【作者】 何燕；

【导师】 赖小平；

【作者基本信息】 广州中医药大学 ， 中药学， 2011， 博士

【摘要】 落新妇苷是一种二氢黄酮类化合物,可以作为一种新的免疫抑制剂用于免疫相关疾病的治疗,具有巨大的社会利益和经济价值,其研究对医药领域有着重要的意义。但落新妇苷为水难溶性药物,不易被机体吸收,生物利用度低,在临床应用上受到了一定限制。因此,如何增加落新妇苷溶解度及生物利用度是扩大其临床应用的重点也是目前药剂学研究的难点之一。本文旨在运用固体分散技术改善落新妇苷溶解度及生物利用度,为扩大落新妇苷应用提供依据。关于此方面的内容,目前国内外少见报道。1、落新妇苷提取分离纯化工艺研究本文对落新妇苷的提取纯化工艺进行了较系统的探讨和研究。首先以土茯苓总黄酮、落新妇苷的含量为指标,考察乙醇浓度、提取时间、提取次数、溶剂用量4个因素对土茯苓中落新妇苷的提取工艺的影响。研究结果表明,各因素对试验结果的影响的大小顺序为乙醇浓度>提取时间>提取次数>溶剂用量。确定最佳提取工艺条件为12倍量60%乙醇溶液回流提取2次,每次2小时。土茯苓中提取的落新妇苷并不稳定存在于土茯苓中,而因不同来源、产地、药材的不同而有差异,土茯苓中落新妇苷含量差异为1.1-25.13mg/g（0.11%-2.51%）。然后应用大孔吸附树脂分离纯化落新妇苷,通过测试它们对落新妇苷黄酮的吸附率、吸附速率、解吸率和产物得率及其黄酮含量,研究6种不同类型吸附树脂对土茯苓中落新妇苷黄酮的吸附特性。结果发现HPD400树脂对落新妇苷黄酮吸附量大,吸附快、解吸容易,吸附后产物黄酮含量高。可见HPD400、AB-8弱极性树脂是一种性能良好的土茯苓黄酮吸附剂,可用于从土茯苓提取物中富集总黄酮和落新妇苷,制备含量达78%以上的总黄酮和35%以上落新妇苷。样品经聚酰胺柱层析,用纯化水、20%、40%、60%乙醇洗脱,得到落新妇苷含量为80%以上的精制品。聚酰胺对落新妇苷有较好的吸附力,其水洗脱物中几乎不含落新妇苷。虽然95%乙醇可将残留在聚酰胺柱上的杂质洗去一些,但随着吸附与洗脱次数的增加聚酰胺对黄酮的吸附力逐渐下降,洗脱物中落新妇苷的含量均有明显下降。因此应根据需要使用几次后对聚酰胺进行有机溶剂再生处理或更换新的聚酰胺。精制品过葡聚糖凝胶柱色谱经80%甲醇洗脱纯化,制得落新妇苷的纯品,样品经过理化性质和波谱性质的测定、化学结构鉴定,证实得到纯度为95%以上的高纯度的单体化合物落新妇苷。该提取纯化工艺稳定性较好,科学可行。2、落新妇苷固体分散体制备及质量评价本文从提高难溶性药物溶解度的技术入手,证明了将落新妇苷制成固体分散体的可行性和科学性,深入探讨了落新妇苷固体分散体的制备方法和工艺。在处方前研究中,首先建立了完善的分析方法体系,并对原料药的理化性质进行考察,为固体分散体可行性方案的制定提供了科学的依据,此外还建立了完整的固体分散体评价指标。在落新妇苷固体分散体的制备中,采用溶剂法和溶剂-熔融法作为成型技术,以PVP K30/C15、吐温80- PVP K30/C15作为载体材料,制备出的各种溶出度都较落新妇苷原药有显著提高的固体分散体,最终确定处方（落新妇苷：吐温80:PVPK30=1:1.5:3）最优。对优化后的的固体分散体进行存在状态、稳定性等质量评价。通过采用热力学分析法、X晶体衍射、显微镜法等研究方法对固体分散体的物相进行鉴别,发现落新妇苷在固体分散体以微晶态的形式存在。采用影响因素稳定性实验,以高温、高湿、强光照为三个因素,对自制的固体分散体进行质量评价。结果显示固体分散体在常温条件下稳定性良好。因此自制出落新妇苷固体分散体的增溶效果理想、质量稳定、工艺可行。本文以自制不同时间灯盏花素缓释片累积释放百分比数据为例,探讨多种溶出度释药模型拟合的简便方法。结果发现用Excel软件对统计数据进行处理,已经大致全部包含我们平时所需的各种模型,数据准确,图文并茂,且同以往的手工处理和查表计算相比大大加快了数据处理速度,操作简便,便于推广。采用此法对落新妇苷原料药和固体分散体的释药曲线进行拟合,结果表明Weibull方程拟合度最高,落新妇苷、固体分散体分别为0.9944、0.9986。通过增溶及溶出度增加机制探讨,可见固体分散体恰恰是在于载体抑制了已被固体分散法高度分散的粒子聚集趋势（抑晶性）,表面积扩大；另外载体本身对药物的溶出有促进作用,因此固体分散体较原药物的溶解度大大提高,改善了其溶出速率,促进了药物的吸收和生物利用度。3、首次采用Caco-2细胞模型评价固体分散体载体系统对落新妇苷转运影响研究建立了Caco-2细胞模型。细胞生长良好均匀,边界清晰,细胞间形成紧密连接且完整。跨膜电阻随着培养时间的增加而增大,在培养21天后TEER>500Ω·cm-2,已形成完整的细胞单层。AP侧与BL侧的碱性磷酸酶活性（ALP）分别为46.371±5.902、5.286±0.86,腔面的碱性磷酸酶是基底侧的8.77倍,两者有显著性差异（P<0.01）,说明细胞生长已经形成了极性,碱性磷酸酶已经大部分集中于刷状缘一侧（AP侧）,即朝向培养基的一侧.普萘洛尔的Papp值为（2.193±0.170）×10-5 cm/s（参考值为（2.397±0.630）×10-5 cm/s）。该模型具备单细胞层紧密性与完整性,完全符合药物转运实验的要求,可用以模拟体外小肠吸收特征的研究。对于落新妇苷和固体分散体,细胞存活率随着药物浓度的增加逐渐下降,细胞增殖抑制作用明显增强,呈现剂量依赖性。在5～400μg/mL浓度范围内,两者的细胞存活率在80%以上,固体分散体的细胞存活率均比原料药的低但无显著性差异,5-400μg/ml是Caco-2细胞的安全浓度范围。本研究运用Caco-2细胞模型考察了固体分散系统中PVP的浓度,PVP和表面活性剂（Tween-80）共存等因素对落新妇苷吸收的影响。不同剂量落新妇及自制固体分散体中药物的表观渗透系数随着剂量的增加而增加,不同落新妇苷用量对表观渗透系数的影响无显著性差异,固体分散体中不同落新妇苷用量对表观渗透系数的影响有显著性差异（P=0.005）。载体及表面活性剂对药物的表观渗透系数有显著性影响,PVP k30促进药物渗透效果最佳（P=0.017）,FEG4000、F68作用不明显。固体分散体载体用量对药物的表观渗透系数的影响有显著性差异（P=0.006）,载体量增加到一定程度,药物表观渗透系数不再增加,其中药物：载体为1:3时促进药物通透效果最佳,药物：载体为1:6时次之,药物：载体为1：1时最差。载体联用表面活性剂能显著改善药物表观渗透系数（P=0.008）,用量增加到一定程度,表观渗透系数反而下降,其中表面活性剂用量为1.5时最佳,表面活性剂用量为3时次之,表面活性剂用量为0时最差。固体分散载体类型、载体与药物比例、表面活性剂是影响落新妇苷表观渗透系数的关键因素。可见将落新妇苷制成固体分散体能改善药物渗透,提示可能提高药物体内吸收。5、首次对落新妇苷及其固体分散体的beagle犬体内药物动力学研究本试验采用液相色谱一串联质谱（LC/MS/MS）分析方法测定血浆样品中落新妇苷含量,并进行了方法确证。落新妇苷和内标葛根素[M-H]-去质子峰分别为m/z 449.0和m/z 414.5,落新妇苷和内标葛根素生成的主要碎片离子分别为m/z 284.9和m/z266.9,将其作为定量分析时监测的产物离子。