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石榴皮多酚大孔树脂纯化工艺及其抗炎机制研究
来海中
学位类型博士
导师阿吉艾克拜尔·艾萨
2017-06-03
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词石榴皮 多酚 大孔吸附树脂 动力学 热力学 放大研究 药理实验 抗炎机制
摘要

石榴皮(Pericarpium Granati)为石榴科植物石榴Punica granatum L.的干燥果皮,收载于《中国药典》2015版一部。石榴原产西域(指敦煌以西诸国),汉代传入中国,在我国很多地方都有栽培,以新疆、河南等地为多。石榴皮作为药材炮制始载于《雷公炮炙论》,在稍后的《本草经集注》、《本草图经》、《本草衍义》、《本草纲目》等历代本草多有记述。中医认为石榴皮味酸、涩、性温、有小毒。归大肠、肾经。传统中医用来治疗久泻,久痢,便血,脱肛,滑精,崩漏,带下,虫积腹痛,疥癣等疾病。石榴皮富含多酚,石榴皮多酚能够通过化学键与细胞表面膜蛋白质结合或者借助疏水键镶嵌于脂质双分子层,从而具有广谱抗菌、抗病毒、驱虫等药理作用,可以治疗腹泻、痢疾等胃肠道疾病及口腔溃疡、龋齿等疾病;具有多羟基酚结构,因此具有较强的抗氧化、抗自由基的作用,具有治疗心脑血管疾病(高血压、冠心病、脑卒中等)、糖尿病、恶性肿瘤等疾病的作用。石榴皮约占石榴重量5-8 %, 石榴皮干物质含多酚10-20 %,新疆现有石榴种植面积大约50多万亩,按年产10万吨计算,新疆每年产生5-6千吨石榴皮,目前大多被废弃。如果其广谱抑菌作用及抗氧化作用得到合理的开发和利用,将产生很好的社会效益和经济效益。为此本研究以开发石榴皮多酚治疗妇科炎症的5类新药为尝试,开展了石榴皮多酚大孔树脂纯化工艺及其抗炎药理作用及机制的研究,其结果如下:1. 通过热力学方程和动力学模型开展了HPD-300大孔树脂对石榴皮多酚的静态吸附研究,发现在本研究设定的温度和实验条件下,Langmuir等温方程能较好地描述HPD-300树脂对石榴皮多酚的吸附过程,其等量吸附焓变ΔH、自由能变ΔG及熵变ΔS在低浓度均为负值,说明反应可以自发进行,等量吸附焓变的绝对值在20 kJ·mol-1以下,说明HPD-300树脂对石榴皮多酚的吸附属于物理吸附过程。其自由能变ΔG的绝对值在3 kJ·mol-1之间,说明石榴皮多酚较易从HPD-300树脂洗脱。Kannan粒子内扩散模型与Lagergren一级动力学模型显示其吸附受到膜扩散和颗粒内扩散影响,控制步骤为颗粒内扩散过程。理论平衡吸附量为65.64 mg/g,实测值为64.58 mg/g (湿树脂),比较接近,说明本热力学动力学研究对实际有一定的指导意义,可以为进一步放大研究提供一定的基础数据和参考。2. HPD-300大孔树脂对石榴皮多酚的动态吸附研究按国家食品药品监督管理局关于大孔树脂的使用要求,对HPD-300大孔树脂动态吸附石榴皮多酚进行了工艺考察,得到吸附多酚较佳的条件为:上样浓度为5 g/L,上柱流率2 BV/h,除杂用水量为4 BV,洗脱剂采用60 %的乙醇,用量为4 BV。经过提取工序,多酚的含量为32.90 %,转移率为72.29 %;鞣花酸含量为0.35 %,转移率为83.92 %;经过纯化工序,多酚的含量提高到了71.68 %,鞣花酸的含量提高到4.78 %,纯度提高13.7倍。得膏率为9.67 %-11.47 %。3. HPD-300大孔树脂提取、纯化石榴皮多酚中试放大研究按新药注册法对中试工艺的的要求,我们在小试及工艺优化的基础上进行中试放大,设计并新建了符合 2010 版 GMP 要求的全自动提取及纯化生产线,目前已规模化生产 3 批,3 批大孔树脂多酚含量变化均不大,石榴皮粗提物多酚得率达到32.90 %以上,鞣花酸得率达到0.35 %以上,干膏重量为52.56 kg,石榴皮多酚的转移率为72.29 %,为准确鉴定中试工艺所得石榴皮粗提物和小试工艺的吻合性,对石榴皮中四种主要组分(柯里拉京,紫云英苷,没食子酸和没食子儿茶素)进行了标准品制备且用高频率核磁共振进行了结构鉴定,连同安石榴苷、安石榴林、鞣花酸,分别用于测定中试和小试级别大孔树脂工艺所得石榴皮提取物中七种多酚的含量。结果显示,中试工艺得到的石榴皮提取物,七种多酚类化合物的含量与小试工艺中的含量高度接近,直接证明了小试至中试工艺转化的成功。根据已有的文献报道表明以上七种化合物都具有较好的抗氧化性及抗炎活性,意味着这七种化合物不仅是石榴皮提取物的主要组分,亦是潜在的活性组分,为其生物活性机制提供了研究基础和借鉴作用。而七种主化合物的总含量测定,继药典记载的以鞣花酸为标记物的多酚类含量测定方法外,亦有望成为石榴皮提取物鉴定合格的重要指标之一。上述实验结果表明此方法操作简便,适用性强,稳定可行,适合于工业化生产。4. 石榴皮多酚抑菌抗炎药理学试验及抗炎机制的研究本研究证实石榴皮多酚能够抑制由二甲苯引起的小鼠耳廓肿胀和由角叉菜胶引起的大鼠足跖肿胀及大鼠棉球肉芽肿的形成,还能够提高豚鼠的致痒阈,减轻阴道炎模型家兔的病变,对霉菌生长有抑制作用。实验结果表明石榴皮多酚对妇科炎症动物模型有很强的抑菌抗炎效果。本研究还揭示了在Raw264.7细胞中石榴皮多酚对脂多糖 (LPS) 引起的炎症反应的作用效果和机制。研究表明,其发挥抗炎作用可能通过抑制促炎介质和细胞因子在体外脂多糖激活巨噬细胞介导中产生,抑制NF-κB 的活性激活蛋白激酶和p38丝裂原活化蛋白激酶的信号通路。本研究首先对HPD-300大孔树脂纯化石榴皮多酚的规律进行了研究,在热力学、动力学研究的基础上,进行石榴皮多酚小试、中试放大工艺的研究,优化了石榴皮多酚的纯化工艺参数,这种生产规模可以满足新药临床研究所需,这种放大接近工业化生产,为将来新药上市质量及生产工艺的一致性提供了保障;同时进行了石榴皮多酚抑菌抗炎动物实验并对其抗炎机制进行了研究,为石榴皮多酚开发为治疗妇科炎症的5类新药提供了主要药效学和药理学支持;不管是石榴皮多酚粗提物还是纯化物,我们都建立了制备工艺和质量标准,可以作为抗氧化、降糖、降脂、辅助治疗心脑血管的保健品原料和新食品原料、标准提取物供应市场,也可在化妆品领域作为美白及抗紫外线辐射的功能性添加剂使用。本研究为石榴皮资源在医药、保健品、化妆品等健康领域的应用和工业化开发提供有益的借鉴。

其他摘要

