【作者】 欧水平；

【导师】 杨明；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 博士

【摘要】 毛茛科乌头属植物乌头(Aconitum carmichaeli Debx.)是我国常用中药和四川道地药材川乌和附子的原植物。根据传统用药习惯,子根(附子)和母根(川乌)是乌头的主要收获和药用的部位,而占全植物生物量40%以上的茎叶通常都被遗弃不用。本文以“一体多用”为理论基础,探寻茎叶作为川乌头新药用部位资源的可行性,首先对全植株各部位的化学成分、毒性、药效等进行系统研究,其次以治疗类风湿性关节炎为药效评价指标,以总生物碱和六种酯型生物碱为成分评价指标,对乌头新药用部位及制剂进行设计及体内外评价研究。本文的具体研究内容和结果如下：1.茎叶作为新药用部位的可行性研究首先研究不同发育期全植株各部位(母根、子根、须根、茎、叶)化学成分的动态差异性,综合考虑各种成分含量及产量等因素,将各部位采收期统一为6月底至7月初,此时各部位的总生物碱和双酯型生物碱含量顺序为：须根>子根>母根>叶≈茎,茎叶的总生物碱含量约为子根、须根的二分之一；TLC、HPLC、 HPLC-MS研究各部位生物碱类成分差异性的结果提示各部位所含生物碱类成分的种类大体相似,均含有尼奥灵,宋果灵,六种酯型生物碱等,但含量有所差异,茎叶可能含有较多的尼奥灵,宋果灵等成分,根中酯型生物碱的含量较茎叶高;进而初步拟定茎叶的质量标准。其次研究各部位的药效和毒性,各给药组均能够显著抑制冰醋酸所致小鼠扭体次数,对二甲苯所致小鼠耳廓肿胀及角叉菜胶所致大鼠足肿胀均有抑制作用,表明各部位均具有良好的镇痛和抗炎作用,且茎叶与母根、子根的组间差异不明显；药材急性毒性以须根最大,子根与母根接近,茎与叶相近,明显小于根部位；根部位有明显的心脏毒性,茎叶水提液组的TD50明显大于根。综合化学成分、药效、毒性的研究结论,考虑生物产量、环境污染等因素,说明茎叶作为乌头新药用部位研究具有可行性和必要性。2.茎叶总生物碱的制备及性质研究在茎叶可用的基础上,以总生物碱及六种酯型生物碱含量为指标,对提取、纯化工艺进行优化,制得了总生物碱含量大于60%的有效部位,酯型生物碱以单酯型为主,有效成分BMA含量约为32mg/g, HA约为6.19mg/g; HPLC指纹图谱表明各批次间的相似度较高,批间差异小,茎叶总碱的制备工艺稳定可行。LC-MS的研究表明茎叶总碱主要含有尼奥灵,BMA,HA,宋果灵,AC, MA等成分。酯型生物碱在酸性条件下相对稳定,在碱性条件下易发生水解,在中性条件下水解,能“减毒存效”,故将水解条件定为pH=7,120℃水解2h,所得水解产物中总生物碱含量大于60%,双酯型生物碱的含量极小,单酯总碱含量约为35.65mg/g。对水解前后、口服和外用茎叶总碱的LD50进行研究,总碱水解产物的安全系数较未水解的大,适合口服给药,而未水解总碱提取物外用给药,在最大给药量(1.92g/kg)时,小鼠仍未死亡,可见茎叶总碱外用的毒性明显远远低于口服,此为药效学研究奠定了基础。3.茎叶总碱对大鼠佐剂性关节炎防治作用及机理研究在茎叶总碱制备工艺稳定可行的基础上,在安全用量范围内,对其抗炎、镇痛、抗类风湿性关节炎的作用及机理进行研究。结果表明茎叶总碱有显著的抗炎镇痛作用,对完全弗氏佐剂所致的佐剂性关节炎具有很好的防治作用,能显著抑制大鼠足肿胀及炎细胞、纤维组织、巨噬细胞等滑膜增生性病变。口服和外用均有效,但口服组体重增长缓慢,且精神萎靡,外用组大鼠活跃,摄食积极,体重增长和正常组比较无显著性差异。口服的中毒量和有效最接近,而外用治疗窗大,说明茎叶总碱往治疗佐剂性关节炎上更适合外用给药。茎叶总碱能降低佐剂性关节炎大鼠血清中炎性介质IL-1β/TNF-α以及血清中IgG的含量,说明其可能通过抑制炎症因子以及B细胞分化、增殖等发挥其抗风湿作用。4.茎叶总碱透皮贴剂的制备及体内外评价在茎叶总碱外用安全有效的基础上,以经皮渗透性和黏附能力等为评价指标,筛选出总碱透皮贴剂的处方及制备工艺为：将Duro-Tak87-4098压敏胶(75.5%)和茎叶总碱提取物(20%)混匀,加入丙二醉(4.5%),真空脱气10min后静置脱气至无气泡,进行涂布，涂布厚度为0.5mm,使含药压敏胶均匀涂布在离型膜上,70℃加热20min固化,覆以背衬膜,剪切,即得。该制备工艺简单易行,可行性强。体外评价表明,制得的茎叶总碱贴剂外观美观,易于揭贴,黏附性和经皮渗透性良好,对家兔正常皮肤无刺激性,对损伤皮肤应慎用,有轻度刺激性。体内评价方面,首先建立了同时测定大鼠血浆中六种酯型生物碱(BMA、 BAC、BHA、MA、AC、HA)含量的HPLC-MS-MS方法,该方法专属性强,灵敏度高,精密度、回收率、稳定性均符合生物样品的分析要求。其次以口服茎叶总碱为参比,进行了大鼠贴剂透皮给药的体内药动学研究；用非隔室模型(统计距法)计算体内药动学参数。结果表明,口服给药吸收快,给药5min就可以在血浆中检测到药物,达峰浓度较高,体内滞留时间短,消除速率快,生物半衰期短；茎叶总碱透皮贴剂吸收平稳,消除时间、体内保留时间明显延长,MRT达到20h左右,能长时间维持体内有效血药浓度。经过统计分析,表明除Cmax外,透皮贴剂的六种酯型生物碱的其余各药动学参数如Tmax,AUC(0-t), AUMC(0-t), MRT(O-t), t1/2z,CLz/F,Vz/F等与口服给药相比,均具有显著性差异(P<0.05)。说明茎叶总碱透皮贴剂更有利于发挥安全、有效、持久的药效作用。更多还原

【Abstract】 Aconitum (Aconitum carmichaeli Debx.) is the original plant of Chuan Wu and Fu Zi which are commonly used in traditional Chinese medicine. According to the people’s long-term drμg habit, the sub-root (Fu Zi) and mother root (Chuan Wu) are the harvest and medicinal parts of Aconitum carmichaeli Debx.. And the stem and leaf which account about40%of the total plant biomass are often abandoned Therefore, this thesis is based on the theory of "A integrated plant but mμlti-medicine"to explore the feasibility of the stem and leaf as a new drμg with a site resource Aconitum. First, We systematically research the chemical composition, toxicity, efficacy of the various parts of the whole plants. Secondly, selecting the efficacy of rheumatoid arthritis and the content of total and ester alkaloids(including aconitine, mesaconitine, hypaconitine, benzoylmesaconine, benzoylaconine, benzoylhypaconine)"as indexes, we study the stem and leaf and preparations.1.The feasibility study of stem and leaf as new medicinal partsFirst, we study the dynamic differences of chemical composition between the mother root, sub root, fibrous roots, stem and leaf at different developmental