本方法具有快速、准确、灵敏度高的特点,落新妇苷和内标葛根素的保留时间分别为1.7 min和1.8 min,线性范围为2.5ng/ml-1000 ng/ml,定量下限为2.5 ng/ml,适合测定人血浆中落新妇苷的浓度,并成功应用到落新妇苷制剂的犬体内药代动力学研究。空白血浆中的内源性物质不干扰落新妇苷和内标葛根素的测定,精密度（RSD）均小于7.7%,相对偏差（RE）小于12.0%,准确度符合相关规定,落新妇苷在血浆浓度为5.0、50.0、500.0 ng/ml时的提取回收率分别为81.6±9.3%,82.2±3.6%,80.0±4.5%,内标的提取回收率为86.9±4.5%,低、中、高三种浓度下的基质效应分别为93.4±5.8%,96.1±3.4%,97.8±7.9%,内标的介质效应为102.1±3.2%,符合相关规定。本方法具有快速、准确、灵敏度高的特点,此分析方法符合中华人民共和国药典人体生物利用度指导原则和生物样品分析国际规范的有关要求,适合测定人血浆中落新妇苷的浓度,并成功应用到落新妇苷制剂的犬体内药代动力学研究。通过Beagle犬给药后学药浓度参数的比较,首次考察了落新妇苷及其固体分散体在Beagle犬体内的药动学行为及其生物利用度,血药浓度经时过程可经非隔室模型描述。原料药Tmax、Cmax、AUC0-t、Ti/2、Ke分别为1.667±0.258（h）、71.050±34.251（ng/mL）、133.186±49.071（ng/mL·h）、3.38±3.19（h）、0.31±0.16（1/h）;固体分散体Tmax、Cmax、AUC0-t、T1/2、Ke分别为2.583±0.665（h）、122.550±52.740（ng/mL）、298.642±173.673（ng/mL-h）、1.106±0.218（h）、0.65±0.12（1/h）。这部分研究内容充实了落新妇苷药代动力学方面的资料,为进一步临床药代动力学研究提供了参考依据。药动学方差分析结果表明,原料药及固体分散体之间AUC0-t、Tmax、Cmax间均有显著性差异,T1/2、Ke间均无显著性差异。说明同等药物浓度条件下,固体分散技术能显著改善原料药的C_max和生物利用度,延长达峰浓度时间(T_max),但对药物的消除速率常数和半衰期无影响。通过两者Cmax、AUC（0-t）、AUC（0-∞）分别计算相对生物利用度分别为1.8、212.4%、192.0%,提示固体分散技术能提高落新妇苷的生物利用度,落新妇苷固体分散体采用较低的剂量可以达到同样的血药浓度和疗效。更多还原

【Abstract】 Astilbin (Ast,3,3’,4’,5,7-pentahydroxyflavanone 3-(6-deoxy-(L-mannopyranoside))) is a flavanone compound isolated from many plants. In recent years, much attention has been paid to its immunosuppressant activity. But according to the biopharmaceutics classification system, astilbin is an extreme example of a class IV compound due to its low permeability and poor oral absorption, thus it becomes a limiting step in clinical application. This present study aimed to solve the poor solubility of Astilbin, study the effects of dispersion carriers and surfactant on astilbin transport behavior in the caco-2 monolayer model and the pharmacokinetics of astilbin complex dispersion system. The research laid the foundation of further development.1. Extraction and purification research of AstilbinThis systematic research studied the optimum extraction and purification process for Astilbin. The Lg(34) orthogonal experiment was designed with content of flavonoids in Smilax glabra and Astilbin as indicators. The experimental results showed that the influence factors were in the following order:ethanol concentration> extract time> extract number> solvent amount. The optimal extractive conditions obtained was:12 times the amount of solvent,60%ethanol,2 hours for two times. Astilbin content is not stable in Smilax glabra, and differs with different origin, sources and herbs ranging within 1.1～25.13mg/g (0.11%-2.51%).Further, the purification technology was studied by using 6 kinds of macropomus adsorption resin with their Astilbin flavonoids adsorption rate, adsorption rate, desorption rate and product yield and flavonoid content as index. The results showed that resin HPD400 had the largest absorption capacity to Astilbin flavonoids with characters of fast adsorption and easy desorption, and its product had a higer flavonoid content after adsorption. AB-8, a weakly polar resin, is also a good kind of sorbents for Smilax glabra flavonoids, and can be used to enrichment of total flavonoids and