【作者】 何明珍；

【导师】 杨世林；

【作者基本信息】 黑龙江中医药大学 ， 中药学， 2014， 博士

【摘要】 Saponins PD,为五坏三帖皂苷类化合物,存在于白头翁(Pulsatilla chinensis)、洋常春藤(Hedera helix)、黑种草(Nigella sativa)、黄褐毛忍冬(Lonicera fulvotomentosa)等多种植物中,现代药理研究表明,Saponins PD具有抗肿瘤、抗炎、保肝、抗利什曼原虫等药理活性。其抗肿瘤活性越来越多的受到国内外学者的关注。课题组前期预试验发现Saponins PD具有明显抗肿瘤作用,但其口服生物利用度极低(<1%)。基于目前有关Saponins PD的研究多集中在其含量测定、药理及生物活性研究等方面,本课题组以开发Saponins PD抗肿瘤新药为目的,探索其口服生物利用度低的原因,对Saponins PD的提取分离、吸收、药代动力学、体内药物代谢等进行了系统的研究,为进一步开发研究Saponins PD提供科学依据。本文主要研究内容分如下几部分：第一部分Saponins PD的提取分离及鉴定为了获得稳定可靠的提取分离工艺及质量可靠的原料,本文对Saponins PD的提取分离工艺进行了研究,采用硅胶柱层析及重结晶的方法进行Saponins PD的分离制备,再运用核磁波谱技术和HPLC法进行结构的确证和含量测定,得到含量达98%以上的Saponins PD,为后续理化性质、药理活性、药物体内过程评价的研究提供物质保障。第二部分Saponins PD抗肿瘤活性研究为了对Saponins PD的抗肿瘤活性进行筛选,采用MTT法测定了Saponins PD对10种肿瘤细胞的生长抑制活性,结果表明,Saponins PD对10种肿瘤细胞均有一定的抑制作用,与文献报道一致。利用小鼠H22荷瘤、S180荷瘤、Lewis荷瘤实验,初步分析了Saponins PD对小鼠的肿瘤抑制情况。结果表明,灌胃给药高、中、低剂量Saponins PD对H22荷瘤的抑瘤率均达40%以上；高、中剂量对S180荷瘤的抑瘤率达40%以上；高、低剂量对Lewis荷瘤的抑瘤率达40%以上,说明对三种荷瘤均具有一定的抑制作用。腹腔注射高、中、低剂量Saponins PD对Lewis荷瘤有明显的抑制作用抑瘤率分别为72.9%、70.5%、47.7%,与阳性药物顺铂的抑瘤率相当。实验还考察了能够反映机体免疫功能状态的脾脏和胸腺脏器指数以及能反映机体骨髓造血功能的血细胞计数指标,结果表明：Saponins PD在抑制肿瘤的同时对机体免疫系.统影响不大,不会造成类似环磷酰胺那么显著的免疫抑制,对机体骨髓造血功能无明显抑制作用。第三部分Saponins PD理化性质研究Saponins PD的主要理化性质是影响其药效及制剂设计的关键因素,本文采用HPLC法对Saponins PD在常温下的长期稳定性、平衡溶解度和表观油水分配系数进行了测定。采用摇瓶法进行平衡溶解度及表观油水分配系数实验,用高效液相色谱法测定SaponinsPD在水、有机溶剂和缓冲液中的质量浓度。结果显示：常温常压下放置,Saponins PD在16个月内稳定；在水中的平衡溶解度为0.01mg·mL-1,在有机溶剂正丁醇中的溶解性最好,为86.10mg·mL-1;在正辛醇-水中的表观油水分配系数为24.73(logP=1.39),SaponinsPD的油水分配系数在适宜的范围内,但是其水溶性很差,可能会影响Saponins PD的吸收。第四部分Saponins PD及Saponins PD钠盐的在体肠吸收研究大鼠在体肠单向灌流模型是目前研究药物吸收机制应用最多,国际上公认的、经典的、成熟的模型和工具。为进一步探讨SaponinsPD的溶解性对吸收的影响,本节实验对Saponins PD及其钠盐的肠吸收机制进行研究。采用大鼠在体肠单向灌流模型进行实验,利用HPLC法对药物的质量浓度进行检测,考察不同肠段、药物不同质量浓度、药液不同pH、肠道菌群以及P-gp抑制剂对药物吸收的影响。结果表明,Saponins PD在不同肠段的吸收速率常数Ka大小顺序为回肠>结肠>空肠>十二指肠；Saponins PD在碱性环境下吸收较好：在肠道的吸收机制可能为被动扩散；肠道菌群对Saponins PD吸收有显著性影响；Saponins PD不是P-gp底物。对Saponins PD钠盐在不同肠段和不同质量浓度下进行在体肠吸收实验的结果表明,Saponins PD钠盐在不同肠段均有吸收,其吸收速率常数K。大小顺序是空肠>十二指肠>回肠>结肠。Saponins PD钠盐不同浓度下在大鼠空肠的吸收参数没有显著性差异,提示Saponins PD钠盐在小肠的吸收没有浓度依赖性,吸收机制可能为被动扩散。上述研究揭示,Saponins PD肠道吸收较差,制成钠盐后,吸收有所增加,主要吸收部位发生改变,说明药物的溶解性对吸收有影响。同时,通过实验推测化合物本身的水溶性差及肠道菌群的代谢可能是导致其吸收差的重要原因。第五部分Saponins PD、Saponins PD钠盐、Saponins PD环糊精包合物大鼠体内药代动力学研究药物动力学研究对于药物生物利用度、疗效、毒性的评价及由此延伸出的药物剂型设计等方面具有十分重要的作用。为了深入了解Saponins PD在大鼠体内吸收情况以及Saponins PD的药理活性机制,本文对大鼠灌胃和静脉给药Saponins PD、灌胃给药SaponinsPD钠盐和Saponins PD环糊精包合物后在大鼠体内的药代动力学进行全面的考察和研究。首先建立了生物样品中检测Saponins PD的HPLC-MS/MS方法,检测了大鼠血浆中的药物浓度,并进行了药代动力学参数的计算。结果表明,Saponins PD、Saponins PD环糊精包合物、Saponins PD钠盐在大鼠体内的吸收非常慢,吸收总量小,口服生物利用度极低,Saponins PD制成钠盐后口服生物利用度有所提高,说明增加其在水中的溶解度,有利于吸收。第六部分Saponins PD、Saponins PD钠盐、Saponins PD环糊精包合物大鼠体内组织分布研究基于前述体内吸收实验推测,Saponins PD可能与某些组织有特殊结合,为了探索药物进入生物体内在各组织中的分布情况,以及不同的制剂是否会影响体内药物的分布。本文采用HPLC-MS/MS法分别对灌胃Saponins PD、Saponins PD钠盐、Saponins PD环糊精包合物后,大鼠心、肝、脾、肺、肾、脑、肌肉、脂肪中药物的浓度进行检测。结果显示,在检测的8个组织中均有分布,SaponinsPD、Saponins PD钠盐、Saponins PD环糊精包合物的分布趋势基本一致,对肝、肺有一定的靶向性。第七部分Saponins PD大鼠体内排泄研究在药代动力学实验研究中,发现Saponins PD的生物利用度极低,为了寻找生物利用度低的原因,本文采用HPLC-MS/MS法测定大鼠灌胃给予Saponins PD后,不同时间段尿液和粪便中Saponins PD的浓度。结果表明,大鼠灌胃给予Saponins PD后,72h内在尿和粪中的累积总排泄率达77.03%,说明主要以原型药物排泄。第八部分Saponins PD在大鼠体内的代谢研究Saponins PD药代动力学实验研究表明,其口服生物利用度极低(<1%)。但前期抗肿瘤活性实验中,其口服仍能表现出良好的抗肿瘤活性,推测可能某些代谢物具有较强的抗肿瘤活性；另对Saponins PD灌胃后大鼠粪便和尿液的排泄研究发现,其在尿和粪中的总排泄率为77.03%,主要以原型排泄,但有22.97%的Saponins PD则去向不明,可能以代谢物的形式排泄。本文采用UHPLC-TripleTOF-MS/MS对大鼠在体肠孵育样品及灌胃给药后胆汁、尿液、粪便和血液样品进行了分析,推测了Saponins PD在大鼠体内的代谢产物。经分析得到各代谢物的二级质谱图,通过与原型药物的二级质谱图及质谱裂解规律进行比较,结合Peak View软件进行分子式的拟合,共推测鉴定了除原型药物及其同分异构体外的26个代谢产物,其中包括了一些代谢物的同分异构体。从代谢物的结构来看,原型药物在体内主要发生了氧化、还原和结合反应。本文对Saponins PD的提取分离、抗肿瘤活性、理化性质、在体肠吸收、大鼠体内药代动力学及体内代谢产物进行了系统研究,得到了分离Saponins PD的稳定可靠的工艺路线,阐明了Saponins PD的溶解特性,Saponins PD在体肠吸收的方式以及其在大鼠体内的吸收、分布、代谢、排泄的规律,为Saponins PD的进一步研究提供了科学依据。更多还原

