中国乌头的研究——ⅩⅩ.赣皖乌头的研究

本文报道了从赣皖乌头Aconitum finetianum Hand-Mazz的根中分到三个二萜类生物碱A.B.c。经鉴定B和C分别为lappaconitine和ranaconitine。碱A,C₃₂H₄₄N₂O₁₀,熔点220～221℃,[α]_D²²+44.7°(甲醇),为一个新生物碱,暂称赣乌碱finaconitine,经光谱推定化学结构为10-β-羟基-冉乌头碱。     

中国乌头之研究 ⅩⅨ 四川雪上一枝蒿中生物碱及其结构

从四川金沙江畔雪上一枝蒿(Aconitum pendulum)中分出四个结晶性的生物碱,证明其中三个为已知生物碱,即次乌头碱(hypaconitine),乌头碱(aconitine)和3-乙酰乌头碱(3-acetylaconitine);另一种为新生物碱,暂称为雪乌碱(penduline)熔点166～167℃,分子式C₃₄H₄₇NO₉,结构式初步推定如下式所示。雪上一枝蒿中主要生物碱为乌头碱,得率约为0.14%;雪乌碱对动物具有镇痛局麻等作用。     

中国乌头之研究——ⅩⅪ.赣皖乌头的研究

从赣皖乌头(Aconitum finetianum Hand-Mazz)中又分得五个生物碱,经光谱及化学方法鉴定,其中二个为新的二萜类生物碱,即脱乙酰冉乌头碱(N-deacetylranconitine),C₃₀H₄₂N₂O₆,熔点125～127℃,和脱乙酰赣乌碱(N-deacetylfinaconitine),C₃₀H₄₂N₂O₉,熔点121～123℃,及三个已知生物碱,即阿娃乌头碱(avadharidine)、牛扁次碱(lycoctonine)和脱乙酰刺乌头碱(N-deacetyllappaconitine)。经药理试验表明,脱乙酰冉乌头碱,脱乙酰赣乌碱和脱乙酰刺乌头碱均具有良好的镇痛效果。     

口服制川乌提取物后大鼠体内乌头类生物碱的组织分布

目的建立HPLC法测定口服制川乌提取物后大鼠体内乌头类生物碱的方法。方法用Waters 2690@996 PAD系统，Halsil 100 C₁₈柱(250 mm×4.6 mm ID, 5 μm)，水-甲醇-二乙胺(75∶25∶0.1)为流动相，流速0.9 mL·min^-1，检测波长240 nm。结果乌头碱在心脏、脾脏、肺脏、肾脏的线性范围：0.4-100 μg·mL^-1，r分别为0.997 2,0.998 6,0.999 3,0.999 4；在肝脏的线性范围：2-200 μg·mL^-1，r为0.999 0。次乌头碱在心、肝、脾、肺、肾、脑和脊髓的线性范围：5-100 μg·mL^-1，r分别为0.999 4,0.999 7,0.999 8,0.998 4,0.999 8,0.999 8和0.999 7。乌头碱及次乌头碱的检测限(S/N=3)为0.4 μg·mL^-1。各组织中的回收率：乌头碱为88.7%-102.2%，次乌头碱为865%-101.3%。结论本法为确证乌头类生物碱的中毒提供了科学有效的依据。     

反相离子对色谱法测定附子中生物碱成分

目的测定附子中乌头碱、新乌头碱、次乌头碱、北乌碱、苯甲酰乌头原碱和苯甲酰新乌头原碱等6种生物碱的含量。方法用反相离子对HPLC法，使用AichromBond-1 C₁₈柱(250 mm×4.6 mm ID)；流动相：乙腈-5 mmol·L^-1 NaH₂PO₄溶液，磷酸调至pH 4.5(50∶50)，内含7 mmol·L^-1 十二烷基硫酸钠(SDS)；检测波长：235 nm；流速：1.0 mL·min^-1；柱温：35 ℃。结果以上6种生物碱可以完全分离，准确测定。结论该方法准确度高，可应用于附子中生物碱的含量测定。     

