【作者】 刘晓丽；

【导师】 孙菁华；

【作者基本信息】 山西医科大学 ， 药学（专业学位）， 2024， 硕士

【摘要】 第一部分紫萝卜花青素的提取对其溃疡性结肠炎的保护作用目的:本研究旨在从紫萝卜中提取出的花青素(Purple radish anthocyanins,PRA),探究其理化性质、稳定性和抗氧化性能,并进一步研究紫萝卜花青素对溃疡性结肠炎(UC)的治疗效果,并评估了PRA的生物安全性及潜在治疗应用价值。方法:1、材料制备:采用了溶剂提取法与超声辅助提取法相结合的方法,从紫萝卜中成功提取出PRA,并通过大孔树脂纯化工艺得到PRA。2、体外表征:采用高效液相色谱(HPLC)、紫外可见吸收光谱验证了提取物PRA的特性。同时,在胃模拟液中测定其稳定性,并通过测量其对超氧阴离子清除率、DPPH自由基清除率及总抗氧化能力,测定了PRA抗氧化性能。3、体内表征:采用葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱导构建急性溃疡性结肠炎(UC)小鼠模型,随后以50、100及200 mg/kg三种剂量的PRA进行口服治疗。在实验过程中,记录并观察小鼠一般活动状态、大便状况、潜血情况、组织病理学以及疾病活动指数评分等指标。此外,通过酶联免疫吸附法检测小鼠肠道中的促炎因子-肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、白介素6(interleukin-6,IL-6)和白介素1β(interleukin-1β,IL-1β),及氧化应激标志物-谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)及丙二醛(malondialdehyde,MDA)水平。最后,通过免疫荧光检测小鼠结肠组织中紧密连接蛋白ZO-1表达水平来评估PRA对小鼠结肠黏膜屏障的影响。为确保该材料的生物安全性,我们将小鼠连续17天以高剂量喂养PRA,观察其对肝肾功能和组织病理学的影响,来验证材料的生物安全性。结果:研究结果表明,成功地从紫萝卜中分离并提取了PRA,并通过高效液相色谱和紫外可见光吸收光谱对其组分及特征峰进行测定,说明提取物为花青素。值得注意的是,PRA表现出强大的抗氧化能力,并且在胃液模拟液中保持了良好的稳定性。体内实验结果表明,PRA对于改善急性溃疡性结肠炎小鼠模型的症状显示出显著的效果,并且结果表现,PRA具有明显抗氧化和抗炎能力,并且这种效应呈现剂量依赖关系,尤其是给予高剂量200 mg/kg的PRA,治疗效果最为突出。结论:紫萝卜提取物PRA不仅具有良好的稳定性,而且具有优良的生物安全性,通过对小鼠溃疡性结肠炎模型的干预实验发现,PRA通过抑制炎症反应、缓解氧化应激、修复肠道屏障,表现出对溃疡性结肠炎的强大治疗潜力,因此,PRA作为一种源自天然的候选物质,为治疗溃疡性结肠炎提供了新策略。第二部分花青素蒙脱土纳米药物治疗溃疡性结肠炎的研究目的:本研究旨在解决花青素利用率低的问题,通过将其负载至蒙脱土上,以提高口服后的抵抗胃酸能力,使其能够更有效地进入肠道靶向炎症部位,并延长药物在体内炎症部位的滞留时间。因此,我们合成了PRA-MMT纳米颗粒,并对其理化性质进行了测定,同时验证了其抗氧化性能。进一步研究了纳米颗粒PRA-MMT在急性溃疡性结肠炎小鼠体内的治疗效果,通过近红外成像验证了靶向治疗效果,并进行了生物安全性研究。方法:1、材料制备:采用酸性溶液对MMT进行剥离,并通过搅拌的方式将PRA负载到MMT层间,最终制备出PRA-MMT纳米颗粒(制备过程在避光条件下进行)。2、体外实验:取适量的纳米颗粒PRA-MMT进行理化性质的研究,包括透射电镜(TEM)、红外吸收光谱、热重分析(TGA)、X射线衍射(XRD)、Zeta电位、紫外吸收光谱等,此外,特别关注了纳米颗粒在模拟胃液环境下的紫外吸收行为,以评估其在生理条件下的稳定性。在细胞水平上,通过DCFH-DA探针检测PRA-MMT纳米颗粒对肠上皮细胞及单核巨噬细胞内活性氧(ROS)生成的影响,以评估其抗氧化活性。3、体内实验:通过DSS诱导构建UC小鼠模型,系统评估PRA-MMT纳米颗粒在体内的治疗效果。实验过程中,在给药后的预定时间节点,收集小鼠的心、肝、脾、肺和肾主要脏器,并进行了病理组织学和血液生化分析,以此作为生物安全性检测的重要组成部分,以评估其生物安全性。利用近红外二区荧光成像技术实时追踪了PRA-MMT纳米颗粒在UC小鼠肠道内的分布及代谢动态,以直观展示其在活体内的靶向输送和疗效表现。结果:纳米材料的表征:TEM和热重分析(TGA)结果表明MMT层距增大,PRA成功的吸附至MMT层间;红外吸收光谱、紫外吸收曲线(UV)、X射线衍射(XRD)证实了PRA负载到MMT上合成了PRA-MMT;热重分析(TGA)进一步表明PRA在MMT内的负载率为18%,Zeta电位结果表明PRA-MMT具有负电荷,为肠炎的靶向给药奠定基础。体外实验表明:PRA-MMT纳米颗粒具有良好的抗氧化能力,能够清除多种ROS。采用DCFH-DA标记PRA-MMT纳米颗粒,与NCM460细胞和RAW 264.7细胞共孵育,抗氧化实验显示PRA-MMT在减轻氧化损伤方面表现出显著潜力,为进一步的体内实验奠定了理论基础。体内实验表明:通过近红外二荧光成像能实时监测小鼠UC模型中PRA-MMT在肠道的累积及代谢情况。研究表明相较同剂量的PRA,PRA-MMT改善急性溃疡性结肠炎小鼠模型的症状的能力尤为显著,并具有较为明显抗氧化、抗炎能力,说明PRA经过负载于MMT上形成的PRA-MMT有更加明显的效果。结论:本研究成功合成了PRA-MMT纳米颗粒,并证实其具有优良的抗氧化性能,在胃液模拟液中表现出良好的稳定性。体内实验结果显示,相较于相同剂量的PRA,PRA-MMT对改善急性溃疡性结肠炎小鼠模型的症状表现出更显著的效果,并具有更强的抗氧化和抗炎能力,说明PRA-MMT可明显改善PRA生物利用度低的问题。特别值得注意的是,PRA-MMT能够实现对溃疡性结肠炎部位的靶向给药,从而实现对UC小鼠的协同治疗(抗氧化和抗炎)。因此,PRA作为一种强效的抗氧化剂,通过MMT作为其纳米载体可有效改善PRA生物利用度低的问题,并具有靶向递送的特性,为治疗UC提供了具有广阔前景的方法。更多还原

【Abstract】 Part One The protective effect of purple radish anthocyanin extraction on ulcerative colitis Objective:The purpose of this study was to explore the physicochemical properties,stability and antioxidant properties of anthocyanins(PRA)extracted from purple radish,and to further study the therapeutic effect of purple radish anthocyanins on ulcerative colitis(UC).The biological safety and potential therapeutic application value of PRA were also evaluated.Methods:1.Materials preparation: PRA was successfully extracted from purple radish by solvent extraction and ultrasonic assisted extraction,and PRA was obtained by macroporous resin purification process.2.In vitro characterization: The characteristics of the extract PRA were verified by high performance liquid chromatography(HPLC)and UV-visible absorption spectroscopy.At the same time,its stability was determined in the gastric simulated solution,and the antioxidant properties