【Abstract】 Saponins PD, the pentacyclic triterpenoid saponins is widely distributed in many plants, such as Pulsatilla chinensis, Lonicera fulvotomentosa, Hedera. helix, Nigella sativ’a, etc. Many studies of modern pharmacology revealed saponins PD has a wide spectrum of biological properties such as anti-tumor, anti-inflammatory, hepatoprotective effect and antileishmanial activity. Its antitumor activity has received more and more attention of scholars at home and abroad. Through the preliminary trials, we found that the effect against cancer, but the oral bioavailability very low. So far, based on the research of saponins PD focused on its content analysis, pharmacological and biological activities. In order to develop saponins PD antitumor drugs and explored the causes of low oral bioavailability, we will study the extraction and separation of saponins PD, its absorption, pharmacokinetics, drug metabolism, and will provide scientific basis for the further research and development of saponins PD.The main content of this paper is divided as follows:Part1Extraction, separation and identification of saponins PDIn order to obtain a stable and reliable extraction and separation process and raw material of reliable quality, we investigated the isolation process. This process combined with silica gel column chromatography and recrystallization was used for extracting saponins PD of herbs. We used NMR spectroscopy and HPLC method for the determination of confirmation and content structure, the content is more than98%. It provided the material safeguard for the study of the drug during the subsequent physicochemical properties, pharmacological activity.Part2Studies on antitumor activity of saponins PD In order to study the antitumor activity of the saponins PD, MTT method is used to determine the saponins PD of10kinds of tumor cell growth inhibitory activity. The results showed that saponins PD of10kinds of tumor cells have certain inhibition, consistent with literature reports. Using mouse tumor-burdened H22, tumor-burdened S180and tumor-burdened Lewis in vivo tumor-burdened experiment, preliminarily analyzed the tumor suppressor of saponins PD in mice. The results show that after intragastric administration of low, middle and high dose of saponins PD have a certain inhibitory effect on the three kinds of tumor, the tumor inhibitory rate to H22over40%, the tumor inhibitory rate of S180was40%, the average tumor inhibition rate of Lewis was40%; Intraperitoneal injection of saponins PD of low, middle and high doses has obvious inhibiting action to Lewis tumor. Spleen and thymus are two important immune of organ, the organ index reflect the immune function to a certain extent. This experiment investigated on the mice spleen, thymus index, the results showed that saponins PD in inhibiting tumor at the same time had a little effects on the body’s immune system and no significant immunosuppressive similar as CXT. Blood cell count is an important index to