【作者】 何晓燕；

【导师】 吴纯洁；

【作者基本信息】 成都中医药大学 ， 中药学， 2014， 博士

【摘要】 槟榔来源于棕榈科植物槟榔Areca catechu L.的干燥成熟种子,具有杀虫消积,行水,降气,截疟的功效,主要用于治疗绦虫病、蛔虫病、姜片虫病、虫积腹痛、积滞泻痢、里急后重、水肿脚气、疟疾等疾病。历代本草记载,在使用本药时,有生、熟之分,传统炮制理论认为：生槟榔力峻,长于治疗虫积、水肿、脚气、疟疾；炒槟榔可缓和药性,以免克伐太过而耗伤正气,适用于夹虚患者；焦槟榔长于消食导滞,用于治疗食积不消、泻痢后重；槟榔炒炭后具有消积止血作用,用于治疗血痢。中国药典2010年版收载了炒槟榔和焦槟榔两个炮制品,《中华药海》收载了槟榔炭的制备方法。目前的已有研究探讨了槟榔生品、炒黄样品以及焦品的生物碱、鞣质、氨基酸、微量元素的含量变化,但单从以上成分含量的变化无法反映出槟榔炮制过程中物质基础的变化,并且未与生物效应研究相结合,无法阐明槟榔炮制的原理。目的运用在线式非接触红外测温系统、自动炒药机、色彩色差计等对槟榔的炒制过程进行实时监控,借鉴动力学思路及方法,结合HPLC、MS、NMR等研究技术,探讨槟榔炒制过程中物质基础与生物效应(药效、毒性)的变化规律及其相互关联性,为槟榔炒制工艺优化及饮片质量标准的制定提供科学依据,阐明槟榔炒制原理,丰富中药炮制的科学内涵。方法1、采用在线式非接触红外测温系统、自动炒药机、色彩色差计等对槟榔的炒制过程进行实时监控,为规范槟榔炒制工艺参数、保证炒制品的质量均一提供可能。2、采用HPLC法测定槟榔炒制过程中槟榔碱、槟榔次碱、去甲基槟榔碱的含量,以及测定醚溶性生物碱和鞣质的含量,探讨槟榔在炒制过程中各化学成分的变化规律,为槟榔饮片及其炮制品的质量标准的制定提供科学依据。3、采用HPLC法,建立槟榔生品至炭品的水溶性成分HPLC指纹图谱,探讨槟榔在炒制过程中指纹图谱的变化规律,找出焦槟榔新增成分,为阐明焦槟榔“长于消食导滞”的传统理论提供科学依据。4、采用硅胶柱、0DS柱、制备型高效等方法对新增成分进行分离,并采用HPLC、LC-MS、1H-NMR法等对新成分进行结构鉴定,并对其生物效应进行评价。5、采用离体胃肠平滑肌收缩实验、在体胃排空、肠推进等实验对槟榔生品及其炒制品、去除生物碱部分、生物碱单体以及新增成分的促胃肠运动作用进行考察,评价生物效应与物质基础的相关性,并进行槟榔生品、炒槟榔、焦槟榔和槟榔炭的小鼠急性毒性试验,阐明槟榔炒制缓和药性的理论。6、探讨炒制过程中物质基础与药效(毒性)随炒制程度(时间)的变化规律。结果1、采用在线式非接触红外测温系统、自动炒药机、色彩色差计对槟榔的炒制过程进行了实时监控,保证了炒制品的质量均一,规范了槟榔炒制的工艺参数,为制定合理、统一、规范的全国性槟榔炮制工艺提供数据支持。2、采用HPLC、MS、1H-NMR等研究技术,揭示了槟榔炒制过程中物质基础的变化规律。通过对比研究三批槟榔炒制过程中槟榔碱、槟榔次碱、去甲基槟榔次碱的含量变化、醚溶性生物碱的含量变化以及鞣质的含量变化,揭示了槟榔主要化学成分的变化规律：槟榔生物碱和鞣质在炒制过程中随着炒制程度的加深而逐渐降低,至炭品生物碱含量微乎其微,槟榔次碱在炒制过程中先降低而后升高再降低,推测可能与槟榔碱的分解有关。建立了槟榔炒制过程中不同炒制品的水溶性成分HPLC指纹图谱,通过对比分析,首次发现了槟榔在炒制过程中新增的3个新成分；指纹图谱中,除新增的三个成分外,其他成分在炒制过程中逐渐降低。以上研究为槟榔饮片质量标准的制定提供数据支持。3、首次采用制备型高效液相色谱法对3个新成分进行了有效分离,并采用HPLC法、UHPLC-MS、TOF-MS.1H-NMR法等对新成分进行结构鉴定,确定新增的三个成分依次为2-羟基-5-羟甲基-2-呋喃甲醛、5-羟甲基糠醛、麦芽酚,并对其进行生物效应评价。4、研究了焦槟榔“长于消食导滞”科学内涵。胃肠平滑肌试验、胃排空、肠推进试验结果表明,槟榔碱是槟榔促胃肠运动的物质基础之一,槟榔碱在0.5×10-4～8×10-4mg/ml范围内对肠平滑肌作用逐渐增强,在0.5×10-4～1.6×10-3mg/ml范围内对胃平滑肌作用显著增强,高于8×10-4mg/ml后对肠平滑肌作用逐渐下降,高于1.6×10-3mg/ml对胃平滑肌作用逐渐下降；槟榔次碱和去甲槟榔次碱无促胃肠运动作用。焦品去除生物碱组具有显著的促胃排空作用和肠推进作用以及焦品的促胃排空和肠推进作用最强表明,在炒焦过程中产生了具有促胃肠运动作用的新物质,使焦槟榔的作用最强。因此,可以确定槟榔促胃肠运动的物质基础为槟榔碱和槟榔炒制后产生的新物质两部分。5、研究了槟榔炒制缓和药性的相关“毒性”。槟榔随着炒制程度的加深,毒性逐渐降低,槟榔饮片(生品)的LD50为65.69g/kg,炒黄后LD50为67.18g/kg,焦品的LD50为71.83g/kg,炭品由于未有小鼠死亡,无法得出LD50,急性毒性大小顺序依次为槟榔饮片(生品)>槟榔炒黄>焦槟榔>槟榔炒炭。6、探讨了有效成分含量变化与生物效应变化的相关性。以炒制时间为横坐标,以槟榔碱含量、药效强度为纵坐标,建立了炒制程度——槟榔碱含量——药效强度曲线图,由图可知,炒焦是槟榔炒制过程中槟榔碱与药效发生显著性变化的拐点；由小鼠急性毒性试验可知,槟榔碱是槟榔的毒性成分,随着炒制过程中槟榔碱含量的逐渐降低毒性逐渐降低。结论：本课题研究从槟榔炒制的全过程和规律性出发,系统揭示了槟榔炒制过程中物质基础(外观性状、化学成分、指纹图谱)与生物效应(离体胃肠平滑肌运动、在体胃肠运动、毒性)的变化规律,为规范、优化槟榔炒制工艺和质量标准的制定提供数据支持,为阐明槟榔炒制原理和丰富中药炮制的内涵提供了科学依据,为其他中药的炮制原理研究提供新的思路和方法借鉴。首次发现了槟榔在炒制过程中产生的3个新成分,成功对其有效分离和结构确证,并对其进行了生物效应评价。槟榔碱是槟榔促胃肠运动的物质基础,但不是唯一的物质基础,槟榔在炒制中产生的某类新成分,与槟榔碱协同作用,使焦槟榔的生物效应远远高于槟榔生品及其他炒制品；槟榔及其炒制品水煎液的急毒试验结果表明,槟榔生物碱是槟榔的毒性成分,槟榔及其炒制品作为药品进行临床使用,安全性较高。创新点1、思路创新——动态观的炒制全过程研究设计与观察本课题立题与思路强调研究炒制以“动态观、全过程、规律性”出发,即从揭示槟榔饮片物质基础与生物效应在炒制过程中的变化规律出发,研究炒制过程中槟榔生品与不同炒制品的物质基础和生物效应以及炒制原理的思路(与现有研究思路相比明显有别)具创新性。2、技术与方法创新一新技术与新方法的运用对于槟榔炒制过程的动态监控及相关数据采集与研究,本课题创新性集成了在线式非接触红外测温新技术、自动炒药机、色彩色差计、HPLC、UHPLC-MS、 TOF-MS、1H-NMR等技术,并借鉴动力学的方法,故本课题研究与应用的技术(与现有的数据采集技术相比明显有别)和方法具创新性。更多还原

【Abstract】 Betel nut is the dry mature seed of Areca catechu L, which belongs to the Palmaceae family, and used in traditional Chinese medicine as the antiscolic, antimalarial drugs, emictory, and agents for removing food retention and descending Qi. In particular, it is used commonly for treating of tapeworm disease, ascariasis, fasciolopsiasis, abdominalgia caused by parasitosis, diarrhea, tenesmus, beriberi edema and malaria. It’s recorded that the effects of raw and cooked betel nut are different in the past dynasties medical books. According to the traditional processing theory, the effects of raw betel nut is powerful and commonly used for the treatment of ascariasis, edema, beriberi, malaria; whereas the effects of