反相高效液相色谱法测定参附注射液及市售黑附片中乌头碱的含量

目的　测定参附注射液及市售黑附片中乌头碱的含量。方法　用RP HPLC ,流动相为乙腈 醋酸铵缓冲液 (pH10 5 ) ,紫外检测波长 2 3 0nm ,流速 0 8ml·min-1。结果　对照品的线性范围 1 3 9～ 2 2 2 4 μg(r =0 9997) ;市售黑附片中的乌头碱比参附注射液中的高 3倍。结论　方法简便、快速、准确、重现性好 ,可以作为样品的检验方法     

The relationship between growth of the aerial part and alkaloid content variation in cultivated <Emphasis Type="Italic">Aconitum carmichaeli</Emphasis> Debeaux

Processed root of aconite, Aconitum carmichaeli Debeaux—known as bushi in Japan—is indispensable for treating diseases among elderly persons in Japanese and Chinese traditional medicine. Its active component is bushi diester alkaloid (BDA), which consists of aconitine (ACO), mesaconitine (MES), hypaconitine (HYP), and jesaconitine (JES). Since an overdose of BDA results in severe side effects, the BDA content should be within safe limits. However, the BDA content of raw aconite root, even that produced by standard cultivation procedures, varies greatly. In this study, to clarify the cause of BDA variation, we examined the weight and BDA content of each part of cultivated A. carmichaeli: the aerial part, the mother tuberous root (MT), the daughter tuberous root (DT), and the rootlet (RL). We found the following positive relationships: between aerial part weight and DT weight, aerial part weight and BDA content in stem of apex, and BDA content in stem of apex and total BDA of DT attached to the plant. Furthermore, DT belonging to a higher weight group showed less BDA content variation. In addition, BDA of DT and those of MT and RL differ in both content and composition. In conclusion, it was suggested that the weight or the size of the aerial part was a good marker for monitoring BDA content and its variation in the tuberous root, and it was found to be desirable to prevent mixing MT and RL at harvest.     

附子配伍原则的电喷雾质谱研究

目的从化学角度探讨附子配伍的原则。方法利用电喷雾质谱分析和比较生附子及其复方中药煎煮液中二萜类生物碱的变化规律；测定附子及其他中药煎煮液的pH值并用电喷雾质谱考察酸性对药材中乌头碱水解的影响。结果生附子单煎煮及与甘草共煎后双酯型乌头生物碱大部分发生水解，生附子与半夏或五味子共煎生物碱水解受抑制，生附子和甘草单煎液的酸性远低于半夏或五味子单煎液，中乌头碱在酸性条件下不水解。结论酸性是影响乌头碱类双酯型生物碱水解的重要因素，强酸条件下水解受抑制，附子煎煮液毒性增加。     

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。     

复方附子口服液中黄芪甲苷含量测定和乌头碱限量检查方法研究

目的建立复方附子口服液中黄芪甲苷含量测定和乌头碱限量检测方法。方法采用HPLC法对黄芪甲苷含量、乌头碱限量进行检测。结果黄芪甲苷在0.047 12⁰.471 2 mg·mL-1与峰面积线性关系良好(r=0.999 7),高、中、低浓度平均加样回收率分别为99.9%、99.9%、97.6%,RSD分别为4.4%、1.9%、2.2%;乌头碱在0.005 970.471 2 mg·mL-1与峰面积线性关系良好(r=0.999 7),高、中、低浓度平均加样回收率分别为99.9%、99.9%、97.6%,RSD分别为4.4%、1.9%、2.2%;乌头碱在0.005 97⁰.597 0 mg·mL-1与峰面积线性关系良好(r=0.999 9);高、中、低浓度平均加样回收率分别为101.3%、98.3%、98.7%,RSD分别为2.1%、1.0%、1.6%。结论该方法专属性强,灵敏度高,重现性好,可用于复方附子口服液中黄芪甲苷含量和乌头碱限量的检测。     

Counter effects of higenamine and coryneine, components of aconite root, on acetylcholine release from motor nerve terminal in mice

The counter effects of higenamine and coryneine, components of aconite root, on acetylcholine (ACh) release from motor nerve terminals in the mouse phrenic nerve-diaphragm muscle preparation were studied by a radioisotope method. Both nerve-evoked release and spontaneous release of [³H]-ACh from the preparation preloaded with [³H]-choline were measured. The change in the tetanic tension of muscle was simultaneously recorded in the same preparation. Higenamine (10 μM) augmented both the nerve-evoked and spontaneous ACh releases, and the muscle tension. The effects were inhibited by pretreatment with propranolol (10 μM), a β-adrenoceptor antagonist. Coryneine reduced the nerve-evoked release of ACh, accelerated the decay of tetanic tension (tetanic fade) at 30 μM, and it depressed the peak amplitude of tetanic tension at a higher concentration of 100 μM. These results suggest that of the two components contained in aconite root, higenamine increases ACh release via activation of β-adrenoceptor, and conversely coryneine depresses ACh release by preferentially acting at motor nerve terminal.     