reflect the hematopoietic function of bone marrow, at the same time is also the body inflammation diagnosis reference. Through observed the white blood cells, erythrocytes and platelets in mice, the results show that saponins PD without inhibition on the bone marrow hematopoietic function.Part3Study on the physicochemical properties of saponins PDIn order to investigate the main physical and chemical properties of saponins PD, in this paper, the long-term stability, equilibrium solubility and apparent oil-water partition coefficient of saponins PD were studied. First of all, we established the content determination method of saponins PD. Shake flask method was adopted to improve the equilibrium solubility and apparent oil-water partition coefficient of experiments, used HPLC method for determination of saponins PD in organic solvent and water quality concentration in the buffer. Results displayed that under atmospheric pressure saponins PD stable in16months, the equilibrium solubility in water of0.01mg mL-1, solubility in n-butyl alcohol was best,86.10mg mL-1and in octanol-water apparent.oil-water partition coefficient of24.73(log P=1.39).The oil-water partition coefficient of saponins PD in appropriate range, but the poorly water-soluble may be one of the factors influence drug bioavailability.Part4Saponins PD and saponins PD sodium absorption in the intestineRat single pass intestinal perfusion model is the model and the tool of international mature and classical studies of drug in vivo absorption mechanism. In order to explore the absorption method and absorption properties of saponins PD and saponins PD sodium, In this article, we used the rat single pass intestinal perfusion model and HPLC method to detect the quality of the drug concentration, study drug absorption, parts mass concentration, pH, intestinal flora and the influence of P-gp inhibitors to absorption respectively. The results showed that, Saponins PD at different intestine absorption rate constant Ka and the size of the order as colon ileum> colon> jejunum>duodenum; Saponins PD was well absorbed in alkaline environment; No concentration dependence in the small intestine absorption, and absorption mechanism in intestinal may belonged to passive diffusion; The intestinal flora affected absorption of saponins PD significantly; saponins PD was not a P-gp substrate. Research revealed that the poor intestinal absorption of saponins PD, the saponins PD has low bioavailability was not only concerned with the physicochemical properties of itself, and the intestinal absorption difference has direct correlation, and intestinal flora metabolism was an important factor leaded to the low bioavailability.Part5Study on the pharmacokinetics of saponins PD, saponins PD sodium and saponins PD cyclodextrin inclusion compoundPharmacokinetic study plays a very important role and thus extends evaluation degree, efficacy, toxicity and biological-pharmaceutical dosage form design. In order to understand of saponins PD in rats and bioavailability etc., we studied the pharmacokinetics and bioavailability of rats that after oral administration of saponins PD and administration inject of saponins PD, oral administration of saponins PD sodium and saponins PD cyclodextrin inclusion compound. At first, we establishmented HPLC-MS/MS method