fried yellow betel nut were weaker than raw betel nut and was used for patient whose body is weaker.Fried brown betel nut has a strong effect of relieving dyspepsia, which is used for food digestion disorder and tenesmus. Charring betel nut has the effects of relieving dyspepsia and stopping bleeding, which is used for the treatment of blood dysentery.The research has been studied on the variation of the content(such as alkaloid, tannin, amino acid, and trace elements) of crude betel nut, fried yellow betel nut and fried-brown betel nut, but the single change from the above content does not reflect the changes of the material basis in the processing of betel nut, and the study not combining with the biological effect of betel nut, so it fails to explain the theory of betel nut of processing.PurposesSo far, the principle of frying process of betel nut was still vague and the material basis of betel nut was unclear. Therefore, this work is designed to clear the principle of frying process of betel nut and provide scientific basis for formulating the quality standard and optimization process of betel nut, rich scientific connotation of traditional Chinese medicine.Methods1. Using the on-line non contact infrared temperature measurement system (NCTS) to monitor the frying process of betel nut by using automatic frying machine (AFM), and provide process parameters and to ensure the quality of fried products is uniform. 2. Using the HPLC method to determinate the content of arecoline, arecaidine, guvacine, guvacoline of betel nut product and UV for the determination of total alkaloids and tannins, in order to explore the chemical composition changes in the frying process of betel nut and reveal the material of the biological effects of betel nut.3. Using the HPLC to set up the water soluble constituents HPLC fingerprint of betel nut and its fried products, and find out the new components of fried betel nut through the comparative analysis of the above maps, which provided scientific basis for frying.4. Using silica gel column, ODS column, preparative HPLC to separate the new appeared compounds of the processing, and using the method of HPLC, LC-MS,1H-NMR for the identification of structure of new components and to evaluate its biological effect.5. Using the gastrointestinal smooth muscle experiment in vitro, gastric emptying, intestinal propulsion experiments in vivo to evaluate the promotive effects of raw betel nut, raw betel nut without alkaloids, fried-yellow betel nut, fried-dark-yellow betel nut, fried-brown betel nut, fried-brown betel nut without alkaloid, charring betel nut, arecoline, arecaidine, guvacine and new appeared compounds on gastrointestinal movements. Additionally, the correclation between biological effects and material basis was also evaluated, as well as the the acute toxicities of the raw betel nut and its processed products.6. Establishing a kinetic model to quantitative analysis of the changes of index components’contents and biological effects (toxicities) with the dynamic changes of frying degree (time), and calculate the kinetic parameters.Result1. The NCTS for betel nut frying process with AFM standardized the frying process parameters of betel nut and ensure the quality of fried products homogeneity.2. In the frying process, the contents of alkaloids were gradually decreased in the frying process, excepting the contents of arecaine were decreased first, then increased, and decreased finally, and it might be related to the decomposition of the arecoline.3. The fingerprint HPLC map of water soluble components of Betel nut and its fried products were established, and three new