高效液相色谱法测定延胡索药材中7种异喹啉类生物碱的含量

本文建立了同时测定延胡索中巴马汀、小檗碱、去氢紫堇碱、四氢巴马汀、异紫堇球碱、紫堇碱和四氢黄连碱7种主要异喹啉生物碱含量的高效液相色谱方法,并考察了不同来源延胡索中异喹啉生物碱的含量。采用Agilent SB C_(18)柱色谱柱(4.6×250 mm,5μm),流动相为乙腈-0.1%的醋酸水溶液(三乙胺调pH至5.0),梯度洗脱,流速为1.0 m L/min,检测波长为280 nm。巴马汀、小檗碱、去氢紫堇碱、四氢巴马汀、异紫堇球碱、紫堇碱和四氢黄连碱在2.0~40.1、2.0~39.5、5.1~101.3、5.0~99.8、2.1~41.2、5.0~100.1μg/m L和2.0~39.7μg/m L浓度范围内线性良好,平均加样回收率分别为95.6%、96.1%、96.5%、101.4%、101.9%、97.3%和102.3%,RSD分别为2.77%、2.50%、3.33%、4.18%、2.93%、2.86%和2.60%。不同来源延胡索样品中7种异喹啉类生物碱含量差异较大,研究表明该方法准确、可靠,可用于延胡索原药材质量控制。     

紫金龙低极性部位的生物碱成分研究

采用柱色谱等分离方法,对紫金龙Aconitum vilmorinianum var.altifidum W.T.Wang低极性部位的生物碱类化学成分进行分离纯化,依据其理化性质及波谱数据分析进行结构鉴定。从中分离、鉴定了7个二萜生物碱类化合物和1个二萜生物碱类复合物,分别为:黄草乌碱丁(1)、hemsleyaconitine G(2)、hemsleyaconitine F(3)、hemsleyaconitine A(4)、14-O-veratroylneoline(5)、塔拉胺(6)、lipoyunaconitine(7)和hemsleyadine(8)。化合物1~8均为首次从该植物中分离得到。     

丁三七的生物碱成分

丁三七即大花荷包牡丹(Dicentra macranlha Oliv.) 为紫堇科荷包牡丹属植物。分布于我国云南、四川、贵州和湖北西部。在云南昭通地区的彝良和大关县海拔1500—2600米山地有较丰富的资源。民间用其根茎治疗支气管炎,并有祛瘀消肿的作用。从同属植物中曾分离到多种异喹啉生物碱。丁三七的化学成分迄今未见报道。本文报道从云南昭通地区大关县产的丁三七根茎中分离得到的5个异喹啉生物碱的结构鉴定。     

大花地不容块根生物碱成分的研究

为研究大花地不容块根的生物碱成分,该研究采用酸提碱沉法从大花地不容块根中提取出总生物碱,通过硅胶柱色谱法和制备液相色谱技术对大花地不容块根的总生物碱提取物进行分离纯化,并利用波谱学手段对化合物进行结构鉴定。结果表明:(1)从总生物碱提取物中分离得到11个化合物,分别鉴定为青藤碱(1)、青风藤碱(2)、斯帝酚灵碱(3)、瑞枯灵(4)、异紫堇定(5)、紫堇单酚碱(6)、巴婆碱(7)、sukhodianine(8)、荷包牡丹碱(9)、7-氧代克班宁(10)和巴马汀(11)。(2)体外细胞毒性测试显示,大花地不容总生物碱和其主成分青藤碱对人类肺癌细胞A549的IC₅₀值分别为7.5×10^-4 g·mL^-1和6.59×10^-9 g·mL^-1。化合物2、3、4、7、8、9和10系首次从大花地不容中分离得到。大花地不容块根中含有吗啡烷、原阿朴啡、阿朴啡、苄基四氢异喹啉和原小檗碱5种类型的生物碱。     