小活络丸中乌头类生物碱成分含量测定方法的建立

目的：建立小活络丸中生物碱成分乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱与苯甲酰新乌头原碱的含量测定方法，以完善其质量标准。方法：采用高效液相色谱法， 选择PICKERING C₁₈（4.6 mm×250 mm，5μm）色谱柱，以甲醇、乙腈和0.1%磷酸水为流动相，梯度洗脱，流速1.0 mL·min^-1，柱温25 ℃，检测波长232 nm，进样量15 μL。结果：乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱及苯甲酰新乌头原碱分别在各自范围内线性关系良好（r=0.9999），平均加样回收率分别为99.8%（RSD=0.8%）、99.9%（RSD=1.0%）、100.6% （RSD=1.4%）、100.8%（RSD=1.5%）、100.9%（RSD=1.5%）、100.6%（RSD=0.8%）。5批样品中上述6个成分含量范围分别为0.5~2.9、5.4~27.4、0.5~10.5、18.8~24.6、18.8~30.2及142.4~181.6 μg·g^-1。 结论：所建立的同时测定6个生物碱含量的测定方法可准确测定小活络丸中生物碱成分的含量，有助于提高小活络丸的质量标准。     

露蕊乌头生物碱的研究(Ⅰ)

从藏药露蕊乌头(Aconitum gymnandrum Maxim)中分得四个生物碱,经化学和光谱鉴定,其中两个为已知生物碱atisine.Hcl(A)和talatisamine(B),另两个为新的二萜类生物碱,分别命名为露乌碱,(C)(gymnaconitine)和甲基露乌碱(D)(mgthyl gymnaconitine),这两种新的生物碱均联接有二甲基咖啡酸酯:在乌头生物碱中是首次发现。     

两个新的双去甲二萜生物碱高乌宁碱丁和高乌宁碱戊的结构研究

目的：研究高乌头Aconitum sinomontanum Nakai根的化学成分。方法：采用硅胶柱色谱及离心薄层色谱分离化学成分,IR, ¹HNMR, ¹³CNMR, ¹H-¹H COSY, HMBC, HMQC, MS等方法进行结构鉴定。结果：得到两个双去甲二萜生物碱高乌宁碱丁(sinomontanine D, 1)和高乌宁碱戊(sinomontanine E, 2)。结论：化合物1,2为新的双去甲二萜生物碱。     

基于膜片钳技术的丹红注射液对人诱导多能干细胞衍生心肌细胞电生理的影响研究

目的 通过全细胞膜片钳实验,从心肌电生理层面探索丹红注射液(DHI)治疗乌头碱诱导心律失常的可能机制。方法 基于人诱导多能干细胞衍生心肌细胞(hiPSC-CMs)膜片钳技术,即时给药并观察乌头碱不同浓度(3、9、27 μmol·L^-1)对钠通道、hERG钾通道的抑制效果,进行造模条件的筛选;设置对照组、模型组(乌头碱1 μmol·L^-1长时间造模)、DHI (3、9、27 μL·mL^-1)组,乌头碱与DHI同时加药,记录动作电位幅度(APA)、放电频率、动作电位幅度下降50 %时的时程(APD₅₀)和动作电位幅度下降90 %时的时程(APD₉₀),并以对照组为标准,计算相对值;设置对照组、模型组、DHI (3 μL·mL^-1)组,乌头碱与DHI同时加药,观察3~5 min,记录钠离子、hERG钾离子、钙离子电流密度。结果 给予即时药物处理,3、9、27 μmol·L^-1的乌头碱对钠电流的抑制率分别为17.94 %、31.07 %、60.67 %,对hERG通道的抑制率分别为5.02 %、10.59 %、28.59 %;在短期内给乌头碱对hERG通道的抑制效果较为有限,而实际实验中,长时间的高浓度乌头碱孵育使钠电流被完全抑制,导致离子通道功能受损,因此选择乌头碱1 μmol·L^-1长时间给药制备模型。与对照组比较,模型组hiPSC-CMs相对APA、相对APD₅₀和相对APD₉₀显著下降(P<0.01、0.001),相对放电频率显著升高(P<0.001);钠电流在一定时间内减小,抑制率为30.18 %;hERG钾电流明显减小,抑制率为72.33 %;钙电流明显增大,电流增大191.35 %。与模型组比较,DHI 3、9、27 μL·mL^-1组相对放电频率显著降低(P<0.05、0.001)、相对APD₉₀显著升高(P<0.05、0.01),3 μL·mL^-1组相对APD₅₀显著升高(P<0.001);DHI组钠电流、hERG钾电流的减小及钙电流的增大均有一定缓解。结论 DHI可能可以通过影响钠、钾、钙通道来缓解乌头碱导致的心律失常现象。     

