement of test. The HPLC system built for testing quercetin in plasma and organs separated well, has a good precision, reproducibility and method recovery as well.The drug half-life of Quercetin within the body of the liver injury model rat is10min, but the drug half-life of the nano-granulated quercetin liposome drug within the body of the model rats reaches to2h. This is because the Liposome prolongs the circulation time of Quercetin in the body of the model rat and the nano-granulated quercetin liposome drug has some slow release effects. The Quercetin within the body of the model rats can accumulate in the liver after being injected nano drug for5minutes, and it will become more apparent if exceeding10minutes, which is also obviously high than the control group(p<0.05). The nano drug made of Quercetin improved its topotaxis towards liver and spleen as well as decreases the topotaxis towards heart and lung; meanwhile, the nano granulated quercetin liposome has very good hepatic targeting, when the doses of the nano-granulated quercetin liposome increases to100mg/kg, the model rat dies. The chromatography and mass spectrometry conditions measured in the Quercetin metabolites within the plasma of the model rat can meet the experiment requirements, and using liquid chromatography-mass spectrometry can confirm that the plasma metabolites of the model rat contains Quercetin, Tamarixetin, Isorhamnetin and some other medical components. The normal situation of rats in the nano-granulated quercetin liposome group and quercetin group are significantly better than the hepatectomined model control group and blank nano-liposomes group but is a bit worse than the normal control group. Compared with the injury control group, the Quercetin group and blank nano-liposome group, the nano granulated quercetin liposome in serum such as AST,ALT, TBiLi, TBA, TP, ALB and liver indexes such as MDA, GSH-Px, SOD, LN, HA, PCⅢ, Ⅳ-C increases to some extent.The protection of liver function for liver injury model rats from nano-granulated quercetin liposome, Quercetin and blank liposome nano drug in the field of AST, ALT, TBiLi and TBA presents a high to low dose-effect dependable relationship. According to the light microscopic observation of livers from each model rat, it can be recognized that if it using the same dose, the liver lesion situation of the nano granulated quercetin liposome group is much better than the Quercetin group and blank nano-liposomes group. Namely, the liver lesion situation of high dose of the nano-granulated quercetin liposome group is much close to the normal control group. At the same time, the blank nano-liposomes also improves the liver lesion situation for the hepatectomined rats. The expression of protein Bcl-2of the liver within the model rat in the injury model control group, the high dose of nano-granulated quercetin liposome group, the high dose of Quercetin drug group, the high dose of the blank nano-liposomes and the normal control group increases successively, while the expression of protein Bax decreases in turn.
Conclusions The granularity of the prepared nano-granulated quercetin liposome is distributed evenly with high drug load and encapsulated efficiency. The Nanometer-Granulated Quercetin Liposome prepared in this experiment has very strong affinity with the experimented rat of liver injury model and very good hepatic targeting, so it is a very ideal targeting administration system. The nano-granulated quercetin liposome prepared in this experiment also has very good spleen targeting with a certain slow release effects, which can maintain a long period of plasma concentration. The nano-granulated quercetin liposome exists some medical ingredients including Quercetin, Tamarixetin and Lsorhamnetin in the plasma metabolites within the body of normal rats. The protection of the nano-granulated quercetin liposome towards the model rat is superior to the Quercetin and blank liposome. What's more, it represents a positive phasor effect relationship. At and over100mg/kg of the nano-granulated quercetin liposome may produce lethal toxicity. The mechanism of the protection towards liver by the nano-granulated quercetin liposome aims to decrease the MDA level of the liver, strengthen the activity of SOD and GSH-px towards the liver, increase the expression of protein Bcl-2and lessen the expression of protein Bax. The blank liposome drug also has efficacy of protecting liver and represents a positive phasor effect relationship as well.