迭裂黄堇生物碱的研究

从藏药迭裂黄堇的根中分得5个生物碱,经光谱分析和化学方法鉴定为四氢紫堇萨明(Ⅰ)、四氢黄连碱(Ⅱ)、紫堇球茎碱(Ⅲ)、紫堇萨明(Ⅳ)和黄连碱(Ⅴ)。     

药用地不容中的生物碱

从药用地不容(Stephania officinarum Lo et Yang)的块根中分得8种生物碱,根据其理化性质、波谱分析及化学方法鉴定为异紫堇定碱(Ⅰ),1-四氢巴马汀碱(Ⅱ)、青风藤碱(Ⅲ)、克班宁(Ⅳ)、异波尔定碱(Ⅴ)、紫堇块茎碱(Ⅵ)、巴马汀碱(Ⅶ)和 N-β-甲基四氢巴马汀碱硝酸盐(Ⅷ)。其中Ⅰ为主要生物碱,得率为1.24%。Ⅷ为天然界中新的季铵型生物碱,并首次证实以硝酸盐形式存在于植物体中。     

房山紫堇地上部分化学成分及其抗乳腺癌细胞增殖作用研究

对房山紫堇Corydalis fangshanensis地上部分化学成分及其体外抗乳腺肿瘤细胞活性进行研究。采用各种色谱分离技术对乙酸乙酯和正丁醇两个萃取部位进行系统分离纯化,通过多种波谱数据(1D、2D NMR、MS、IR、UV)鉴定化合物结构,从房山紫堇乙酸乙酯和正丁醇两个部位中鉴定了17个化合物,分别为7-acetyl thalifoline(1)、pycnarrhine(2)、corynoxidine(3)、trans-isocorypalmine N-oxide(4)、四氢巴马汀(5)、四氢药根碱(6)、liriotulipiferine(7)、异波尔定碱(8)、乌药碱(9)、脱氢甲卡维丁(10)、feruloylputrescine(11)、木黄酮(12)、二甲基咖啡酸(13)、4-羟基-3-甲氧基桂皮酰基-β-D-葡萄糖苷(14)、水仙苷(15)、丁香脂素-4-O-β-D-葡萄糖苷(16)、异落叶松脂醇9-O-β-D-葡萄糖苷(17)。其中化合物1作为天然产物首次报道,2～17是首次从房山紫堇中分离得到。结合体外MTT法评价单体化合物对人乳腺癌肿瘤细胞(MDA-MB-23...     

山豆根和苦参生物碱类成分UPLC/Q-TOF MS^E比较研究北大核心CSCD

使用UPLC/Q-TOF MSE对山豆根和苦参的生物碱类成分进行详细的比较研究。色谱柱:Acquity UPLC BEH C18(2.1 mm×150 mm,1.7μm);流动相:乙腈-5 mmol/L甲酸铵水溶液;流速为0.5 m L/min;检测器:四极杆串联飞行时间质谱(Q-TOF),正离子检测模式。根据离子流色谱图中色谱峰的分子量及其二级质谱碎片离子的裂解规律,结合参考文献进行结构推定;利用主成分分析及正交偏最小二乘法进行聚类分析。结果从山豆根总生物碱中鉴定12个主要成分,从苦参生物碱中鉴定了11个主要成分,其中有10个是相同的结构,但相同成分在两种药材中的含量差异很大;聚类分析表明山豆根和苦参具有显著的差异。本研究表明山豆根和苦参的主要生物碱成分虽然结构相同,但含量差异很大,二者临床疗效差异,可能由于生物碱含量的差异,或生物碱以外的其他成分所引起。     

山豆根和苦参生物碱类成分UPLC/Q-TOF MS~E比较研究

使用UPLC/Q-TOF MSE对山豆根和苦参的生物碱类成分进行详细的比较研究。色谱柱:Acquity UPLC BEH C18(2.1 mm×150 mm,1.7μm);流动相:乙腈-5 mmol/L甲酸铵水溶液;流速为0.5 m L/min;检测器:四极杆串联飞行时间质谱(Q-TOF),正离子检测模式。根据离子流色谱图中色谱峰的分子量及其二级质谱碎片离子的裂解规律,结合参考文献进行结构推定;利用主成分分析及正交偏最小二乘法进行聚类分析。结果从山豆根总生物碱中鉴定12个主要成分,从苦参生物碱中鉴定了11个主要成分,其中有10个是相同的结构,但相同成分在两种药材中的含量差异很大;聚类分析表明山豆根和苦参具有显著的差异。本研究表明山豆根和苦参的主要生物碱成分虽然结构相同,但含量差异很大,二者临床疗效差异,可能由于生物碱含量的差异,或生物碱以外的其他成分所引起。     

