【作者】 魏巍；

【导师】 张寒琦； 金永日； 李绪文；

【作者基本信息】 吉林大学 ， 分析化学， 2011， 博士

【摘要】 毛莨科(Ranunculaceae)乌头属(Aconitum L.)植物为多年生、二年生或一年生草本,全世界约有300多种,主要分布于北半球的高海拔地区。我国乌头属植物超过200种,除海南外各省均有分布,我国已有76种乌头属植物被应用于中药处方中,该属植物的根和块茎普遍地应用于治疗多种疾病,例如阳虚汗出,昏厥,风湿热,关节疼痛,肠胃炎,痢疾,水肿,支气管哮喘,肿瘤,以及一些内分泌紊乱引起的疾病如月经不调等。乌头属植物主要含有生物碱、黄酮和多糖类成分。国内外学者已从乌头属植物中分离鉴定了400多种生物碱类化合物,主要为二萜类生物碱,其中普遍存在且含量较高的是乌头碱、新乌头碱和次乌头碱。研究结果显示,这三种生物碱是乌头属植物中主要的药效成分,具有抗炎、镇痛等活性。同时这三种生物碱也是毒性成分。文献指出这三种双酯型生物碱易发生水解,生成单酯型乌头碱和乌头原碱,单酯型乌头碱仍具有抗炎镇痛活性,但毒性大大降低。有关单酯型乌头碱活性的报道比较少,为了进一步研究单酯型乌头碱单体的活性,本文对附子中主要的双酯型乌头碱进行了提取、分离和鉴定,得到四个化合物,利用1D NMR、MS和IR等波谱学手段确定了它们的结构。分别为脱氧乌头碱(deoxyaconitine)、次乌头碱(hypaconitine)、乌头碱(aconitine)和新乌头碱(mesaconitine)。将上述四种双酯型乌头碱水解,共得到11个化合物,利用1D NMR、2D NMR、MS和IR等波谱学手段鉴定了它们的化学结构,其中包括四种单酯型乌头碱分别为苯甲酰乌头碱(benzoylaconine)、苯甲酰新乌头碱(benzoylmesaconine).苯甲酰次乌头碱(benzoylhypaconine)和苯甲酰脱氧乌头碱(benzoyldeoxyaconine);三种乌头原碱分别为乌头原碱(aconine)、新乌头原碱(mesaconine)、次乌头原碱(hypaconine);四种焦乌头碱分别为焦乌头碱(pyroaconitine)、焦新乌头碱(pyromesaconitine)、焦次乌头碱(pyrohypaconitine)和焦脱氧乌头碱(pyrodeoxyaconitine)。另外,本文还对苯甲酰次乌头碱、乌头原碱、新乌头原碱和次乌头原碱、焦乌头碱、焦新乌头碱、焦次乌头碱的1H和13C化学位移进行了全归属,纠正了原有文献对苯甲酰乌头碱和苯甲酰新乌头碱的归属错误。本论文应用反相高效液相色谱法,使用Agilent Zorbax Eclipse plus-C18 (5μm,250 mm X 4.60 mm),流动相为甲醇(A)和水(包含5mmol/L的醋酸铵和0.2%的乙酸)(B)(A：B=40：60),流速1.0 mL/min,检测波长为235 nm,柱温35℃,对制草乌中新乌头碱,乌头碱,次乌头碱,苯甲酰新乌头碱,苯甲酰乌头碱和苯甲酰次乌头碱进行了含量测定。结果显示：六种化合物进样量在0.01～10.00μg范围内均有良好的线性关系,相关系数r均在0.9995以上(n=8)；有很好的稳定性,测定重复性的RSD值均在1.84-3.17%之间；平均回收率(n=6)在96.83%-104.73%之间,RSD值均小于2.61%,本方法能够准确测定制草乌中的6种生物碱的含量,为草乌类药材的质量控制提供了依据。本文还建立了一种新的提取生物碱的方法,即基质固相分散法(MSPD),并对MSPD法影响提取率的几个因素如分散剂、洗脱剂、物料比、净化剂、助溶剂和洗脱体积等条件进行了优化。当采用碱性氧化铝为分散剂、样品与分散剂比为1：2、80%的乙腈水溶液(pH=12)作为洗脱剂和洗脱体积为10 mL,由MSPD法提取的新乌头碱、次乌头碱、乌头碱和脱氧乌头碱的提取率分别为2214.44,1769.55,1299.28,275.40μg g-1,各化合物含量的RSD值均小于1.20%,而由回流法得到的四种生物碱的提取率分别为1949.08,1239.75,1152.61,149.03μg g-1,由此可见,MSPD法的提取率较高,且四种生物碱的回收率都在93.16-102.73%之间,回收率良好。此外,MSPD法还具有提取时间短、操作步骤简单、溶剂用量少的优点,因此MSPD法更适合提取提取乌头类药材中生物碱类化合物。本文还研究了三种单酯型乌头碱对大鼠佐剂性关节炎的作用。结果显示,苯甲酰乌头碱、苯甲酰新乌头碱和苯甲酰次乌头碱均能降低大鼠原发性和继发性足肿胀率,能够明显降低佐剂性关节炎大鼠血沉和白细胞数量,降低MDA含量,升高SOD活性。更多还原

