【作者】 杨晓宇；

【导师】 万江陵； 杨祥良；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2021， 博士

【摘要】 蛋白多肽类活性成分正越来越广泛地被应用于养生保健、疾病预防与诊疗等领域。虽然口服途径具有方便、顺应性好的优势,但是蛋白多肽的口服吸收、分布面临的多重屏障导致其生物学效应仍有待提高。对消化酶较为稳定的蛋白多肽,例如核桃粕经酶解得到的多肽混合物,其口服吸收的主要屏障是胃肠道的物理屏障;对于易被消化酶降解的蛋白多肽,例如胰岛素,其口服吸收的主要屏障是胃肠道的酶屏障和物理屏障。具有改善记忆功效的核桃多肽还在分布上面临血脑屏障。因此,针对以上蛋白多肽所面临的不同吸收、分布屏障,需要采用差异化的策略来提高其口服吸收效率和靶组织分布能力。促渗剂能够提高活性物质突破吸收或分布物理屏障的能力,其中中药促渗剂来源于天然产物,在长期的临床应用中已被证明其毒副作用较小。功能相近、配伍使用的中药成分往往对活性物质的吸收、分布具有促进作用,而中药成分促进蛋白、多肽吸收分布的研究仍较少。本文旨在深入挖掘中药宝库,从中筛选能促进活性蛋白多肽突破生物屏障的中药成分,或与纳米载体联用来促进其口服吸收分布。本文的主要研究内容和结论如下:(1)核桃多肽的结构鉴定及活性多肽的筛选。核桃粕水解物组成复杂,通过液质联用和de novo测序的方法鉴定获得约2000个多肽组分。基于de novo测序结果和核桃粕水解物的氨基酸组成,通过构建科学打分标准,筛选得到8个多肽组分。进一步通过体外抗氧化能力、促神经细胞抗氧化损伤能力的筛选,得到在中性p H下带不同电荷、结构与核桃多肽整体相似、活性较强的多肽P1(VEGNLQVLRPR,等电点为10.31)和P18(HNLDTQTESDV,等电点为4.01),用于口服吸收分布的研究。(2)龙眼多糖促核桃多肽口服吸收。行为学实验表明,核桃多肽和龙眼多糖分别能更有效增强小鼠被动学习和主动学习的能力,因此两者配伍使用在改善记忆方面可以增效。大鼠药动学实验表明,龙眼多糖能显著提高带负电多肽P18的血药达峰浓度。人克隆结肠腺癌细胞(Caco-2)实验表明,龙眼多糖能显著增强肠上皮细胞对P18的摄取,并改变P18的入胞途径,从而有利于其吸收。等温滴定量热实验表明,龙眼多糖与P18之间存在较强的单一相互作用力,促使两者形成形态均一的纳米粒。而带正电多肽P1与龙眼多糖之间的作用力较复杂,形成的纳米粒均一性较差,不利于肠上皮细胞摄取和口服吸收。小鼠组织分布实验表明,龙眼多糖对多肽P1和P18的脑分布均无显著促进作用。(3)茶多酚促核桃多肽脑分布。行为学实验表明茶多酚能增强核桃多肽逆转小鼠记忆障碍的作用,两者配伍使用可增强改善记忆效果。大鼠药动学实验和Caco-2细胞摄取、转运实验均表明口服茶多酚对多肽P1和P18的口服吸收效率无影响。小鼠组织分布实验表明,口服茶多酚可以显著促进核桃多肽口服后P1和P18的脑分布。BCEC细胞摄取实验表明,茶多酚可以增强P1和P18穿透血脑屏障的能力。(4)冰片促核桃多肽口服吸收和脑分布。行为学实验表明,常用的中药来源促渗剂冰片能增强核桃多肽逆转小鼠记忆障碍的作用。药动学和组织分布实验表明,冰片既能显著促进核桃多肽口服后P1和P18的吸收又能提高其脑分布。细胞实验表明,冰片可以抑制Caco-2细胞和脑血管内皮细胞(BCEC)表面P-糖蛋白介导的外排,从而显著增强P1和P18的口服吸收和入脑。(5)冰片修饰的纳米载体促胰岛素口服吸收。采用乳化溶剂挥发法制备得到冰片修饰载胰岛素纳米粒,平均粒径约110 nm、电位约-5.1 m V。糖尿病大鼠降血糖评价表明,相对于未修饰冰片的纳米粒或将纳米粒与冰片物理混合,表面修饰冰片的纳米粒口服后具有更强的降血糖作用。体外稳定性及释药行为实验表明,该纳米粒在含酶模拟胃肠液中能保护胰岛素,提高其克服酶屏障的能力。Caco-2细胞实验表明,冰片修饰能通过抑制P-糖蛋白介导的外排或增加内吞途径的方式,提高胰岛素突破物理屏障的能力,且提高作用显著强于将纳米粒与冰片物理混合的策略。综上所述,本文针对消化酶稳定和消化酶敏感的蛋白多肽口服吸收面临的不同生理屏障,分别采用与中药活性成分或促渗剂物理混合、包载入中药来源促渗剂修饰的纳米粒的策略,有效地提高了其口服吸收效率。本研究对新型促渗剂的发现和配伍机制的阐释具有重要的意义。更多还原