Aconitum Alkaloid Poisoning Related to the Culinary Uses of Aconite Roots

Aconite roots (roots or root tubers of the Aconitum species) are eaten as root vegetables and used to prepare herbal soups and meals, mainly for their purported health benefits. Aconite roots contain aconitine and other Aconitum alkaloids, which are well known cardiotoxins and neurotoxins. To better understand why Aconitum alkaloid poisoning related to the culinary uses of aconite roots can occur and characterize the risks posed by these “food supplements”, relevant published reports were reviewed. From 1995 to 2013, there were eight reports of aconite poisoning after consumption of these herbal soups and meals, including two reports of large clusters of cases (n = 19–45) and two reports of cases (n = 15–156) managed by two hospitals over a period of 4.5 to 5 years. The herbal formulae used did not adhere to the suggested guidelines, with regarding to the doses (50–500 g instead of 3–30 g per person) and types (raw instead of processed) of aconite roots used. The quantities of Aconitum alkaloids involved were huge, taking into consideration the doses of aconite roots used to prepare herbal soups/meals and the amounts of aconite roots and herbal soups/meals consumed. In a large cluster of cases, despite simmering raw “caowu” (the root tuber of A. kusnezoffii) in pork broth for 24 h, all 19 family members who consumed this soup and boiled “caowu” developed poisoning. Severe or even fatal aconite poisoning can occur after consumption of herbal soups and foods prepared from aconite roots. Even prolonged boiling may not be protective if raw preparations and large quantities of aconite roots are used. The public should be warned of the risk of severe poisoning related to the culinary and traditional medicinal uses of aconite roots.     

Distribution of Aconitum alkaloids in body fluids and tissues in a suicidal case of aconite ingestion

A case involving a suicidal ingestion of Aconitum tubers is presented. A 40-year-old woman in Hokkaido, Japan ingested ground aconite and died of aconite intoxication about 4 h after ingestion. The Aconitum alkaloids were quantitated using gas chromatography-selected ion monitoring from extracts of the body fluids and organs. The blood and urine concentrations of jesaconitine, the main alkaloid of the aconite in this case, were 69.1 ng/mL and 237.8 ng/mL, respectively. Higher values of the alkaloid were demonstrated in the kidneys, the liver, and in the bile rather than other organs or serum, suggesting the alkaloids were eliminated by the liver and kidneys. In the gastrointestinal tract, the highest value of jesaconitine (471.3 ng/g) was in the ileal contents. These findings show that Aconitum alkaloids were found in the liver and kidneys in much higher concentrations than in serum and suggest that they were eliminated not only via urine but also in feces. Feces may be useful to detect Aconitum alkaloid if other biological samples are not available.     

3种乌头类中药在大鼠体内外的神经毒性

目的 考察几种常见乌头类中药的神经毒性.方法 采用体内、外给药的方法,大鼠连续90 d经口给予生川乌和生草乌,检测其对大鼠神经行为、血液、血液生化及脑组织的影响;采用大鼠胚胎海马神经元体外培养的方法,研究生川乌、生草乌和生附子对神经细胞的体外毒性.结果 经口给予生川乌和生草乌后,大鼠的神经行为、血液与血液生化指标无变化,脑组织检查未见异常;体外试验结果表明,生川乌、生草乌及生附子对大鼠海马神经元具有一定毒性,呈剂量效应关系,毒性强度为生草乌＞生川乌＞生附子.结论 乌头类中药在大鼠整体动物试验中未见神经毒性作用,但在海马神经元体外培养模型中,表现出神经毒性,这可能与血脑屏障及体内代谢有关.     