基于药物代谢酶CYP3A4的乌头碱配伍人参皂苷、甘草苷的减毒机制研究

目的 考察乌头碱配伍人参皂苷Rb₁、甘草苷后对HepG2药物代谢酶（Cytochrome P450,CYP450）中3A4亚型的报告基因荧光活性、mRNA转录及蛋白翻译水平的影响。方法 将pGLuc-CYP3A4报告基因质粒与pcDNA3.1-hPXR表达质粒共转染HepG2细胞,检测乌头类生物碱、人参皂苷和甘草的单体成分对CYP3A4的激活效应;并利用实时荧光定量PCR（qRT-PCR）及Western blotting技术检测人参皂苷Rb₁、甘草苷与乌头碱对CYP3A4 mRNA及蛋白水平的影响。结果 报告基因模型检测结果显示,与对照组比较,乌头碱、新乌头碱、次乌头碱和乙酰乌头碱能下调CYP3A4报告基因荧光强度（P<0.05）,其中乌头碱下调能力最强,人参皂苷Rb₁、Rc、Re、Rg1以及甘草苷、异甘草苷、甘草素和甘草酸均能上调报告基因的荧光强度（P<0.05、0.01）,其中人参皂苷Rb₁和甘草苷上调能力最明显;同时乌头碱能下调CYP3A4 mRNA与蛋白表达水平（P<0.05、0.01）,人参皂苷Rb₁和甘草苷能逆转乌头碱下调CYP3A4的能力（P<0.05、0.01）。结论 人参皂苷Rb₁、甘草苷与乌头碱配伍后可上调CYP3A4的表达,减少乌头碱在体内蓄积时间,起到减毒的作用。     

中国乌头的研究 ⅩⅢ.北草乌中的生物碱

从内蒙古赤峰产的北草乌中共分得五种结晶性的生物碱,并证明其中四种为已知物,即去氧乌头碱(deoxyaconitine),次乌头碱(hypaconitine),乌头碱(aconitine)和新乌头碱(mesaconitine);另一种为新生物碱,暂称北草乌碱(beiwutine),熔点196～198℃,分子式C₃₃H₄₅NO₁₂,结构式推定如图(1)。此草乌中的主要生物碱为新乌头碱。     

乌头中主要生物碱的高效液相色谱测定

用高效液相色谱分离、测定乌头及附子中一些主要生物碱。生药的乙醚提取物在十八烷基键合相柱上,用甲醇—水—氯仿—三乙胺(70:30:2:0.1)作流动相,中乌头碱、乌头碱及次乌头碱与其它杂质能很好分离。以β-甲基萘作内标化合物,用峰高比测定各生物碱含量。曾测定了不同品种、不同产地及不同加工炮制方法的一些样品,其生物碱组成及比例相差较大。     

生物碱的安培滴定 Ⅲ.四苯硼钠法

本文研究了在不同pH的底液中,一些生物碱(乌头碱、小蘖碱、番木鳖减、辛可宁、白屈菜碱、那可汀、奎宁)与四苯硼钠间反应条件,观察了二者形成沉淀的关系.通过试验,提出在进行生物碱滴定时,根据生物碱的碱性强弱,可计算出底液适宜的pH,并提出以四苯硼钠滴定生物碱的安培方法.     