stages. Considering the factors such as the content of a variety of composition and yield, the harvest period of various parts is unified at the end of June to early Jμly when the order of total alkaloids and ester alkabids in various parts is fibrous roots>sub root> mother root> leaf and stem. The total alkaloid content of stem and leaf is half of sub-root and fibrous root.The resμlts of TLC, HPLC, HPLC-MS sμggest that the species of alkaloids contained in various parts are broadly similar. They contain neoline, songorine and six ester alkaloid, etc. But the content of each component is different. The stem and leaf may contain more neoline and songorine, and the ester alkaloid content is less than the roots. Secondly, We study the efficacy and toxicity of various parts. The methods of chemical stimμlation was used to observe the function of relieving pain on the mouse. Result indicated that the various parts can obviously reduce the times of body sprain caused by acetic acid; It can obviously lighten the swelling and distention caused to the rat’s feet by carrageenan. Total the various parts has both anti-inflammation and analgesic effects obviously. And there is not significantly different between the group of stem, leaf and root. The toxicity of fibrous roots was stronger than the other parts, and stem showed similar toxicity to leaf which were far less than the root. The water extracts of root has obvious cardiotoxicity, the TD50of stem and leaf is much larger than the root. Complexing the conclusions of chemical composition, efficacy and toxicity, Considering the biological yield, environmental pollution, and other factors, We can explain the feasibility and necessity of stem and leaf as aconitum new parts for medicine.2.The preparation and properties of total alkaloidsOn the basis of availability, selecting the content of total and ester alkaloids as indexes, we have optimized the extracts and purification process and obtained the effective parts which the content of total alkaloid is greater than60%. The monoester type is the main of ester alkaloids. The content of benzoylmesaconine and hypaconitine is32mg/g and6.19mg/g, respectively.The HPLC fingerprint show the similarity and small differences between batch extracts, And the preparation process is stable and feasible. By LC-MS, we know the extracts contain neoline, songorine and six ester alkaloid, etc.The ester alkaloids are relatively stable under acidic conditions and are prone to hydrolysis under alkaline conditions. The ester alkaloids hydrolysis under neutral conditions, which can reduce toxicity and save efficacy. The hydrolysis conditions is pH=7,120℃hydrolysis2h. The total alkaloid content of is greater than60%and the content of monoester total alkaloids about35.65mg/g. Then.we determine the toxicity of the extracts. The extracts after hydrolysis which are less toxic are suitable for oral administration. And the extracts not-hydrolysis are suitable for transdermal administration. And the transdermal administration is obviously far below the oral. It help to lay the foundation for the pharmacodynamic study. 