【作者】 张琳；

【导师】 李永吉；

【作者基本信息】 黑龙江中医药大学 ， 药学（专业学位）， 2015， 硕士

【摘要】 金雀根为豆科植物锦鸡儿Caragana sinica(Buc’ hoz)Rehd的根或根皮,我国大多数省区均有分布,有清肺益脾、活血通脉等功效,用于治疗虚损劳热、咳嗽、高血压、关节痛风、跌打损伤等症。自上世纪90年代起,针对金雀根的化学成分、药效学、作用机理的研究已取得大量的研究成果,证明金雀根中的二苯乙烯类成分可以有效地预防和治疗骨质疏松症,促进骨细胞生长,增加骨密度,调节骨代谢。本论文拟在已有研究基础上,从金雀根中提取纯化具有治疗骨质疏松的二苯乙烯类有效部位,并对其质量标准、稳定性、制剂及其吸收机理等内容进行研究。本论文主要包括以下五部分研究内容:1金雀根二苯乙烯有效部位提取纯化工艺研究采用两相溶剂萃取法纯化金雀根二苯乙烯有效部位,以乙酸乙酯为萃取剂,以金雀根二苯乙烯有效成分carasinolB、kobophenolA、(+)-α-viniferin总含量及萃取后的保留率为指标,采用单因素考察结合正交试验优选最佳纯化工艺。最终确定最佳提取纯化工艺为:70%乙醇加热回流提取2次,每次2h,溶剂用量第一次为药材重量的10倍,第二次为8倍量;减压浓缩回收乙醇,得到浓度为0.25g.mL-1的萃取原液,加入1倍量乙酸乙酯萃取2次,每次萃取20min。2金雀根二苯乙烯有效部位提取物质量标准与稳定性研究以三批金雀根二苯乙烯有效部位提取物的中试样品为研究对象,对金雀根二苯乙烯有效部位提取物的性状、鉴别、检查(水分、炽灼残渣、重金属、砷盐)、含量测定、指纹图谱等项目进行了研究,起草了金雀根二苯乙烯有效部位质量标准草案。通过影响因素试验和加速稳定性试验,对金雀根二苯乙烯有效部位中间体的稳定性进行了研究。结果表明,金雀根二苯乙烯有效部位中间体的性质稳定。3金雀根二苯乙烯片剂研究采用湿法制粒压片法,以片剂的硬度、崩解时限和有效成分溶出度作为考察指标,通过单因素考察结合正交试验对金雀根二苯乙烯片的处方进行了考察,优选出片剂的最佳处方(以制备金雀根二苯乙烯片1000片计):金雀根二苯乙烯提取物100g,微晶纤维素185g,羧甲基淀粉钠15g,50%乙醇20ml,硬脂酸镁1.5g。对所得片剂进行了性状、鉴别、检查(外观、重量差异、硬度、崩解时限、溶出度)、含量测定等项目的研究,结果显示,片剂外观完整,色泽均匀无杂斑,重量差异小于5%,硬度在60～70N之间,崩解时限小于 1min,45min 溶出度以 carasinolB、kobophenolA、(+)-α-viniferin三个成分总量计,均大于70%。并根据样品测定结果起草了金雀根二苯乙烯片剂的质量标准草案。加速稳定性试验和初步稳定性试验的结果显示,金雀根二苯乙烯片剂性质稳定。4 Caco-2细胞模型研究金雀根二苯乙烯有效成分kobophenol A吸收机理建立了 Caco-2细胞模型,首次考察了 kobophenol A的Caco-2细胞摄取及跨膜转运过程。结果表明:kobophenol A在50～300μg.ml-1浓度范围内,细胞摄取量与时间、浓度呈正相关,高浓度时出现饱和现象;pH、温度均对摄取有显著影响,pH7.4时摄取量最大,37℃较4℃摄取量高;P-gp抑制剂对kobophenol A的细胞摄取及转运均无显著影响(p>0.05);二苯乙烯有效部位提取物中其他成分对kobophenolA吸收无明显促进作用;表观渗透系数Papp约为10-6cm·s-1,属吸收中等的药物,表明kobophenolA在肠道上的转运方式非单纯的被动扩散,可能存在载体介导的主动转运。5在体肠灌流模型研究金雀根二苯乙烯有效成分kobophenol A吸收机理建立大鼠在体单向肠灌流模型,对金雀根二苯乙烯有效成分kobophenol A的肠吸收机理进行研究。结果表明:kobophenol A在各肠段均有吸收,而回肠段的吸收优于十二指肠、空肠、结肠;具有浓度依赖性,随着浓度的增加,其Ka及Papp值均逐渐下降(P<0.05);kobophenol A在4个肠段的Papp为1.61×10-3～3.60×10-3cm.min-1,属中等程度吸收;中、高浓度提取物组的Ka及Papp值与kobophenol A组相较无显著性差异(P>0.05),低浓度组的Ka及Papp值较kobophenol A组低且存在显著性差异(P<0.05)。大鼠在体单向肠灌流试验进一步验证了 Caco-2细胞试验的结果,即kobophenol A的吸收方式并非单纯的被动扩散,可能存在主动转运或促进扩散。综上,本文以金雀根二苯乙烯有效部位为研究对象,对其中间体及制剂的制备工艺、质量标准、稳定性进行了全面系统的研究,并结合Caco-2细胞模型和在体肠灌流模型对金雀根二苯乙烯指标性成分kobophenol A的体内外吸收机理进行了详尽研究,为口服制剂的开发提供实验和理论依据。更多还原