海南萝芙木季胺碱化学成分的研究

从海南萝芙木生产“降压灵”的母液,经盐析得到生物碱部分,用硅胶柱层,制备薄层和葡聚糖凝胶过滤法,分得六种生物碱。碱Ⅰ(ajmaline)、碱Ⅱ(vellosimine)、碱Ⅲ(spegatrine)、碱Ⅳ(verticillatine)为已知碱,碱Ⅴ和碱Ⅵ为两个新的二聚体生物碱。根据红外、紫外、质谱、氢谱及碳谱等光谱分析,推定碱Ⅴ的化学结构为1式,命名为双斯配加春(dispegatrine)。并用半合成方法进一步确证了其结构.碱Ⅵ的结构待定.药理研究表明碱Ⅴ和碱Ⅵ均具有α-肾上腺素能受体阻断作用。     

Structure-dependent inhibitory action of the Aconitum alkaloids 14-benzoyltalitasamine and talitasamine in rat hippocampal slices

In the present study the effects of the two Aconitum alkaloids 14-benzoyltalitasamine and talitasamine on neuronal activity were investigated in order to obtain further insight into structure-dependent effects of this group of alkaloids on central nervous activity. Both alkaloids are closely related to aconitine, the main alkaloid of plants of Aconitum species. However, they have shortened side chains at position C3 and C8 of the molecule. The experiments were performed as extracellular recordings of orthodromically and antidromically evoked population spikes as well as of field excitatory potentials (EPSPs) from the CA1 region of rat hippocampal slices. 14-Benzoyltalitasamine exerted a reversible inhibition of the field potentials in a concentration-dependent manner. The orthodromic population spike was attenuated at concentrations higher than 1 μM, while the field EPSP was already affected at a concentration of at least 0.3 μM. Both responses were completely blocked at a concentration of 30 μM. The alkaloid failed to affect the presynaptic fiber spike at concentrations less than 10 μM. There was only a up to 30% decrease in the antidromic population spike (10–100 μM). The inhibition of the antidromic spike was increased by using a higher stimulus frequency. In contrast to 14-benzoyltalitasamine, the alkaloid talitasamine which is lacking the benzoylester side chain was a less effective inhibitor of the orthodromic population spike and even failed to affect the antidromic spike. Furthermore, the effects of the alkaloids on experimentally induced epileptiform activity was examined. While talitasamine was lacking any significant effect at concentrations less than 100 μM, 14-benzoyltalitasamine reversibly reduced both stimulus-triggered epileptiform activity in area CA1 elicited by omission of Mg²⁺ from the bathing medium as well as spontaneously occurring epileptiform activity in CA3 elicited by omission of Mg²⁺ and elevation of K⁺ to 5 or 8 mM. The antiepileptiform efficacy of this compound was concentration-dependent (0.3–10 μM) and manifested itself as a decrease in burst frequency as well as in burst amplitude and was significantly increased by the higher K⁺ concentration. Received: 25 August 1997 / Accepted: 15 February 1998     

露蕊乌头的二萜生物碱

从露蕊乌头(Aconitum gymnandrum Maxim)中分离到11个二萜生物碱,利用光谱方法确定结构,证明其中一个为新生物碱,命名为露乌定,其余10个分别鉴定为14-乙酰基-8-O-甲基-塔拉胺(tal-atisamine,Ⅱ)、acoforine(Ⅲ)、非洲防己碱(columbidine,Ⅳ)、乌头碱(aconitine,Ⅴ)、ranaeonitine(Ⅵ)、塔拉定(talatizidine,Ⅶ)、异塔拉定(isotalatizidine.Ⅷ)、露乌碱(gymanaconitine,Ⅸ)、塔拉胺(talatisamine,Ⅹ)和阿替辛盐酸盐(atisine.HCl)。其中碱Ⅱ为首次在自然界中发现,碱Ⅲ～Ⅶ为首次从该植物中分离得到。     

Mode of antinociceptive and toxic action of alkaloids of Aconitum spec.