多刺绿绒蒿中的一个新生物碱类化合物（英文）

多刺绿绒蒿在藏区是一种非常重要的民间用药。该植物中已发现多种生物碱,其中很多都具有显著的生物活性。用正相硅胶、ODS柱色谱从多刺绿绒蒿的地上部分分离得到了三个生物碱,根据其质谱、红外、核磁等波普数据鉴定结构,分别为(6a-S)-des-N-methyl-(+)-cryprochine(1),华紫堇碱(2)及原鸦片碱(3),其中化合物1为新化合物,化合物2为首次在该植物中分离得到。     

Quinoline alkaloids from Acronychia laurifolia.

Bioassay-directed fractionation of a root extract of Acronychia laurifolia (Rutaceae) using the KB-V1+ human tumor cell line led to the isolation of six quinoline alkaloids. One of these alkaloids is novel, namely, 2,3-methylenedioxy-4,7-dimethoxyquinoline and the other five were identified as the known compounds, evolitrine, gamma-fagarine, skimmianine, kokusaginine and maculosidine. Two known bis-tetrahydrofuran lignans, sesamolin and yangambin, were also identified. The structure of the new alkaloid was determined by spectroscopic methods. All of the isolates were evaluated against a panel of human cancer cell lines; four of the alkaloids showed weak cytotoxic activity.     

雷公藤总生物碱对粘虫幼虫神经系统酶活性及神经递质含量的影响

【目的】明确雷公藤Tripterygium wilfordii Hook. f.生物碱对粘虫Mythimna separata (Walker)神经系统的影响,为阐明其杀虫作用机制提供依据。【方法】采用载毒叶片法测定粘虫5龄幼虫经雷公藤总生物碱处理后体内乙酰胆碱酯酶（AChE）、Na⁺, K⁺-ATPase、Ca²⁺, Mg²⁺-ATPase、谷丙转氨酶（GPT）和谷氨酸脱羧酶（GAD）等重要神经系统酶活性及乙酰胆碱（ACh）、谷氨酸（Glu）和γ-氨基丁酸（GABA）等神经递质的含量。【结果】雷公藤总生物碱处理对粘虫5龄幼虫AChE无明显影响,麻醉期处理粘虫幼虫体内ACh相对含量与同期对照无显著差异。处理粘虫幼虫在轻度麻醉期、深度麻醉期和复苏期体内GABA和Glu含量显著升高,GABA含量分别升高了89.86%, 49.28%和20.29%,Glu含量分别升高了24.55%, 23.33%和8.13%。处理粘虫幼虫GPT活性明显受到抑制,而GAD活性无明显变化。处理明显抑制粘虫幼虫头部Na⁺, K⁺-ATPase和Ca²⁺, Mg²⁺-ATPase活性,但对中肠两种ATPase活性影响不大。【结论】研究结果有助于了解雷公藤生物碱对昆虫神经系统的影响,也为进一步阐明其作用靶标奠定了基础。     

Phytochemical complementarities among endophyte-infected tall fescue, reed canarygrass, birdsfoot trefoil and alfalfa affect cattle foraging