Pericarpium Granati, recorded in 2015 edition of Chinese Pharmacopoeia, is the dry peel of Punica granatum L. which are sour, warm and astringent when is regarded as a medicine that having function including astringent intestinal antidiarrheal, hemostasis and deworming. In traditional Chinese medicine, it is used to cure lasting diarrhea, dysentery, blood in the stool, prolapse of the anus, uterine bleeding, leucorrhea and insect plot abdominal pain.Polyphenols are main active ingredient in Pericarpium Granati having various biological activities such as antioxidant effect, broad-spectrum antibacterial, inhibited virus and microorganism, anti-cancer, anti-aging and suppression of cholesterol. The total content of polyphenols is approximately 10-20% in Pericarpium Granati which account about 5-8% in Punica granatum L. Pericarpium Granati is produced 5-6 kiloton per year due to the planting area of Pomegranate is up to 50 million mu in Xinjiang province. Most of them were abandoned and needed rational exploitation, which would generate considerably social and economic benefits.The systematic study on polyphenols, the active composition of pomegranate peel, would provid scientific basis for the exploitation of Xinjiang pomegranate industry.Four parts of this thesis are detailedly discussed as following:1. on the study of adsorption of polyphenols of Pericarpium Granati on HPD 300 macroporous resin. It is found that the adsorption of pomegranate polyphenols on HPD 300 macroporous resin is similar to that of multi-molecular layer and the isothermal adsorption process is descripted well by Freundlich equation. The adsorption process belongs to spontaneous physical adsorption confirmed by the adsorption enthalpy change (ΔH) ranging from -75 to -5 KJ.mol-1 and the free energy (ΔG) ranging from -5 to -2 KJ.mol-1. The entropy change (ΔS) is ranging from -0.08 to -0.01kJ.mol-1K and the characteristic separation coefficient RL is ranging from 0 to 1. The control step is the membrane diffusion process because of the membrane diffusion and in-particle diffusion being effected by adsorption which is revealed by Kannan particle diffusion model and Lagergren first order kinetic model.2. The study of dynamic adsorption and desorption of pomegranate polyphenol on HPD 300 macroporous resin.According to the requirements of Chinese Food and Drug Association (CFDA), dynamic adsorption of the total polyphenols contained in extract of Pericarpium Granati on HPD 300 macroporous resin was investigated. The optimum condition was listed as follows:, sample volume was 50mL-sample/mL-resin, the loading concentration was 1.5g/L, the column flow rate was kept at 2.5BV/h, the amount of impurity eluent-water was 4 BV, the eluent was 40% ethanol aqueous solution while the dosage was 6BV. The content of total polyphenol increased from 27% to 68.73% and the yield of extract was 34.80%.3. The study of industrialization of polyphenols enrichment from pomegranate peel by HPD 300 macroporous resin.In order to meet the national requirement to develop new drugs with the technology based on macroporous resin, through the small test and basic research and pilot enlargement and verification, the industrialized macroporous resin purified production line matched the standard of GMP were designed and installed. Three batches of large-scale production were generated and the variation of content of polyphenols was kept at a satisfactory level (RSD<1.98%), which proved that the process is stable. To test the coordination of purification technology of small