for detection of Saponins PD in biological samples, measured the content of drug concentration in rat plasma, and calculated the pharmacokinetic parameters. The results showed that the absorption of saponins PD in rat was very slow, high clearance rate, total absorption, and low bioavailability.Part6Study on the distribution of saponins PD and saponins PD sodium and saponins PD cyclodextrin inclusion compound in tissues of ratsIn order to explore the distribution of the drug into the body in different tissues, and different preparation will affect the distribution of biological drugs. This paper used the HPLC-MS/MS method to detect drug concentration in heart, liver, spleen, lung, kidney, brain, muscle, fat of rat who oral administration of saponins PD, saponins PD sodium and saponins PD cyclodextrin inclusion compounds. The results showed that saponins PD were distributed in 8organizations detection, distribution trend in all preparations were basically the same.Part7Saponins PD excretion of ratsIn the study of dynamical experimental pharmacokinetics, we found that the bioavailabil ity of saponins PD was very low. In-order to find the reasons of low bioavailability, we used HPLC-MS/MS method determined the concentration of saponins PD in different time of urine and feces. The results showed that, the total cumulative excretion was77%of urine and feces in72h after oral administration of saponins PD, and mainly excreted by the prototype drug.Part8Saponins PD in the metabolism of rats in vivoSaponins PD pharmacokinetic studies have shown that the oral bioavailability was very low (<1%). But the early cancer screening experiments, we found saponins PD can still showed good antitumor activity and presumed some metabolites exhibiting significant antitumor activity. The study found that the total excretion of urine and feces of rats after intragastric administration of saponins PD was77.03%, mainly excreted unchanged, but22.97%of saponins PD is unknown and may be excrete as metabolites. We used UHPLC-TripleTOF-MS/MS technology analyzed the samples of in situ incubation and bile, urine, feces and blood samples after oral administration of saponins PD, and speculated the saponins PD metabolites in rats. This study adopted UHPLC-TripleTOF-MS/MS technology analyzed biological samples of rat after oral dosing saponins PD. Analysised the two grade mass spectrum of each metabolite and compared with the original drug mass spectra and fragmentation pattern, combined PeakView software fitted molecular formula, we identified26metabolites in addition to the original drug and its isomers, included some of the metabolites of isomers. From the perspective of the structure of metabolite, prototype drugs mainly the combined, oxidation and reduction reaction in the body.In this paper, we systematic researched the extraction and separation of saponins PD, antitumor activity, the physical and chemical. properties, intestinal absorption mechanism, pharmacokinetics and metabolic products. We obtained the stable and reliable isolation process of saponins PD, clarified the dissolving properties of saponins PD, the way of intestinal absorption in rats in vivo and the’law of distribution, absorption, metabolism and excretion in mice, it will provide scientific basis for the further study of the saponins PD更多还原