使用特殊的载体是目前实现肝靶向药物运输的主要方式,而纳米载体可以改变药物的体内分布,显示出体内分布的靶向性。载药纳米颗粒进入人体内迅速被网状内皮系统(RES)摄取,而RES主要分布于肝,因而可借此种被动靶向作用达到肝靶向给药的目的。脂质体(liposome)是磷脂依靠疏水缔合作用在水中自发形成的一种分子有序组合体,为多层囊泡结构,每层均为类脂双分子膜,层间和脂质体内核为水相,双分子膜为油相。载药脂质体纳米粒具有与细胞结构相似,有生物膜的特性和功能,进入体内被生物降解,免疫原性小、毒性极低；已广泛用于运载水溶性和脂溶性药物。
槲皮素(Quercetin)及其衍生物是自然界分布最广的一种黄酮类化合物,其具有抗癌、抗炎症、抗纤维化、抗氧化和清除自由基、抗病毒、逆转某些肿瘤的耐药性、扩张血管、降低血压的药理活性,并对机体免疫有增强作用；它可对肝癌细胞显出较强的细胞毒作用,能有效抑制肝癌细胞的生长；对肝损伤具有良好的保护作用及一定的抗肝纤维化的作用,在治疗肝脏疾病中有着广阔的应用前景。但由于其普通制剂不溶于水,肝靶向性差,直接应用可影响凝血功能,限制了其在肝病治疗中的应用。根据上述思想,以纳米脂质体为药物载体包裹中药成分槲皮素制成纳米脂质体槲皮素,改善给药系统水溶性,实现肝靶向给药,使纳米药物在肝脏中更加聚集。
本课题目的是首先制备出水溶性较好的纳米脂质体槲皮素,验证其肝脏靶向性及体内代谢的药效成分,然后应用于肝损伤模型动物考察其对肝脏的保护作用。
方法采用高压均质-喷雾干燥法制备纳米脂质体槲皮素,使用透射电镜、粒度仪观察其形态及测量其粒径,HPLC法测量药物包封率。使用CCL4加猪血清配合乙醇饮食建立复合大鼠肝损伤模型,同时建立测定模型大鼠血浆及各组织中槲皮素的HPLC法,肝损伤大鼠分组后分别给予槲皮素和纳米脂质体槲皮素,观察各组模型大鼠体内槲皮素的时间空间分布,并验证其靶向性。给予正常大鼠纳米脂质体槲皮素,使用高效液相色谱-质谱法(LCMS)分析大鼠血浆中的药物代谢产物是否存在药效成分,为药效学研究提供依据。将肝损伤模型大鼠按体重随机分为正常对照组、损伤模型对照组、纳米脂质体槲皮素组、槲皮素药物组,空白纳米脂质体组后按不同剂量给药,比较各组大鼠血清ALT、AST、TBiLi、TBA、TP;各组大鼠肝脏系数；各组大鼠肝脏的病理改变；各组大鼠肝脏MDA、GSH-Px、SOD、LN、HA、PCⅢ、Ⅳ-C。同时通过各组大鼠肝脏Bcl-2和Bax蛋白表达的比较,探讨纳米脂质体槲皮素保护肝脏的机制。
结果采用本实验方法制得的纳米脂质体槲皮素粒径为123±31nm,纳米粒子呈类圆形,表面有厚度均匀的包衣包裹,且粒度分布均匀。为测定槲皮素纳米药物所建立的HPLC法色谱分离良好,精密度、重现性、方法回收率良好。纳米脂质体槲皮素的包封率为91.18±0.78%,载药量为0.61±0.08mg/mg。本实验所建立的复合肝损伤模型大鼠达到实验要求。用于测定血浆及各组织中槲皮素的HPLC法色谱分离及专属性良好,精密度、重现性、方法回收率良好。槲皮素在肝损伤大鼠体内的药物半衰期为10min,而纳米脂质体槲皮素在大鼠体内的药物半衰期达到2h,脂质体延长了槲皮素在大鼠体内的循环时间,纳米脂质体槲皮素具有一定的缓释性。模型大鼠体内槲皮素由注射纳米药物后5min起即可较浓集于肝,以10min后更为明显,且明显高于对照组(P<0.05),槲皮素被制成纳米药物后提高了其对肝脏、脾脏的趋向性,降低了其对心脏、肾脏的趋向性；纳米脂质体槲皮素较槲皮素有很好的肝靶向性。当纳米脂质体槲皮素药物剂量增加到100mg/kg时,大鼠出现死亡。大鼠血浆中槲皮素代谢产物测定的色谱及质谱条件可满足实验需要,使用液相色谱-质谱联用法可确认大鼠血浆代谢产物中含有槲皮素、柽柳素、异鼠李素等药效成分。纳米脂质体槲皮素组及槲皮素组大鼠的一般状况明显优于损伤模型对照组和空白纳米脂质体组,但较正常对照组稍差,同剂量纳米脂质体槲皮素组在血清AST、ALT、TBiLi、TBA、TP、ALB及肝脏MDA、GSH-Px、SOD、LN、HA、PCⅢ、Ⅳ-C等指标总体较损伤对照组、槲皮素组及空白脂质体纳米药物组有明显的提高。纳米脂质体槲皮素、槲皮素及空白脂质体纳米药物对肝损伤大鼠肝功能保护在AST、ALT、TBiLi、TBA上呈现出由高到低的剂量-效应依赖关系。各组大鼠肝组织光镜观察可见相同剂量的槲皮素纳米脂质体药物组的肝组织病变情况好于槲皮素组和空白脂质体纳米粒组及损伤模型组,特别是高剂量的纳米脂质体槲皮素组大鼠的肝组织病理情况接近正常对照组；同时,空白脂质体纳米粒对肝损伤大鼠的肝脏病理情况亦有所改善。损伤模型对照组、纳米脂质体槲皮素高剂量组、槲皮素药物高剂量组,空白纳米脂质体高剂量组及正常对照组的大鼠肝脏Bcl-2蛋白的表达依次升高,而Bax蛋白表达则是依次降低。
结论所制备的纳米脂质体槲皮素粒度分布均匀,载药量、包封率高。本实验制备的纳米脂质体槲皮素与肝损伤模型大鼠肝脏有较强的亲和力,具有良好的肝靶向性,是十分理想的靶向性给药系统。本实验制备的纳米脂质体槲皮素同时具有较好的脾靶向性,有一定的药物缓释性,可较长时间维持血药浓度。纳米脂质体槲皮素在正常大鼠体内的血浆代谢产物中存在药效成分槲皮素、柽柳素及异鼠李素。纳米脂质体槲皮素对模型大鼠的肝脏保护优于未纳米化的槲皮素及空白脂质体纳米药物,且呈正相量效关系,100mg/kg以上的纳米脂质体槲皮素会产生致命的毒性反应。纳米脂质体槲皮素保护肝脏的机制是降低肝脏MDA水平,增强肝脏SOD和GSH-Px的活性及增强Bcl-2蛋白的表达和减弱Bax蛋白的表达。空白脂质体纳米药物亦有保护肝脏的功效,亦呈现正相量效关系。
Objective Currently metabolic diseases and infectious diseases,as well as primary and secondary tumors, are easily found in liver. China is hepatopathy high risk area and more than one-tenth of the population has HBV infection, with a significantly higher occurrence of positive hepatitis B than negative HBsAg. Primary liver cancer ranking the second leading cause among all tumor deaths in China, it is of great significance to study therapeutic drug of hepatic targeting property.
The major mechanism of achieving its drug delivery is to use special carriers, of which nano-carrier is able to change the distribution of drugs inside,thus showing the targeting property of drug distribution.