3.Research on the pharmacodynamic action and mechanism of total alkaloids to treat the rheumatoid arthritisOn the basis of stability and feasibility of the preparation process of total alkaloids, in the safe dosage range, we research on the pharmacodynamic action and mechanism of total alkaloids to treat the rheumatoid arthritis, and anti-inflammatory, analgesic effects. The resuμlts show that the total alkaloids have significant anti-inflammatory and analgesic effects, and have a good role in the prevention and treatment for the complete Freund’s adjuvant-induced adjuvant arthritis. The total alkaloids have the obvious inhibitory effect of Oral and transdermal administration for the AA model rat’s paw swelling and, also have prevention function for inflammatory cells, fibrous tissue, macrophages and synovial hyperplasia. But the rat’s weigh in the oral group grow slowly, the rats in the transdermal group feed actively and gain weight, show no significant difference to the normal group. The toxic dose and effective amount of the oral administration is close, so the total alkaloids are more suitable for transdermal administration in the treatment of adjuvant arthritis.Total alkaloids can reduce inflammatory mediators IL-1β, TNF-α, and IgG levels of rats with rheumatoid arthritis, and play its anti-rheumatic role may by inhibiting inflammatory cytokines, and B-cell differentiation, proliferation.4.The preparation and evaluation in vitro-in vivo of transdermal patchSelecting percutaneous permeation in vitro and adhesion ability as the main evaluation index, we investigated the prescription and preparation process. The prescription and preparation process are follow:we weigh the prescribed amount (75.5%) of pressure-sensitive adhesive Duro-Tak87-4098, add the appropriate amount (20%) of extracts, then stir, and join the propylene glycol (4.5%), and remove the air bubbles by vacuum degassing10min and standing. We control the coating thickness to0.5mm, then the drμg-containing pressure-sensitive adhesive is uniformly applied in the release film. Then solidify at70℃and heat for20min. Cover with the backing film, shear. The preparation process is simple and feasible. The evaluation in vitro showed that the transdermal patch look beautifμl,and is easy to mortgage and paste. It has good adhesion and percutaneous permeability, and non-irritation In rabbit normal skin, but there is a mild irritation in damage skin, it shoμld be used with caution.For the evaluation in vivo, We first establish the HPLC-MS-MS method for the simμltaneous determination of the six ester alkaloid (BMA、BAC、BHA、 MA、AC、HA) content in rat plasma. The method has high specificity and sensitivity, and the precision, recovery and stability are in line with the analysis of biological samples requirements.Secondly, reference to the oral administration, We study the pharmacokinetics in vivo of the patch for transdermal administration in rats. Pharmacokinetic parameters calcμlate using non-compartment model (the method of statistics). The resμlts show that the oral administration absorb rapidly. The drμg can be detected in the plasma when the oral administration has been used5min. The peak concentration is high, The residence time and biological half-life are short in vivo, and the elimination rate is fast. The resμlts showed that transdermal administration absorb smoothly, eliminating and retention time of transdermal administration extend obviously. The transdermal administration can maintain the effective blood concentration for a long time. Throμgh statistical analysis, in addition to the Cmax, six ester alkaloids remaining pharmacokinetic parameters such as Tmax, AUC (0-t), AUMC (0-t), MRT (0-t), t1/2z, CLz/F, Vz/F compared with the oral administration, has a significant difference (P<0.05). The transdermal patch is more conducive to play safe, effective and long-lasting pharmacodynamic effect.更多还原