We determined whether plant diversity and sequence of plant ingestion affected foraging when cattle chose from plants that varied in concentrations of alkaloids, tannins and saponins. We hypothesized cattle that ate high-alkaloid grasses (endophyte-infected tall fescue (TF) or reed canarygrass (RCG)) would prefer forages high in tannins (birdsfoot trefoil, BFT+) or saponins (alfalfa, ALF+), because tannins and saponins can bind to alkaloids, presumably reducing their absorption. We further hypothesized that forages with tannins or saponins consumed before, rather than after, foraging on high-alkaloid grasses would promote greater use of those grasses presumably by binding to alkaloids, thereby reducing their absorption. In Phase 1, cattle (n = 32) grazed on either high (+) or low (-) alkaloid grass (TF or RCG) pastures for 30 min each morning at 0600 h and were then offered a choice of BFT+, BFT-, ALF+ and ALF- for 60 min each day for 12 days. In Phase 2, cattle (n = 32) were first offered a choice of BFT+ or ALF+ for 30 min at 0600 h and then placed on grass (TF+ or -, or RCG+ or -) pastures for 60 min for 12 days. In both phases, we had four spatial replications of four treatments with 2 per calves assigned to each of the 16 replications per treatment combinations. Scan samples of individuals at 2-min intervals were used to determine incidence of foraging on each plant species (%). Cattle grazed more on RCG than on TF in Phases 1 (62% v. 27%; P = 0.0015) and 2 (71% v. 32%; P = 0.0005). In Phase 1, cattle that first foraged on RCG+ or TF- subsequently preferred ALF over BFT, whereas cattle offered RCG- or TF+ foraged on ALF and BFT equally. Foraging by cattle on RCG was cyclic during Phase 1, whereas cattle foraging on TF markedly decreased incidence of use of TF from 41% to only 16% by the end of the 12-day trial (P = 0.0029). Contrary to the cyclic (RCG) or steadily declining (TF) use of grasses in Phase 1, cattle steadily and dramatically increased foraging on both RCG and TF throughout Phase 2, when they first grazed BFT+ or ALF+ followed by high-alkaloid grasses (P = 0.0159). Our findings suggest that in plant species the sequence of ingestion influenced foraging behavior of cattle and that secondary compounds influenced those responses.     

Induction of micronuclei by alkaloids extracted from Senecio brasiliensis and stored for 23 years

In the present study, we report the results of an investigation on pyrrolizidine alkaloids extracted from Senecio brasiliensis (Sprengel) Less., which were stored for more than 23 years under variable conditions of temperature and humidity and exposed to light. Both the crude alkaloid (integerrimine+retrorsine+impurities) and pure integerrimine conserved the ability to induce acute toxicity in mice, leading to the death of the animals in less than 24h. The alkaloids also conserved the potential to induce significant increases in micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow compared to the negative control. The administration of alkaloids to lymphocyte cultures blocked with cytochalasin-B showed no significant increase in micronucleus frequency in binucleated cells, probably due to the lack of a metabolic activation mechanism. However, an antimitotic effect was observed.     

Characterization of Opioid Receptor Subtypes in Solution

Stable opioid receptor binding activity that retains distinct subtype specificities (mu, delta, and kappa) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the delta, mu, and kappa sites (the latter subtype measured by the binding of [3H]dynorphin1-8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii greater than 60 A), whereas both those and several small species present (less than 60 A) bind opiate alkaloids and dynorphin1-8.     

Antagonistic effects of simultaneous exposure of ergot alkaloids on kidney adenosine triphosphatase system

Much of the research on fescue toxicosis has concentrated on evaluating animal response to grazing endophyte-infected (E+) versus endophyte-free tall fescue or the effects of single toxins such as ergonovine (EN), ergovaline (EV), or ergotamine (ET) on animal performance. Such approaches have eliminated the opportunity to test the possible additive, synergistic, or antagonistic interactions of one or more ergot alkaloids with the other ergot alkaloids found in E+ tall fescue. This study was conducted to determine the effects of simultaneous exposure of pairs of EN, EV, and ET on the kidney adenosine triphosphatase (ATPase) system in vitro. Tests were performed using three separate rat kidney homogenates and were repeated four times at concentrations of 0, 75, and 200 microM. Individually, EN, EV, and ET induced dose-dependent inhibitions of kidney Na(+)/K(+) ATPase, with EN being most potent, followed by purified EV, and then by ET. The ergot alkaloids inhibited Mg(2+) ATPase to a lesser degree than Na(+)/K(+) ATPase, with EN again being the most potent toxin. Simultaneous exposure to any combination of the ergot alkaloid pairs tested (EV + ET, EV + EN, and ET + EN) resulted in significant interactions (P < 0.05), indicating antagonistic effects on the inhibition of Na(+)/K(+) ATPase and Mg(2+) ATPase for most concentration combinations. These interactions suggest that in studies of the effects of any ergot alkaloid on animal performance, effects of other ergot alkaloids may also be present. Effects may not be additive, as was the case in this study, and the presence of one toxin may enhance or hinder the effectiveness of others.     