Drug-carried nano-particles will be instantly absorbed by RES. With RES mainly spreading in liver, it is possible to achieve hepatic targeted drug delivery based on the effect of passive targeting. Liposome, with multilamellar vesicle, every layer being membrane, interlayer&liposome kernel in water phase, double molecule membrane being oil phase, is a kind of molecular organized assembly formed spontaneously in water,depending on the hydrophobic association of phospholipid.
Drug-carried nano-particles, which can be degraded with low immunogenicity and low toxicity when taken in, are of the qualities and functions of biofilm structure like cell structure,. Therefore, they have been extensively applied in water-soluble and lipophilic drugs.
Quercetin and its derivatives are the most diverse and widespread flavonoids in nature, with the pharmacological activity of anti cancer, anti inflammation, free radical scavenging, antivirus, drug resistance against some tumors, vessels dilation, decreasing blood pressure, and intensifying immunity. Quercetin shows significant cytotoxicity on hepatoma cells, being able to inhibit effectively the growth of hepatoma cells, with good protective effect of liver injury and certain anti-hepatic fibrosis effects.
Since its common preparation with poor quality of targeting is undissolved, the direct application of it has an effect on blood coagulative system,which limits the application of liver disease therapy. On the above basis, nano-granulated quercetin liposome made of Quercetin encapsulated in nano-Liposome as Carrier is able to improve the water solubility of drug delivery system,achieving hepatic targeted drug delivery and aggregating nano-drug in liver.
This paper is first to prepare nano-granulated quercetin liposome, to verify its targeting property and its efficiency in vivo metabolism,and then to apply it to the evaluation of model animal with liver injuries and the protective effect.
Methods High pressure homogeneous and pressure evaporation technology was adopted in preparing nano-granulated quercetin liposome; transmission electron microscope and particle size analyzer were used to observe its morphological feature and measure its particle size; the drug content and entrapment efficiency were measured by HPLC. The hepatectomined mouse modal was made by CCL4plus swain serum and alcoholic diet; HPLC method was established to measure plasma of the modal mouse and quercetin in various tissues. Given quercetin and quercetin nanoliposomes, the grouped hepatectomined mice were observed to detect the time and space distribution of quercetin in their bodies and to testify its targeting index. In order to provide evidence to pharmacodynamics, some normal rats were given nano-granulated quercetin liposome and their metabolites in serum were analyzed to see if there were pharmaceutical components by liquid chromatography masss pectrometry (LCMS). The hepatectomined rats were grouped by weight as normal group, hepatectomined modal group, nano-granulated quercetin liposome group, quercetin group and empty nanoliposomes group, and given different doses of drug. After that, ALT, AST, TBiLi, TBA, TP in the serum, liver coefficient, pathological change in liver and MDA, GSAH-px, SOD, LN, HA, PCⅢ, IV-C of the five groups were compared. At the same time, by comparing Bcl-2of liver and Bax protein expression of the five groups, the liver-protecting mechanism of quercetin nanoliposomes was explored.