Astilbin from Smilax glabra gaining more than 78% content of total flavonoids and 35% above Astilbin.Samples then passes through polyamide column chromatography, eluting with purified water,20%,40%,60% ethanol, obtaining refined products with Astilbin content more than 80%. But with the increase in the adsorption and elution, the adhesive force of polyamide to flavonoids decreased, so it was necessary of the regeneration or replacement of the polyamide after the several uses.Products were further refined through dextran gel column chromatography by 80% methanol elution. The sample was confirmed as the purity compound higher than 95% by determination of physical-chemical and spectral properties and chemical structure identification.The extraction and purification process is stable, easily-operation and suitable for industrial mass production.2. Preparation of astilin solid dispersionIn this paper, the possibility and process of preparation of Astilbin solid dispersion was proved by technicals of improving the solubility of insoluble drugs. In preformulation studies, we first established a sound analysis system, inspected the physical and chemical properties of APIs, and established a complete evaluation indicator of solid dispersion to provide a scientific basis. Researches on the effects of various preparations, carriers, and the ratio between the carriers and drug were carried out. PVPK30/C15 and tweens 80 were used as joint carriers to prepare astilbin solid dispersions by solvent method and Astilin solid dispersion of drug-Tween80-PVP K30 with the proportion of 1:1.5:3 was ideal. The characteristics of astilbin solid dispersions were studied by its in vitro dissolution, solubility, Papp, microscope, X-ray diffraction, and DSC. And experiments on the influence of temperature, humidity and illumination on astilin solid dispersion were also conducted. The results of the microscope, X-ray diffraction, and DSC study showed that astilbin in solid dispersion were molecular or microcrystalline. The content of Astilin in Astilin solid dispersion decreased in high temperature, but which was free from the influence of the humidity and illumination. Solid dispersion can improve the dissolution and solubility of astilin, obviously changed Papp and then promote the in vivo absorption. Astilin content in solid dispersion decreased in high temperature but was free from the influence of the humidity and illumination and it can be kept in normal temperature.The use of Excel software in models fitting of in vitro drug release was discussed, with self-prepared sustained-released Breviscapine tablets as an example. It takes less than lh of time to complete using Excel, containing all of the various models, and the data is accurate and illustrated. The method is easily operated and promoted. This simple data processing method was applied in Astilbin and its solid drug release curve fitting. The results showed that the Weibull equation was highest-fitting with the r of Astilbin, solid dispersions 0.9944, 0.9986 respectively. By mechanism discussion of solubilization, we