appeared constituents were found in the frying process of the Betel nut. What’s more, the3new appeared constituents were isolated and identified as2-hydroxyl-5-hydroxymethyl-2-furan-Formaldehyde.5-hydroxymethyl furfural, maltol. However, the results of our investigation showed that5-hydroxymethyl furfural, maltol have no effect on promoting gastrointestinal movement.4. Gastrointestinal smooth muscle test, gastric emptying, intestinal propulsion test results show that, arecoline is one of the material basis of betel nut on gastrointestinal movement, arecaidine, guvacine has no effect on promoting gastrointestinal movement.Fried-brown betel nut without alkaloid samples showed a significant effect on promoting gastric emptying and intestinal propulsion, and the effect of fried-brown betel nut is the most strong of all, it indicate that some new material had been produced which has the effect of promoting gastrointestinal movement in frying process. Therefore, the material basis of betel nut on gastrointestinal movement are arecoline and the new material which produced in the.frying process of betel nut.5. Color of betel nut was darkening with the degree of frying, and the toxicities were gradually reduced:raw betel nut> fried-yellow betel nut> fried-brown betel nut> charring betel nut; the LDso values of raw betel nut, fried-yellow betel nut and fried-brown betel nut were65.69g/kg,67.18g/kg, and71.83g/kg, respectively, what’ more, the charring betel nut unable to obtain the LDso due to no mice died in our tested doses.ConclusionOur present work systematically studied the relationship and law between the substances basis (characteristics, chemical constituents and finger print) and pharmacological activities (effects on the gastrointestinal movement in vitro and in vivo and toxicology), which will help for establishing the quality standard and processing technology. In our investigation,3constituents were found in the processing for the first time, and they were identified as5-hydroxymethyl furfural,2-hydroxyl-5-hydroxymethyl-2-furan-Formaldehyde, and maltol. Furthermore, we studied the pharmacological activities of these constituents. Arecoline is an important compound of the substances basis of betel nut on gastrointestinal movement, but is not the only constituent. The biological effect of fried-brown betel nut is much higher than that of raw betel nut and other processed products might due to the synergy of arecoline and the new appeared compounds. The LD50values of Betel nut were gradually increased with the levels of frying process, suggesting that alkaloid contents of Betel nut gradually decreased in the frying process, which also indicated that alkaloids in the Betel nut are the toxic components, and demonstrated the traditional theory that Betel nut need to fry to moderate its drug-nature.Innovations1、This paper emphasized the frying process based on "dynamic view, the whole process, the regularity of dynamic change", in other words, our work investigated substances of processed products and processing principle the based on the dynamic changes of chemical constituents in the processing, which possessed significant innovation.2、The new technology of on-line non contact infrared temperature measurement system (NCTS) was used to monitor the frying process of Betel nut; moreover, we established the kinetic model to quantitative analysis of the changes of index components’contents and biological effects (toxicities) with the dynamic changes of frying degree (time), and calculate the kinetic parameters. These methods mentioned above possessed obvious innovation compared to current technology.更多还原