膨大素对川麦冬生长发育的影响研究

目的:通过研究膨大素对川麦冬生长发育的影响,为川麦冬的规范化种植提供一定的理论依据。方法:根据膨大素施用量和施用方式设计随机分组试验,测定植株的株高、冠幅、分蘖数、块根数、单株块根重、块根长、块根直径和百粒重,评价膨大素对川麦冬生长发育的影响;对比各实验组川麦冬块根的鲜重及干重,评价膨大素对川麦冬产量的影响。结果:膨大素能促进川麦冬植株地上部分的生长,明显增加块根数、单株块根重,显著提高川麦冬产量。结论:膨大素能在一定程度上促进川麦冬的生长发育,显著提高川麦冬产量,且与多效唑分段合用时效果更佳。     

Hypaconitine, the dominant constituent responsible for the neuromuscular blocking action of the Japanese-sino medicine "bushi" (aconite root)

The neuromuscular blocking actions of several constituents extracted from Japanese-sino medicine, aconite, were compared in mouse phrenic nerve-diaphragm muscle preparations. Hypaconitine (HAT) was more potent than aconitine (ATN), mesaconitine (MAT) and deoxyaconitine. Lipohypaconitine, coryneine and lipodeoxyaconitine were less effective. Lipoaconitine, benzoylmesaconine, higenamine, kobusine and chasmanine were not effective. The blockades by HAT, ATN and MAT were not recovered by neostigmine. The mechanisms of blockade were similar to that of aconite crude extract. These results suggest that aconite action is dependent on HAT, a main constituent.     

Intestinal transport of pure diester-type alkaloids from an aconite extract across the Caco-2 cell monolayer model

Aconitine (AC), mesaconitine (MA), and hypaconitine (HA) are the active alkaloids identified in aconite tuber, an important traditional Chinese medicine. The study is aimed to investigate their intestinal transport profiles and potential interaction during the intestinal absorption using the Caco-2 cell monolayer model. All three alkaloids had good permeability with P(app) values greater than 1 × 10 (-6) cm · s (-1). However, AC, MA, and HA in a mixture and as an extract, in both cases with the same content of alkaloids, showed higher transport efficiency in the apical to basolateral, and lower transport efficiency in the basolateral to apical directions. Digoxin, as a P-glycoprotein (P-gp) substrate, was substantially effluxed in the basolateral to apical direction but inhibited by the three alkaloids. Furthermore, the backwards transport of MA and HA was inhibited by the P-gp inhibitor verapamil. These observations indicated that the three alkaloids may not only be P-gp inhibitors but also its substrates; they interact with each other and can potentially enhance their own bioavailability when taken concomitantly.     

生、制乌头总生物碱对心脏功能及其毒性的比较

目的　比较生、制乌头总生物碱对离体大鼠心脏收缩功能及对小鼠心率和脑血流的影响。方法　制备大鼠离体心脏 ,恒温灌注不同浓度药物 ,观察心脏收缩幅度和心率 ;小鼠经戊巴比妥麻醉后 ,尾静脉给予不同浓度药物 ,记录脑膜血流量和心率变化。结果　生、制乌头生物碱都能增加离体心脏的收缩幅度 ,加快心率 ,制乌头总生物碱的效强不如生乌头 ,但毒性较小 ;制乌头生物碱 8mg·kg-1、10min时使小鼠心率和脑血流出现明显下降 ,生乌头生物碱则在 4mg·kg-1、5min使之下降直至接近零。结论　从离体心脏和整体动物两个方面 ,证明生乌头经过炮制后 ,虽然效强有所下降 ,但对心脏的毒性明显降低。     

Physicochemical quality evaluation of natural compounds isolated from crude drugs

Aconite root has high toxicity caused by diester alkaloids, thus it was necessary to define the limiting value of diester alkaloids used in medicine formulation. To give the quality of “Processed Aconite Root” and “Powdered Processed Aconite Root” in the Japanese Pharmacopoeia (14th edn, supplement II), we established the official specification and evaluation methods of standard substances. High qualitative grade diester alkaloids, aconitine, hypaconitine, jesaconitine and mesaconitine, which were useful to evaluate the purity of processed aconite root and powdered processed aconite root, were prepared and evaluated for their stability. We studied the physicochemical specification and evaluation methods of these alkaloids. In addition, an “Aconitum diester alkaloids standard solution for purity”, which was used for the purity test, was prepared, and we also studied its physicochemical specification and evaluation methods. In addition, to evaluate the quality of processed aconite root and powdered processed aconite root, a TLC identification test was established. A monoester alkaloid of benzoylmesaconine hydrochloride was used as the reference standard in the latter test, and we also investigated its physicochemical specification and evaluation methods.     

附子对小鼠超氧化物岐化酶活性和丙二醛、NO含量的影响

目的:研究附子不同的临床使用剂量对小鼠心脏超氧化物歧化酶(SOD)活性、丙二醛(MDA)、一氧化氮(NO)含量的影响,探讨其对心脏的毒性机制.方法:制备附子及与甘草配伍的水煎液,将小鼠随机分为9组,分别以附子15,45,75,105g的临床使用剂量按等效换算系数换算成小鼠给药剂量,给小鼠灌胃20 d,测其心脏组织SOD活性及MDA、NO含量.结果:与对照组比较,单附子临床剂量45,75,105 g组小鼠心脏SOD活性明显增加,MDA含量显著降低(P＜0.05).甘草配伍后的各组与对照组比较,SOD含量有增加的趋势,但仅105 g组有明显差异(P＜0.05);除15 g组外,其余各组MDA含量均显著降低(P＜0.05);NO含量均呈下降趋势,但差异无统计学意义(P＞0.05).结论:大剂量使用附子对心脏的SOD活力、MDA和NO含量没有负面影响,相反能减轻小鼠心脏组织氧化反应.     