can see exactly that the solid dispersion carriers suppressed the aggregation trend of highly dispersed particle (suppressing crystalline), and the surface area was expanded. Besides carriers theirselves can promote the dissolution of drug and therefore solid dispersions greatly improved the drug’s solubility, and promote the drug absorption and bioavailability.3. Drug transport in caco-2 monolayerThe established Caco-2 cell were observed good and homogenous growth, Clear boundary, and the formation of tight and complete junctions between cells. TEER values of Caco-2 cell monolayer increased with the incubation time with the value> 500Ω·cm-2 after 21 days of cell culture, indicating the formation of integrate monolayer. The activity of ALP in the AP and BL side were 46.371±5.902,5.286±0.86 respectively with a significant difference (P<0.01). The activity of ALP of the AP side is 8.77 times of the BL side indicating cell polarity had been formed and ALPs were mostly concentrated in the AP side. Papp value of propranolol was (2.193±0.170)×10-5 cm/s with the reference value (2.397±0.630)×10-5 cm/s. The established Caco-2 cell monolayer model was in full compliance with the requirements of drug transport experiments, and could be used to simulate characteristics of intestinal absorption in vitro.For astilbin and its solid dispersion, the cell viability gradually decreased with the increase of the drug concentration, and the cell proliferation was significantly enhanced in a dose dependent manner. The cell viability of both astilbin and its solid dispersion were more than 80% in the concentration range 5～400μg/mL, and latter was lower than that of astilbin with no significant difference. The safety concentration range of astilbin in Caco-2 cell was 5-400μg/mL.This study explored the effect of PVP-tween 80 combined solid dispersion system on the astilbin transportation in Caco-2 cell model. Papp of the drug and drug in solid dispersion increased with dose, and Papp of the latter was significantly different (P=0.005). As to the effects of carriers to promote drug penetration (P=0.017) PVP k30 was best, while PEG4000 and F68 not significant. The effects of drug/carrier ratio on the Papp of astilbin were of significant difference (P=0.006), and the Papp of astilbin increased with the amount of the carrier in a certain range, in which the ratio of drug/carrier 1:3 was best and the ratio of 1:1 worst. Carrier and surfactant combined system can significantly improve drug apparent permeability coefficient (P=0.008), and the effect of surfactant amount on the Papp was of significantly different, in which the carrier/surfactant ratio 1:1.5 best. The carrier and the surfactant in solid dispersion system were the key factors in astilbin transporting behavior in the caco-2 monolayer model. The apparent permeability coefficient of astilbin in the caco-2 monolayer model was improved, suggesting the improved drug penetration and enhanced drug absorption in vivo by solid dispersion system.4. Pharmacokinetics and oral bioavailability in beagle dogThis report