and pilot scale, four kinds of polyphenols including corilagin, astragalin, gallic acid and gallocatechin were prepared and the structure was identified by NMR. Together with punicalagin, punicalin and ellagic acid, the content of seven main polyphenols were then tested being quantified with these standards. The results indicated that the contents of these compounds were highly similar in both small and pilot scale, suggesting that the successful transition of technology from small to pilot scale. Compared to the earlier reports, all of these compounds showed positive anti-oxidant and anti-inflammatory activities, indicating that these compounds were not only the main components, but also the active components, which might present the potential usage in studying the mechanism of bioactivity of pomegranate peel extract. Furthermore, the quantitative study of the seven main compounds was expected to be an important index for evaluating the crude extract prepared. 4. The study of anti-inflammatory effect of pomegranate peel extract on RAW 264.7 cells by suppression of AKT/GSK-3β and NF-κB signalingThe present study aimed at examining and explaining the effect and mechanism of the lipopolysaccharide (LPS) inflammatory response of pomegranate peel extract (PGE) on RAW 264.7 cells s. PGE effectively decreased the production of pro-inflammatory mediators and cytokines. The expression of NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced macrophages significantly decreased at both mRNA and protein levels with the pretreatment of PGE. The inhibition effect of PEG on LPS-induced activation of NF-κB in macrophages was also revealed. Moreover, the degradation of IκBα and NF-κB activity but also the phosphorylation and activation of AKT, JNK and p38 MAPK was inhibited by PEG. Thus, it could be concluded that PEG exerted the anti-inflammatory effects through the suppression of pro-inflammatory mediators and cytokine production in LPS-stimulated macrophages mediated by inhibition of NF-κB activation through AKT, JNK and p38 MAPK signaling pathways. Abbreviations: PEG, pomegranate peel extract; LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; PI3K, phosphoinositide-3-kinase; MARK, mitogen-activated protein kinases; NF-κB, nuclear factor-κB; IκBα, inhibitory factor kappa B alpha. AKT, protein kinase B; JUK, cJun Nterminal kinaseThe results of this research provide the auxiliary for the application of macroporous resin for the purification of polyphenols. According to the requirements of the new drug registration act, we completed the industrialized purification process and amplification of macroporous resin. By designing small test and pilot test, the production line with separation process of macroporous adsorption resin was installed successfully. This provides useful reference in industrial development and application.

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4998
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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来海中. 石榴皮多酚大孔树脂纯化工艺及其抗炎机制研究[D]. 北京. 中国科学院大学,2017.
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