Extracts of the plant Aconitum spec. are used in traditional Chinese medicine predominantly as anti-inflammatory and analgesic agents, the latter allegedly equally potent as morphine but without any habit-forming potential. As the only pharmacologically active compounds, the C₁₉ diterpenoid alkaloid aconitine, and some of its derivatives, have been proven to be antinociceptive in different analgesic assays, but the mode of action is unknown. To elucidate the mode of action, ten aconitine-like derivatives were investigated with respect to their affinity for voltage-dependent Na⁺ channels, the action on synaptosomal Na⁺ and Ca²⁺ homoeostasis and their antinociceptive, arrhythmogenic and acute toxic properties. Since aconitine is known to bind to site II of Na⁺ channels, we determined the affinity of the aconitine-like derivatives in vitro to synaptosomal membranes by the [³H]-batrachotoxinin-binding assay and their properties on intrasynaptosomal concentrations of free Na⁺ and Ca²⁺ ([Na⁺]_i and [Ca²⁺]_i), both the latter determined fluorometrically with SBFI and Fura-2 respectively. Furthermore, the alkaloids’ arrhythmogenic potential was investigated in guinea-pig isolated atria and the antinociceptive action on formalin-induced hyperalgesia and the acute toxic action estimated in mice. The results show that the alkaloids could be divided into at least three groups. The first is characterized by a high affinity to the site II of Na⁺ channels (K _i about 1.2 μM), the ability to enhance [Na⁺]_i and [Ca²⁺]_i (EC₅₀ about 3 μM), a strong arrhythmogenic action that starts at about 30 nM, an antinociceptive effect (ED₅₀ about 0.06 mg/kg) and high acute toxicity (LD₅₀ values about 0.15 mg/kg). To this group belong aconitine, 3-acetylaconitine and hypaconitine. The second group, with lappaconitine as the only member, has an affinity to Na⁺ channels an order of magnitude lower (K _i = 11.5 μM), less acute toxicity (LD₅₀ about 5 mg/kg), and a two orders of magnitude lower antinociceptive action (ED₅₀ about 2.8 mg/kg) and lower cardiotoxicity (bradycardia observed at 3 μM). Additionally, lappaconitine suppresses the increase in [Ca²⁺]_i of aconitine-stimulated synaptosomes and increases the excitation threshold of left atria, indicating an inhibition of Na⁺ channels. The other derivatives, i.e. delcorine, desoxydelcorine, karakoline, lappaconidine, lappaconine and lycoctonine, belong to the third group, which has hardly any effects. They have a low affinity to Na⁺ channels with K _i values in the millimolar range, show no effect on synaptosomal [Na⁺]_i and [Ca²⁺]_i, no arrhythmogenic potential up to 100 μM, no antinociceptive activity and low toxicity with LD₅₀ values greater than 50 mg/kg. For the investigated alkaloids we suggest two different antinociceptive-like modes of action. Aconitine, hypaconitine and 3-acetylaconitine may induce a block of neuronal conduction by a permanent cell depolarisation, whereas lappaconitine might act like local anaesthetics. However, because of the low LD₅₀/ED₃₀ quotients of 2–6, the antinociceptive-like action of the Aconitum alkaloids seems to reflect severe intoxication rather than a specific antinociceptive action. The structure/activity relationship shows that alkaloids that activate or block Na⁺ channels have a benzoyl ester side chain in the C-14 or C-4 positions respectively, whereas the other compounds lack this group. Received: 24 June 1997 / Accepted: 8 September 1997     

平贝母生物碱的分离和鉴定

自平贝母(Fritillaria ussuriensis Maxim)中分得四种生物碱。晶Ⅰ和晶Ⅱ是已知的生物碱,分别为西贝素和贝母辛。晶Ⅲ和晶Ⅳ是二个新的C-去甲-D-异甾体生物碱,分别命名为平贝碱甲和平贝碱乙。经制备衍生物和光谱分析推定平贝碱甲的结构为Ⅲ。平贝碱乙(熔点255～259℃,分子式C₂₇H₄₆O₆N)的结构尚在研究中。     

中国乌头的研究——Ⅸ.川乌、附子中的生物碱

作者对中药川乌的生物碱进行研究,从其中共分得六种结晶性的生物碱,并证明四种是已知物,卽次乌头碱(hypaconitine)、乌头碱(aconitine)、新乌头碱(mesaconitine)和塔拉地萨敏(talatisamine).另二种为新生物碱,暂称为川乌碱甲和川乌碱乙.川岛碱甲,熔点111℃,实验式为C₂₃H₃₇NO₆;川乌碱乙,熔点185℃,实验式为C₂₂H₃₅NO₄。另在附子中亦分得新马头碱及次乌头碱.