of PRA were determined by measuring its superoxide anion scavenging rate,DPPH free radical scavenging rate and total antioxidant capacity.3.In vivo characterization: A mouse model of acute ulcerative colitis(UC)was induced by dextran sulfate sodium(DSS),followed by oral administration of PRA at three doses of 50,100 and 200 mg/kg.During the experiment,the general activity status,stool status,occult blood,histopathology and disease activity index score of mice were recorded and observed.In addition,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in the intestine of mice were detected by enzyme-linked immunosorbent assay.And oxidative stress markers-glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)and malondialdehyde(MDA)levels.Finally,the effect of PRA on the colonic mucosal barrier in mice was evaluated by detecting the expression level of tight junction protein ZO-1 in the colonic tissue of mice by immunofluorescence.In order to ensure the biological safety of the material,we fed mice with high-dose PRA for 17 consecutive days to observe its effects on liver and kidney function and histopathology to verify the biological safety of the material.Results:The results showed that PRA was successfully isolated and extracted from purple radish,and its components and characteristic peaks were determined by high performance liquid chromatography and ultraviolet-visible absorption spectroscopy,indicating that the extract was anthocyanin.It is worth noting that PRA exhibits strong antioxidant capacity and maintains good stability in simulated gastric fluid.The results of in vivo experiments showed that PRA had a significant effect on improving the symptoms of acute ulcerative colitis mouse model,and the results showed that PRA had obvious antioxidant and anti-inflammatory ability,and this effect was dose-dependent,especially given a high dose of 200 mg/kg PRA,the treatment effect was the most prominent.Conclusion:Purple radish extract PRA not only has good stability,but also has excellent biosafety.Through the intervention experiment of mouse ulcerative colitis model,it is found that PRA shows a strong therapeutic potential for ulcerative colitis by inhibiting inflammatory response,alleviating oxidative stress and repairing intestinal barrier.Therefore,PRA,as a natural candidate substance,provides a new strategy for the treatment of ulcerative colitis.Part Two Study on the treatment of ulcerative colitis with anthocyanin montmorillonite nano-drugsObjective:The purpose of this study is to solve the problem of low utilization of anthocyanins.By loading it onto montmorillonite,the ability to resist gastric acid after oral administration is improved,so that it can enter the intestinal targeted inflammatory site more effectively and prolong the retention time of the drug in the inflammatory site in vivo.Therefore,we synthesized PRA-MMT nanoparticles,measured their physical and chemical properties,and verified their antioxidant properties.The therapeutic effect of PRA-MMT nanoparticles in mice with acute ulcerative colitis was further studied.The targeted therapeutic effect was verified by near-infrared imaging,and the biosafety was studied.Methods:1.Material preparation: MMT was exfoliated by acidic solution,and PRA was loaded into the interlayer of MMT by stirring.Finally,PRA-MMT nanoparticles were prepared(the preparation process was carried out under dark conditions).2.In vitro experiments: The appropriate amount of nanoparticles PRA-MMT was used to study the physical and chemical properties,including transmission electron microscopy(TEM),infrared absorption