【Abstract】 Aconitum L. is a large genus of the Ranunculaceae family, which consists of over 300 species distributed all over the world. Most of them grow naturally in high altitudes in the northern hemisphere. The genus Aconitum consist of more than 200 species in China, and is widely distributed in all the provinces of China except Hai nan province. As widely used Chinese herbal medicine,76 Aconitum species in China have been medicinally used. The tubers and roots of Aconitum L (Ranunculaceae) are commonly used for various diseases, such as collapse, syncope, rheumatic fever, painful joints, gastroenteritis, diarrhea, oedema, bronchial asthma, various tumors, and some endocrinal disorders like irregular menstruation.A literature survey revealed that about 400 alkaloids, some flavonoids and polyglycosides have been isolated and identified from Aconitum L. Mesaconitine, aconitine and hypaconitine are not only toxic, but also analgesic and anti-inflammatory constituents in Aconitum L. The acetyl group at the 8-postion in the diestertype alkaloids is easily hydrolyzed into benzoylmesaconine, benzoylaconine and benzoylhypaconine. Those monoester aconitines showed a much lower toxicity than that of the diester-type aconitines. However, the pharmacological studies showed that they also have significant anti-inflammatory and analgesic actions.There are few reports about the pharmacological effects of the monoester aconitines. In order to further study the bioactivities of the monoester aconitines, the extraction, separation and determination of active constituents in Radix Aconiti lateralis praeparata were preformed. Four compounds including mesaconitine, aconitine, hypaconitine and deoxyaconitine were separated and identified by]H NMR and 13C NMR. Eleven compounds were separated from the hydrolysates of the four diester-type alkaloids and identified by 1D NMR,2D NMR, IR and MS. The compounds are benzoyldeoxyaconine, benzoylhypaconine, benzoylaconine, benzoylmesaconine, aconine, mesaconine, hypaconine, pyrodeoxyaconitine, pyrohypaconitine, pyromesaconitine and pyroaconitine. The chemical shift assignment of 1H and 13C were made.To determine the six alkaloids, including mesaconitine,aconitine, hypaconitine, benzoylmesaconine, benzoylhypaconine and benzoylaconine in Aconitum kusnezoffii Reichb., the chromato graphic separation of the compounds was achieved with an Agilent Zorbax Eclipse plus-C18 column(5μm,250 mm×4.60 mm). The elution was carried out using methanol (A) and water containing 5mmol L-1 ammonium acetate and 0.2% acetic acid (B) (A:B=40:60) as mobile phase at a flow rate of 1.0 mL min-1. UV absorption was measured at 235 nm. The column temperature was maintained at 35℃and the sample injection volume was 5μL.The results revealed that the linearity of the six compounds was in the range of 0.01-10.0μg, with the correlation coefficients from 0.9995 to 0.9999 (n=8). The RSD for determining the six compounds ranged from 1.84% to 3.17%. The stability of the analytes were satisfactory. Mean recoveries ranged from 96.83% to 104.73%, with relative standard deviations less than 2.61%. This method is accurate,reliable and sensitive for determination of six alkaloids in Aconitum kusnezoffii Reichb. and provide a standard for quality control of Aconitum kusnezoffii Reichb..The matrix solid-phase dispersion (MSPD) was developed for the extraction of four alkaloids, including aconitine, mesaconitine, hypaconitine and deoxyaconitine, from the roots of Aconitum kusnezoffii Reichb.. The effects of several extraction parameters such as dispersion sorbent, ratio of sample to dispersion sorbent, elution solvent, the volume of elution solvent and clean-up sorbent were examined and optimized. When alkaline alumina was used as dispersion sorbent, acetonitrile-water (80:20, v/v pH=12) was used as elution solvent, the ratio of sample to dispersion sorbent was 1:2 and the volume of elution solvent was 10 mL, the extraction yields of mesaconitine, hypaconitine, aconitine and deoxyaconitine obtained by MSPD were 2214.44,1769.55,1299.28 and 275.40μg g-1, respectively. The RSDS of extraction yields for the compounds were all less than 1.20%. Whereas, those obtained by reflux extraction were 1949.08,1239.75,1152.61,149.03μg g-1, respectively. Mean recoveries ranged from 93.16% to 102.73%, with relative standard deviations from 0.27% to 4.17%. Compared with reflux extraction, the MSPD extraction has the advantages in extraction time, and consumption solvent.This paper also studied the effect of benzoylaconine、benzoylmesaconine and benzoylhypaconine on the adjuvant arthritis profile of rats. The results showed that these alkaloids can inhibited the hind paw edema in rats, decreased erthrocyte sedimentation rate、content of leucocyte and MDA(malon dialdehyde), and increased the activity of SOD(superoxide dismutase).更多还原