正交试验优选大孔树脂吸附纯化乌头碱工艺

目的：优选大孔吸附树脂提取纯化附子中总生物碱的工艺条件。方法：应用正交设计安排试验因素，以干物质量和乌头碱含量为指标，比较不同工艺条件对乌头类生物碱的提取分离效果。结果：不同上样液的pH对生物碱的吸附影响非常显著，洗脱液乙醇浓度对生物碱的洗脱效果有显著影响。综合得率和生物碱含量的影响因素，确定最佳提取条件为：上样液pH为10，浓度为0．0578mg&#183;ml^-1（乌头碱），洗脱液（乙醇）浓度为90％。结论：本法的工艺条件可用于含乌头类生物碱的药材提取或中药制剂的制备。     

中国乌头的研究Ⅶ.乌头生物碱的薄层层析

本文选择七种已知结构的生物碱,用不加粘合剂氧化铝薄层层析法的桑件进行了研究,结果表明此法完全可应用于乌头生物碱的检定.其较好条件是细度小于150号筛孔,活性Ⅳ级碱性氧化铝为吸附剂;石油醚-乙醚(1:10)为展开剂和以碘蒸气为显色剂.将此薄层层析条件应用到从中国乌头中分出的新生物碱检查时,亦获得良好的结果.     

Causes and prevention of herb-induced aconite poisonings in Asia

The recent reports from Hong Kong, Taiwan, China and Korea were reviewed to determine the causes and prospects for prevention of herb-induced aconite poisonings. The contributory factors included overdose (use of greater than the recommended doses), faulty processing (after harvest and during decoction), use of tincture (herbal medicinal wine), use of crude aconite roots (for preparing decoction, proprietary medicines and tincture), lack of standardisation in processing of aconite roots and preparation of tincture and proprietary medicines, unsupervised use of aconite roots and contamination or mix-up with aconite roots. As tincture (herbal medicinal wine) made from aconite roots contains a much larger amount of Aconitum alkaloids, the public should be strongly discouraged from making their own and taking it by mouth. Aconite roots should only be used after post-harvest processing and proper decoction. The public should be educated on the hazards from unsupervised use and improper decoction of processed aconite roots. There should be regular publicity measures to promote awareness among the herbalists and to publicise the risk of serious cardiotoxicity if the recommended doses of processed aconite roots are exceeded. The processing of aconite roots and their proprietary preparations should be standardised. Quality control of processed aconite roots and their proprietary preparations should be strengthened. National reporting or monitoring systems can be used to identify the causes of aconite poisonings and assess the impact of preventive measures.     

乌头安全有效性研究进展及其临床应用前景

目的 分析文献中乌头疗效与安全应用的研究进展、用药特点与注意事项,促进该药材及其复方制剂的临床合理与广泛的应用。方法 通过检索秦汉时期至今的主要书籍和文献,从乌头的药理作用、药效物质、炮制方法、药代动力学、民间应用、临床疗效等多方面进行系统的总结和归纳,并提出乌头安全有效的应用前景和现代制剂研制的基本要求。结果 乌头有抑瘤、镇痛、提高免疫力、抗炎等作用,其药效物质为乌头类生物碱,其毒性大小为乌头碱、新乌头碱、次乌头碱>苯甲酰乌头碱、苯甲酰新乌头碱、苯甲酰次乌头碱>乌头原碱、新乌头原碱、次乌头原碱;体内研究显示乌头在大鼠体内吸收迅速,代谢较快,但不同文献中同一成分生物半衰期数据差异较大,变化幅度分别为：乌头碱0.936~20.130 h,新乌头碱1.490~20.930 h,次乌头碱1.930~17.300 h,导致体内消除速率和蓄积时间明显不同;临床使用中出现的毒副作用主要与合理炮制、恰当配伍、正确服用、控制剂量,避免误服等操作的失当以及个体差异相关。结论 乌头应用的民间基础扎实,镇痛等疗效确切,炮制工艺、药理毒理与临床应用等数据翔实,建议加强体内研究数据的确证,开展临床安全的再评价,进一步阐明乌头的作用机制和量效关系。