【作者】 吴金凤；

【导师】 李学刚；

【作者基本信息】 西南大学 ， 药用资源化学， 2012， 博士

【摘要】 随着“吸烟与健康”的矛盾日益显著,低危害卷烟的开发越来越受到烟草研究者的广泛关注。我国拥有丰富的中草药资源及深厚的中医理论基础,运用中草药减轻卷烟烟气对健康的危害已成为发展低危害中式卷烟的特色之路。本论文通过筛选具有降低卷烟烟气中氰化氢作用的中草药,研制出黄连生物碱复合卷烟滤嘴,并研究了该复合滤嘴对卷烟烟气中其它有害成分的影响；采用离体实验研究了黄连生物碱复合滤嘴降低卷烟烟气对口腔过氧化物酶活性的抑制作用机理；通过检测经烟气熏染的人支气管上皮细胞(BEAS-2B)氧化应激反应,研究了黄连生物碱添加在滤嘴中抑制由卷烟烟气刺激引用的细胞氧化应激。实验方法与结果如下：1.植物生物碱的筛选及提取采用乙醇浸提法对大量中草药进行生物碱提取,使用Dragendorff试剂、Mayer试剂、Wagner试剂等生物碱检测试剂鉴定,筛选出中草药醇提物经去脂处理后含生物碱的10种中草药,分别为黄连、黄柏、川贝母、荷叶、粉防己、八仙草、延胡索、百部、萝芙木和三尖杉,将提取到的以上10种中草药的生物碱以30mg/cig的添加量添加到卷烟滤嘴中,与空白卷烟对照,黄连生物碱以18.3%的清除率成为10种中草药生物碱中对氰化氢清除效果最好的植物生物碱。采用大孔树脂和硅胶柱等天然产物分离纯化技术对黄连生物碱进一步分离纯化。经HPLC鉴定,分离纯化后制得的黄连总生物碱及其单体小檗碱、巴马汀和黄连碱的纯度都在95%以上。2.黄连生物碱复合滤嘴对卷烟烟气中有害成分的影响通过黄连生物碱载体的选择,确定将活性炭纤维(ACF)作为黄连生物碱的载体,经吸附参数优化后,制定出ACF负载黄连生物碱的最佳工艺,并设计了一种黄连生物碱复合滤嘴的制作方法。采用国家标准和行业标准对卷烟烟气进行分析,用15mg ACF负载1.5mg黄连生物碱添加在滤嘴中制成的黄连生物碱复合滤嘴可分别降低卷烟烟气中氰化氢和苯并[a]芘含量20%以上,对烟气自由基含量清除率达50%以上。与同牌号的醋纤滤嘴卷烟进行烟气感官对比评吸,黄连生物碱复合滤嘴可增加烟气细腻柔和程度,对卷烟感官品质没有负面影响。3.黄连生物碱降低卷烟烟气中有害成分的机理研究运用离体清除实验对黄连生物碱复合滤嘴的减害机理进行研究,结果表明,黄连生物碱复合滤嘴可清除氰化钾溶液中的氰离子58.7%,而醋纤滤嘴材料和ACF材料对同浓度氰化钾溶液中氰离子的清除率仅为5.3%和17.4%；采用苯并[a]芘荧光淬灭实验研究黄连生物碱单体对苯并[α]芘的作用,结果表明,黄连生物碱单体的浓度对苯并[α]芘荧光淬灭呈线性相关,相关系数为0.991-0.998,结合常数为2.0×103～9.0×103,三种黄连生物碱单体对苯并[α]芘的荧光淬灭作用强度为巴马汀>小檗碱>黄连碱；在黄连生物碱清除自由基的离体实验中,黄连生物碱表现出对DPPH自由基、氮氧自由基、超氧自由基的清除活性,其抗氧化活性的大小顺序为：巴马汀>黄连总生物碱>小檗碱>黄连碱。4.黄连生物碱抑制卷烟烟气引起的口腔过氧化物酶活性降低的作用通过卷烟烟气对唾液的熏染实验,研究了黄连生物碱添加在滤嘴中对烟气抑制口腔过氧化酶(OPO)活性的影响,并在唾液中分别添加抗氧化剂、烟气成分、螯合剂等成分,以及唾液透析法研究了卷烟烟气中抑制OPO活性的主导因子。实验结果表明,与醋纤滤嘴卷烟比较,黄连生物碱复合滤嘴可降低卷烟烟气对OPO活性的抑制率25%以上；抗氧化剂、单支卷烟烟气中主要羰基化合物和烟碱的量对OPO活性都无显著性影响,通过在唾液中添加氰离子、螯合剂以及透析法说明氰离子是烟气中抑制OPO活性的主要因素；黄连生物碱复合滤嘴通过降低卷烟烟气中的氰化氢而降低了烟气对OPO活性的抑制作用。5.黄连生物碱复合滤嘴减轻卷烟烟气对细胞的氧化损伤选用人支气管上皮细胞(BEAS-2B),研究了卷烟烟气对细胞氧化应激损伤的影响。MTT实验结果表明,醋纤滤嘴组、ACF复合滤嘴组和黄连生物碱复合滤嘴组的烟气溶液(CSS)对BEAS-2B细胞的半致死量LD50分别为5.21×10-3、6.32×10-3、7.26×10-3cig/mL;细胞ROS水平及MDA含量测定结果表明,与醋纤滤嘴组和ACF复合滤嘴组比,黄连生物碱复合滤嘴可降低卷烟烟气对BEAS-2B细胞ROS水平的增高及导致的脂质过氧化。黄连生物碱复合滤嘴组CSS对BEAS-2B细胞的总抗氧化能力、过氧化氢酶(CAT)和谷胱甘肽还原酶(GR)的活性具有诱导作用,对总SOD活性具有抑制作用,与对照卷烟组比较,黄连生物碱复合滤嘴可减轻卷烟烟气对细胞的氧化应激损伤。更多还原