Results The size of nano-granulated quercetin liposome obtained from the experiment is123±31nm, the Nanoparticles are kind of circular which covered by coating of uniform thickness and with a uniform size distribution. The HPLC system built for testing nano-granulated quercetin liposome separated well, has a good precision, reproducibility and method recovery as well.The entrapment rate was91.18±0.78%drugloading was0.61±0.08mg/mg.The hepatectomined rats in this experiment can meet the requirement of test. The HPLC system built for testing quercetin in plasma and organs separated well, has a good precision, reproducibility and method recovery as well.The drug half-life of Quercetin within the body of the liver injury model rat is10min, but the drug half-life of the nano-granulated quercetin liposome drug within the body of the model rats reaches to2h. This is because the Liposome prolongs the circulation time of Quercetin in the body of the model rat and the nano-granulated quercetin liposome drug has some slow release effects. The Quercetin within the body of the model rats can accumulate in the liver after being injected nano drug for5minutes, and it will become more apparent if exceeding10minutes, which is also obviously high than the control group(p<0.05). The nano drug made of Quercetin improved its topotaxis towards liver and spleen as well as decreases the topotaxis towards heart and lung; meanwhile, the nano granulated quercetin liposome has very good hepatic targeting, when the doses of the nano-granulated quercetin liposome increases to100mg/kg, the model rat dies. The chromatography and mass spectrometry conditions measured in the Quercetin metabolites within the plasma of the model rat can meet the experiment requirements, and using liquid chromatography-mass spectrometry can confirm that the plasma metabolites of the model rat contains Quercetin, Tamarixetin, Isorhamnetin and some other medical components. The normal situation of rats in the nano-granulated quercetin liposome group and quercetin group are significantly better than the hepatectomined model control group and blank nano-liposomes group but is a bit worse than the normal control group. Compared with the injury control group, the Quercetin group and blank nano-liposome group, the nano granulated quercetin liposome in serum such as AST,ALT, TBiLi, TBA, TP, ALB and liver indexes such as MDA, GSH-Px, SOD, LN, HA, PCⅢ, Ⅳ-C increases to some extent.The protection of liver function for liver injury model rats from nano-granulated quercetin liposome, Quercetin and blank liposome nano drug in the field of AST, ALT, TBiLi and TBA presents a high to low dose-effect dependable relationship. According to the light microscopic observation of livers from each model rat, it can be recognized that if it using the same dose, the liver lesion situation of the nano granulated quercetin liposome group is much better than the Quercetin group and blank nano-liposomes group. Namely, the liver lesion situation of high dose of the nano-granulated quercetin liposome group is much close to the normal control group. At the same time, the blank nano-liposomes also improves the liver lesion situation for the hepatectomined rats. The expression of protein Bcl-2of the liver within the model rat in the injury model control group, the high dose of nano-granulated quercetin liposome group, the high dose of Quercetin drug group, the high dose of the blank nano-liposomes and the normal control group increases successively, while the expression of protein Bax decreases in turn.
Conclusions The granularity of the prepared nano-granulated quercetin liposome is distributed evenly with high drug load and encapsulated efficiency. The Nanometer-Granulated Quercetin Liposome prepared in this experiment has very strong affinity with the experimented rat of liver injury model and very good hepatic targeting, so it is a very ideal targeting administration system. The nano-granulated quercetin liposome prepared in this experiment also has very good spleen targeting with a certain slow release effects, which can maintain a long period of plasma concentration. The nano-granulated quercetin liposome exists some medical ingredients including Quercetin, Tamarixetin and Lsorhamnetin in the plasma metabolites within the body of normal rats. The protection of the nano-granulated quercetin liposome towards the model rat is superior to the Quercetin and blank liposome. What's more, it represents a positive phasor effect relationship. At and over100mg/kg of the nano-granulated quercetin liposome may produce lethal toxicity. The mechanism of the protection towards liver by the nano-granulated quercetin liposome aims to decrease the MDA level of the liver, strengthen the activity of SOD and GSH-px towards the liver, increase the expression of protein Bcl-2and lessen the expression of protein Bax. The blank liposome drug also has efficacy of protecting liver and represents a positive phasor effect relationship as well.
引文
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