【Abstract】 Genistein root is the root of plant Caragana sinica(Buc’ hoz)Rehd which is widely distributed in China.It has been used in China as a folk medicine for the treatment of asthenia syndrome,vascular hypertension,leukorrhagia,bruises and contused wounds.Since the 1990’s,research results on the chemical composition,pharmacodynamics,mechanism research haveproved that stilbene activeingredients of Genistein root can promote the growth of bone cell,increase bone density,regulate bone metabolism,and thus effectively prevent and treat of osteoporosis.On the basis of the previous results,our present study focuses on the purification of stilbene active ingredients from Genistein root,the quality standard,the preparation of tablet,and theabsorption mechanism of kobophenol A.1 Study on purification of stilbene effective parts from Genistein rootWe take the percentage of active ingredients(carasinol B,kobophenol A,(+)-α-viniferin)in the extraction and extraction ratio was used as index and adopt extraction as the method to purify stilbene active parts.The optimized purification parameters were obtained by single factor and orthogonal experiment.The optimized purification conditions were as follows:extraction solution concentration 0.25 g·ml-1,extract two times with one fold of ethyl acetate,extraction 20 min each time.2 Study on the quality standard andstability ofGenistein root stilbene active partsIn accordance with Chinese Pharmacopoeia 2010(Volume I),the quality standard of Genistein rootstilbene active parts was studied by 3 batches of middle-scale extract.Genistein root stilbene active parts were identified by traits,identification,inspection(water,residue on ignition,heavy metals,arsenic salt,organic residues),content determination,fingerprint.According to the experimental results,the quality standard of Genistein root stilbene effective partsextract has been drafted.Study on the stability of Genistein root stilbene effective parts by stress test and accelerate test.The results show that Genistein root stilbene active parts were stabled.3 Study onGeistein root stilbene effective parts tabletsWith the tablet hardness,disintegration time and dissolution of three active components(carasinolB,kobophenol A,(+)-α-viniferin)as the indexes,single factor and orthogonal experiment was used to optimize tablet formulations.The optimized formation process condition of Caragana sinicastilbene tablet were as follows:medicine powder and MCC control about 1:2;50%EtOH as the adhesive;5%CMS-Na as the disintegrator and 0.5%Magnesium stearate as the lubricant.The quality standard of Genistein root stilbene active parts tablets was established,including identification,examination(appearance,weight difference,hardness,disintegration,dissolution),content determination.The results showed that the tablet appearance complete,uniform color and no clutter,the weight difference is less than 5%,hardness was 60～70N,disintegration time was less than lmin,and dissolution of three active components(carasinolB,kobophenol A,(＋)-α-viniferin)was more than 70%.According to the experimental results,the quality standard of Genistein root stilbenetablet has been drafted.Study on the stability of Genistein root stilbene active parts tablets by preliminary stability test and accelerated stability test.The results show that Genistein root stilbene active parts tablets were stabled.4 Study on the oral absorption mechanisms of kobophenol A by Caco-2 cell modelThe absorption mechanisms of kobophenol Awas studied by uptake and transport experiments performed across Caco-2 cell model.The resultsshowed that in the concentration range of 50～30μg·ml-1,the uptake of kobophenol A was positively correlative with incubation time and drug concentration,and high concentration had saturation phenomenon.PH and temperature had significant effect on the uptake,pH7.4 had the greatest uptake amount,and 37℃ was higher than 4℃,P-glycoprotein inhibitors and stilbene active ingredientsfrom Genistein root had no significant influence on cellular uptake of Kobophenol A.Papp is about 10"6cm.s-1,which indicates the absorption of kobophenol A is secondary.These all suggest thatabsorption of kobophenol A in Caco-2cell modelwas active transport.5 Study on the oral absorption mechanisms of kobophenol A by intestinal perfusion modelThe absorption mechanisms of kobophenol A was studied by single pass intestinal perfusion model.The resultsshowed that kobophenol A can be absorbed in whole intestinal segments,but best in ileum;With the increase of kobophenol A concentration,Ka and Papp decreased(P<0.05);Papp of kobophenol A was(1.61～3.60)×10-3cm·min-1 in 4 intestinal segments,which indicates the absorption of kobophenol Ais secondary.The Ka and Papp in high and middle concentration of extract group had no significant difference compared with kobophenol A(P>0.05),but the low concentration of extract group hadsignificant difference(P>0.05).Single pass intestinal perfusion experiment were further confirmed in vivo intestinal Caco-2cell test,that was the absorption mechanisms of kobophenol A in vivo is similar to active transport or facilitated diffusion.In conclusion,we set the Genistein root stilbene active parts as the research object,carried out a detailed study onthe preparation process,quality standard and stability of Genistein root stilbene active parts and its preparation.Study on the oral absorption mechanisms of kobophenol A by Caco-2 cell model and intestinal perfusion model.All of these studies provide experimental and theoretical basis for the development of oral preparation.更多还原