describes a successful development of LC/MS/MS assay for astilbin in plasma. [M-H]-proton peak was m/z 449.0 for astilbin and m/z 414.5 for internal standard puerarin. The main fragments were m/z 284.9 for astilbin and m/z 266.9 for internal standard as the product ion for quantitative analysis. The retention time of astilbin and internal standard puerarin were 1.7 min and 1.8 min respectively and the method shows a good separation of astilbin and internal standard to the plasma constituents. The Standard profile of astilbin in plasma was linear over the concentration range of 2.5-1000|-ig/ml examined. The regression equation for astilbin was Y=-0.0023578+0.000887034*X (r= 0.9816).The precision was determined at three different concentrations (5.0,50.0,500.0 ng/mL). The relative standard deviation (R.S.D.) for every studied concentration was less than 7.7%, relative deviation (RE) less than 12.0%, indicating the precision of the method for routine purposes. The mean extraction recovery was 81.6±9.3%,82.2±3.6%,80.0±4.5% and 86.9±4.5% for astilbin at 5.0,50.0,500.0ng/mL and internal standard respectively. The medium effect was 93.4±5.8%,96.1±3.4%,97.8±7.9% and 102.1±3.2% for astilbin at 5.0,50.0,500.0ng/mL and internal standard respectively. Based on a signal-to-noise ratio (S/N)=10 (R.S.D.<20%), the limit of quantization of astilbin was 2.5 ng/m.L in plasma and the precision of LLOQ concentration of (RSD) was 3.18% (n=6). The method has been applied to pharmacokinetic studies after oral administration of the compound and its solid dispersion to beagle dogs. The sensitivity of this method appeared satisfactory for monitoring the plasma concentration.This is the first study on the pharmacokinetics and bioavailability of astilbin and its solid dispersions in Beagle dogs. The non-compartmental calculation indicated that the rmax of astilbin solid dispersion (2.583±0.665 h) appeared to be longer than that for API (1.667±0.258 h) of significantly different (P< 0.05). The Ke of astilbin solid dispersion (0.65±0.12 h"1) was significantly different from that of astilbin (0.31±0.16 h’1) (P< 0.01). The Cmax of astilbin solid dispersion (122.550±52.740 ng-mL"1) was significantly different from that of astilbin (71.050±34.251 ng-mL-1) (P< 0.05). Astilbin solid dispersion had a faster elimination rate with a smaller tm (1.106±0.218 h) than astilbin (3.380±3.190 h) (P< 0.05). The AUCo-t of astilbin solid dispersion (298.642±173.673 ng-mLT1) was significantly different from that of astilbin (133.186±49.071 ng-h-mL-1) (P< 0.05), while the AUC0-∞of astilbin solid dispersion (305.994±70.640 ng·h·mL-1) and the AUC0-∞of astilbin (160.048±86.874 ng·h·mL-1) was of no significantly difference (P> 0.05). Indeed, calculated on the basis of the AUC0-∞of each formulation, the oral bioavailability of astilbin solid dispersions evaluated by Cmax、AUC(0-t)、AUC(0-∞)was about 1.8、212.4%、192.0% reapectively as compared to astilbin. Comparing the pharmacokinetic parameters and oral bioavailability data indicates the effects of solid dispersions on the oral absorption of astilbin.更多还原