Spectrometric studies of cytotoxic protoberberine alkaloids binding to double-stranded DNA

The noncovalent complexes of five cytotoxic protoberberine alkaloids, that is, berberine, palmatine, jatrorrhizine, coptisine, and berberrubine with several double-stranded oligodeoxynucleotides were systematically investigated by using electrospray ionization mass (ESI-MS) and fluorescence spectrometric methods, with the aim of establishing the structure-activity relationships. ESI-MS spectrometric studies indicated that these five alkaloids showed both 1:1 and 1:2 binding stoichiometries with d(AAGAATTCTT)(2), d(AAGGATCCTT)(2), and d(AAGCATGCTT)(2). Their relative binding affinities toward these three double-stranded DNA were semi-quantitatively evaluated by measuring the ratios of the complex signals ([ds+alkaloid-5H](4-)+[ds+2alkaloid-6H](4-)) to those of the duplexes ([ds-4H](4-)) and also by ESI-MS competitive binding experiments. These experiments established the relative binding affinities of five protoberberine alkaloids in the order of palmatine>jatrorrhizine>coptisine>berberine>berberrubine with d(AAGAATTCTT)(2), palmatinecoptisine>jatrorrhizineberberine>berberrubine with d(AAGGATCCTT)(2) and palmatine>jatrorrhizinecoptisine>berberine>berberrubine with d(AAGCATGCTT)(2). Significantly, these alkaloids except berberrubine bound to d(AAGGATCCTT)(2) and d(AAGCATGCTT)(2) with the affinities comparable to Hoechst 33258, a typical DNA minor groove binder. The relative binding preferences of berberine, palmatine, and coptisine with these three double-stranded DNA were further quantitatively assessed by their association constants obtained from fluorescence titration experiments. The values revealed the order of relative binding affinities as berberine>coptisine>palmatine with d(AAGAATTCTT)(2) and coptisine>berberine>palmatine with d(AAGGATCCTT)(2) and d(AAGCATGCTT)(2). These results were not in full agreement with those obtained from ESI-MS experiments, maybe due to the different measuring solution conditions. The results from ESI-MS and fluorescence titration experiments indicated that the sequence selectivities of these five alkaloids were not significant and remarkable AT- or GC-rich DNA binding preferences were not obtained, in contrast to the report that berberine binds preferentially to AT-rich DNA. To provide further insight into the sequence selectivities, the association constants of berberine with d(AAGATATCTT)(2), 5'-AAGTAATCTT-3'/5'-AAGATTACTT-3', d(AAGGGCCCTT)(2), d(AAGGCGCCTT)(2), and 5'-AAGGCCGCTT-3'/5'-AAGCGGCCTT-3', that is double helical DNA from AT-rich to GC-rich sequences, were further measured by fluorescence titration methods. No significant differences in their association constants were observed, suggesting that berberine showed no remarkable sequence selectivities.     

Asexual Endophytes in a Native Grass: Tradeoffs in Mortality, Growth, Reproduction, and Alkaloid Production