【Abstract】 The health hazards caused by smoking cigarettes have been established, and the development of less harmful cigarette has been focused on by the researcher of tobacco industrial. Due to the foundation of application of Chinese herb as medicine for thousands of years and the rich resources of herb in China, the application of Chinese herbal medicine and its active substances in making less harmful Chinese cigarette has been researched as the special way to develop Chinese cigarette industry. The objective of this paper was to choose herb with the effect on reducing hydrogen cyanide in the cigarette smoke (CS), and coptis alkaloids were standout. The coptis alkaloids were added in the cigarette filter to make coptis alkaloids filter, and the effects of coptis alkaloids filter on the harmful components in the CS were studied. The effect and mechanism of coptis alkaloids filter on reducing the inhibition of CS on oral peroxidase (OPO) activity were researched. The coptis alkaloids filter cigarettes were compared with cellulose acetate filter cigarette, and the effects of CS from different filter cigarette on oxidative stress were also studied. The methods and results were shown as follows:1. The selection and extraction of plant alkaloidsThe extraction of plant alkaloids were used ethanol, and the extractions were determined by Dragendorff reagent, Mayer reagent and Wagner reagent. The alkaloids of Coptis chinensis Franch, Amur Corktree Bark, Fritillariae Cirrhosae Bulbus, Lotus Leaf, Radices stephaniae tetrandrae, Tender Catchweed Bedstraw Herb, Rhizoma Corydalis, Radix Stemonae, Rauwolfia and Cephalotaxus sinensis were chosen to added in the filter with30mg/cig. The results showed that smoke from cigarettes with filter containing coptis alkaloids had less hydrogen cyanide by18.3%, which was compared with the control CS. Coptis alkaloids were chosen as active component to reduce hydrogen cyanide among the10kinds of herb. The Coptis alkaloids were extracted and isolated by resin column and determined by HPLC. The total alkaloids and monomer of Coptis alkaloids were obtained with purity of over95%.2. The effect of coptis alkaloids multi-filter on the harmful compounds in the CSCompared to different carrier of Coptis alkaloids, activated carbon fiber (ACF) was superior as Coptis alkaloids’carrier to make modified filter. The Coptis alkaloids multi-filter was composed by cellulose acetate part and15mg ACF part containing1.5mg Coptis alkaloids. The cigarettes were smoked by machine under the ISO standard. A series of smoking runs was undertaken. The smoke was collected and groups of constituents were measured in the smoke. The content of hydrogen cyanide (HCN) and benzo[a]pyrene (B[a]P) in the Coptis alkaloids multi-filter group were over20%less than the control group respectively. And the free radicals in the Coptis alkaloids multi-filter group were over50%less than the control one. Little effect on smoking quality was perceived.3. The mechanism of the reduction of harmful components in the CS by addition of coptis alkaloids in the cigarette filterThe multi filter containing Coptis alkaloids was used to scavenge cyanide ion in the solution of potassium cyanide. The reductions of cyanide ion were observed by58.7%with multi filter containing coptis alkaloids,5.3%with cellulose acetate filter and17.4%with ACF. The interactions of Coptis alkaloids with B[a]P were investigated by fluorescence quenching. The correlation coefficients of Coptis alkaloids and B[a]P were0.991-0.987, and the binding constants were(2.0-9.0) x103. The reduction trend for B[α]P by coptis alkaloids was Palmatine> Berberine> Coptisine. Spectrophotometry was used to examine the scavenging effect of Coptis alkaloids on DPPH free radicals, nitric oxide free radical and superoxide anion radical. In all the models tested, Coptis alkaloids showed their ability to scavenge the free radicals, and the ability to scavenge were Palmatine> Total Coptis alkaloids> Berberine> Coptisine.4. Reducing the inhibitory effect of cigarette smoke on the activity of oral peroxidase by addition of Coptis alkaloids in cigarette filterThe effect of Coptis alkaloids which were added in the cigarette filter on the activity of oral peroxidase (OPO) was studied by exposure of saliva to the CS. The mechanism of inhibition of OPO by CS was investigated by adding aldehydes, nicotine, cyanide, which were present in CS, to the saliva. The antioxidants, cyanate chelator and dialysis were also applied to saliva in vitro in the presence and absence of CS. The results showed cyanide was likely to be the species in CS responsible for the loss of salivary OPO activity. The reduction in HCN might be related with Coptis alkaloids in the cigarette filter which reduce the inhibition on OPO activity by CS over25%.5. The inhibitory effect of Coptis alkaloids in the cigarette filter on oxidative stress of cell caused by the CSBEAS-2B cells, a model of normal human bronchial epithelial cells, were investigated on their oxidative stress (OS) caused by CS. MTT assay results showed that LD50values of cellulose acetate filter group, ACF multi-filter group and Coptis alkaloids multi-filter group were5.21×10-36.32X10-3、7.26×10-3cig/mL, respectively. Compared to the control CS group, the level of intracellular reactive oxygen species (ROS) and lipid peroxidation (MDA) caused by CS from Coptis alkaloids multi-filter group were much lower. The total antioxidant capacity, catalase activity (CAT) and glutathione reductase (GR) of Coptis alkaloids multi-filter group were induced, but the total activity of SOD was reduced. Compared with the control CS group, CS from cigarette with filter containing Coptis alkaloids could reduce the OS damage by CS.更多还原