spectroscopy,thermogravimetric analysis(TGA),X-ray diffraction(XRD),Zeta potential,ultraviolet absorption spectroscopy,etc.In addition,the ultraviolet absorption behavior of nanoparticles in simulated gastric juice environment was paid special attention to evaluate its stability under physiological conditions.At the cellular level,the effect of PRA-MMT nanoparticles on the production of reactive oxygen species(ROS)in intestinal epithelial cells and mononuclear macrophages was detected by DCFH-DA probe to evaluate its antioxidant activity.3.In vivo experiments: The UC mouse model was induced by DSS to systematically evaluate the therapeutic effect of PRA-MMT nanoparticles in vivo.During the experiment,the main organs of the heart,liver,spleen,lung and kidney of the mice were collected at the predetermined time point after administration,and histopathological and blood biochemical analyses were performed as an important part of biosafety to evaluate its biosafety.The distribution and metabolic dynamics of PRA-MMT nanoparticles in the intestine of UC mice were tracked in real time by near-infrared two-zone fluorescence imaging technology to visually demonstrate their targeted delivery and therapeutic performance in vivo.Results:Characterization of nanomaterials: TEM and thermogravimetric analysis(TGA)results showed that the interlayer distance of MMT increased,and PRA was successfully adsorbed to the interlayer of MMT;infrared absorption spectroscopy,ultraviolet absorption curve(UV)and X-ray diffraction(XRD)confirmed that PRA was loaded onto MMT to synthesize PRA-MMT.Thermogravimetric analysis(TGA)further showed that the loading rate of PRA in MMT was 18%.Zeta potential results showed that PRA-MMT had negative charge,which laid a foundation for targeted drug delivery of enteritis.In vitro experiments showed that PRA-MMT nanoparticles had good antioxidant capacity and could scavenge a variety of ROS.DCFH-DA-labeled PRA-MMT nanoparticles were incubated with NCM460 cells and RAW 264.7 cells.Antioxidant experiments showed that PRA-MMT showed significant potential in reducing oxidative damage,which laid a theoretical foundation for further in vivo experiments.In vivo experiments showed that the accumulation and metabolism of PRA-MMT in the intestine of mouse UC model could be monitored in real time by near-infrared two-fluorescence imaging.The results showed that compared with the same dose of PRA,PRA-MMT improved the symptoms of acute ulcerative colitis mice model significantly,and had obvious antioxidant and anti-inflammatory ability,indicating that PRA-MMT formed by PRA loaded on MMT had more obvious effect.Conclusion:In this study,PRA-MMT nanoparticles were successfully synthesized and confirmed to have excellent antioxidant properties and good stability in simulated gastric fluid.The results of in vivo experiments showed that compared with the same dose of PRA,PRA-MMT showed a more significant effect on improving the symptoms of acute ulcerative colitis mice model,and had stronger antioxidant and anti-inflammatory ability,indicating that PRA-MMT could significantly improve the low bioavailability of PRA.It is particularly noteworthy that PRA-MMT can achieve targeted drug delivery to the site of ulcerative colitis,thereby achieving photoacoustic self-monitoring and synergistic treatment(antioxidant and anti-inflammatory)for UC mice.Therefore,as a strong antioxidant,PRA can effectively improve the low bioavailability of PRA by using MMT asa nanocarrier,and it also has the characteristics of targeted delivery,which provides a promising method for the treatment of UC.更多还原