Neotyphodium endophytes are asexual, seed-borne fungal symbionts that are thought to interact mutualistically with their grass hosts. Benefits include increased growth, reproduction, and resistance to herbivores via endophytic alkaloids. Although these benefits are well established in infected introduced, agronomic grasses, little is known about the cost and benefits of endophyte infection in native grass populations. These populations exist as mosaics of uninfected and infected plants, with the latter often comprised of plants that vary widely in alkaloid content. We tested the costs and benefits of endophyte infections with varying alkaloids in the native grass Achnatherum robustum (sleepygrass). We conducted a 4-year field experiment, where herbivory and water availability were controlled and survival, growth, and reproduction of three maternal plant genotypes [uninfected plants (E−), infected plants with high levels of ergot alkaloids (E+A+), and infected plants with no alkaloids (E+A−)] were monitored over three growing seasons. Generally, E+A+ plants had reduced growth over the three growing seasons and lower seed production than E− or E+A− plants, suggesting a cost of alkaloid production. The reduction in vegetative biomass in E+A+ plants was most pronounced under supplemented water, contrary to the prediction that additional resources would offset the cost of alkaloid production. Also, E+A+ plants showed no advantage in growth, seed production, or reproductive effort under full herbivory relative to E− or E+A− grasses, contrary to the predictions of the defensive mutualism hypothesis. However, E+A+ plants had higher overwintering survival than E+A− plants in early plant ontogeny, suggesting that alkaloids associated with infection may protect against below ground herbivory or harsh winter conditions. Our results suggest that the mosaic of E−, E+A+, and E+A− plants observed in nature may result from varying biotic and abiotic selective factors that maintain the presence of uninfected plants and infected plants that vary in alkaloid production.     

Insecticidal and antifeedant effects of two alkaloids from Cynanchum komarovii against larvae of Plutella xylostella L

A bioassay‐guided fractionation of Cynanchum komarovii crude alkaloid extract led to the isolation of two alkaloids. The isolated alkaloids were identified as 7‐demethoxytylophorine (1) and 6‐hydroxyl‐2,3‐dimethoxy phenanthroindolizidine (2) based on the comparison of their spectroscopic characteristics with the literature data. Insecticidal, antifeedant and growth inhibitory effects of these two alkaloids against the 3rd instar larvae of Plutella xylostella L. (Lepidoptera: Plutellidae) were examined. The results showed that alkaloid 1 was more toxic than alkaloid 2 against the 3rd instar larvae of Plutella xylostella L., but both alkaloids were less toxic than the total alkaloid fraction. For antifeedant activity, alkaloid 1 showed AFC₅₀ of 1.82 mg/ml at 24 h after treatment, alkaloid 2 showed 3.89 mg/ml, while total alkaloids showed 1.56 mg/ml. In dipping toxicity test, alkaloids 1 and 2 produced 93.3% and 63.3% mortality at 72 h after treatment, respectively, while total alkaloids produced 96.7% mortality. The LC₅₀ values for alkaloids 1, 2 and the total alkaloids were 3.54, 9.21 and 2.63 mg/ml, respectively. The development of larvae was also inhibited, and the growth inhibition rates at the concentration of 15.00 mg/ml were 92.8%, 78.2% and 98.6% for alkaloids 1, 2 and total alkaloids, respectively, at 72 h after treatment. Compared with antifeedant and dipping effect, the alkaloids 1, 2 and total alkaloid fraction revealed weak feeding toxicity, and their corrected mortality rates at the concentration of 15.00 mg/ml were 60.0%, 40.0% and 63.3% at 7 days after treatment. The LC₅₀ values for alkaloids 1, 2 and total alkaloids were 12.58, 32.37 and 8.88 mg/ml, respectively, at 7 days after treatment.     

Effects of ATP and magnesium ions on the fluorescence of harmala alkaloids. Restrictions for the use of harmala alkaloids as fluorescent probes for (Na+ + K+)-ATPase.

1. Harmine and harmaline were investigated as potentially useful fluorescent inhibitors of (Na+ + K+) activated ATPase. 29 From spectroscopic measurements both compounds were shown to form 1 : 1 complexes with ATP, the dissociation constants being 0.65 mM and 1.83 mM for harmine and harmaline respectively. Addition of Mg2+ and enzyme further affected these equilibria. 3. Although it was possible to demonstrate a competitive effect of harmine at the sodium-loading site of the enzyme, other inhibitory effects, including inhibitions of ouabain binding and the ouabain-insensitive ATPase were found. 4. It was concluded that the harmala alkaloids can inhibit (Na+ + K+)-activated ATPase in a complex way involving both Na- and ATP-binding sites. This severely limits their usefulness as spectroscopic probes.