【Abstract】 Bioactive proteins and peptides have been widely used in health care,disease prevention and treatment.Though the oral route has advantages of convenience and good compliance,the low oral bioavailability of proteins and peptides seriously weakens their biological effects.For digestive enzyme-stable proteins and peptides,such as defatted walnut meal hydrolysate(DWMH),the main barrier for oral absorption is the physical barrier of the gastrointestinal tract(GIT).For digestive enzyme-sensitive proteins and peptides,such as insulin,the main barriers for oral absorption are the enzymatic and physical barrier of the GIT.Moreover,walnut peptides with memory enhancing effects also face the blood-brain barrier for distribution.Therefore,in view of the different barriers for oral absorption faced by different types of proteins and peptides,it is necessary to design diverse strategies to improve their oral absorption.Permeation enhancers are effective strategies to improve absorption or distribution.Tranditional Chinese medicine(TCM)permeation enhancers are derived from natural products and have been proved to have less toxic and side effects in long-term clinical application.Several TCM ingredients are reported to have the ability to promote the absorption and distribution of ingredients with similar functions,but few studies focus on the promotion of the absorption and distribution of proteins or peptides by TCM ingredients.The aim of this work is to explore the treasure of TCM and find the effective herbal components of TCM that have potentials to promote the oral absorption and distribution of proteins or peptides.Using these herbal ingredients to enhance the oral absorption and distribution efficiencies of DWMH.Moreover,nanocarrier is used in combination with permeation enhancers derived from TCM to promote oral absorption of insulin.The main research contents and conclusions are listed as follows:(1)Identification of DWMH and selection of bioactive peptides.The composition of DWMH was complex.Nearly 2,000 peptides were identified by HPLC-ESI-MSn and de novo sequencing.Based on the de novo sequencing results and structural information,a scientific scoring criteria for the selection of bioactive peptides was established,and eight peptides were selected.According to antioxidant capacities and protective effects on oxidative damaged nerve cells,two opposite charged peptides(P1-VEGNLQVLRPR,p I =10.31 & P18-HNLDTQTESDV,p I = 4.01),which had similar structure and antioxidant capacity to DWMH,were chosen for further study.(2)Coadministration with Longan polysaccharide(LP)promotes oral absorption of walnut peptides.Behavioral experiments of mice showed that LP had a better effect on improving active learning ability than DWMH,while DWMH was more beneficial to improve passive learning ability.Thus,the combination of LP and DWMH could be meaningful.Pharmacokinetic experiment showed that LP could significantly increase the plasma concentration of negatively charged P18.Moreover,LP could significantly enhance Caco-2 cells uptake amount of P18 and change the endocytosis pathway of P18.Isothermal titration calorimetry(ITC)showed that there was a strong interaction between LP and P18,which promoted the formation of homogeneous nanoparticles.However,the interaction between LP and P1 was complex,resulting in poor homogeneity of nanoparticles,which was not conducive to the cellular uptake of Caco-2 cells.Tissue distribution experiment showed that LP had no effect on the accumulation of walnut peptides in the brain.(3)Coadministration with tea polyphenols(TP)increases brain accumulation of walnut peptides.Behavioral experiments showed that TP could enhance the effect of walnut peptides on reversing memory impairment in mice.Therefore,the combination of TP and walnut peptides could be used for improving memory.Both pharmacokinetic experiment and Caco-2 cells transport experiment showed that TP had no effect on the oral absorption of walnut peptides.Biodistribution experiment showed that TP could significantly increase the brain distribution of walnut peptides.Moreover,TP could promote the cellular uptake of walnut peptides by BCEC cells.(4)Coadministration with borneol(BO)enhances oral absorption and brain distribution of walnut peptides.According to behavioral experiments,as a permeation enhancer derived from TCM,BO was found to be able to enhance the effect of walnut peptides on reversing memory impairment in mice.Pharmacokinetic and tissue distribution experiments showed that BO could significantly improve the oral bioavailability and brain distribution of walnut peptides.Cellular studies showed that BO could inhibit P-gp efflux and significantly enhance the cellular uptake of walnut peptides by Caco-2 cells and BCEC cells.(5)Borneol modified nanoparticles promote oral absorption of insulin.The nanoparticles were prepared by the emulsifying solvent evaporation method,which have an average diameter of about 110 nm and a zeta potential of-5.1 m V.The hypoglycemic experiment showed that BO modified nanoparticles had a better hypoglycemic effect than the unmodified nanoparticles or mixture of unmodified nanoparticles and BO.Results of stability and drug release in vitro showed that BO modified nanoparticles could improve the ability of insulin to overcome the enzyme barrier in simulated gastric and intestinal fluid.Cellular studies showed that BO modification of nanoparticles could improve the ability of insulin to cross the physical barrier by inhibiting P-gp efflux or promoting cell bypass transport.The enhanced effect was significantly stronger than simply mixing nanoparticles and BO.In conclusion,this work proposed different strategies to improve oral absorption and distribution for different types of proteins/peptides.For enzyme-stable proteins/peptides,co-delivery with absorption enhancers derived from TCM ingredients could be a promising method.For enzyme-sensitive proteins/peptides,nanocarriers might be an effective method.In addition,using active ingredients of TCM ingredients to modify the nanoparticles could enhance oral absorption.This study is of great significance for the discovery of new permeation enhancers and their mechanisms.更多还原