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。     

Biodiversity of Convolvulaceous species that contain ergot alkaloids,indole diterpene alkaloids,and swainsonine

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n = 30) for indole diterpene alkaloids, representing four genera, Argyreia, Ipomoea, Stictocardia, and Turbina, that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species.     

Studies on the Sesquiterpene Alkaloids of Tripterygium wilfordii Hook.

This paper reports two sesquiterpene alkaloids isolated from Tripterygium wilfordii Hook. f. Their structures are assigned as Ⅰ and Ⅱ, respectively, by MS, UV, IR, 1HNMR and 13CNMR spectroscopic analysis. Alkaloid Ⅱ is a new alkaloid named wilfornine. These al- kaloids were shown to be immunosuppressives.     

Cellular Localization of Debromohymenialdisine and Hymenialdisine in the Marine Sponge <Emphasis Type="Italic">Axinella</Emphasis> sp. Using a Newly Developed Cell Purification Protocol

Sponges (Porifera), as the best known source of bioactive marine natural products in metazoans, play a significant role in marine drug discovery and development. As sessile filter-feeding animals, a considerable portion of the sponge biomass can be made of endosymbiotic and associated microorganisms. Understanding the cellular origin of targeted bioactive compounds from sponges is therefore important not only for providing chemotaxonomic information but also for defining the bioactive production strategy in terms of sponge aquaculture, cell culture, or fermentation of associated bacteria. The two alkaloids debromohymenialdisine (DBH) and hymenialdisine (HD), which are cyclin-dependent kinase inhibitors with pharmacological activities for treating osteoarthritis and Alzheimer's disease, have been isolated from the sponge Axinella sp. In this study, the cellular localization of these two alkaloids was determined through the quantification of these alkaloids in different cell fractions by high-performance liquid chromatography (HPLC). First, using a differential centrifugation method, the dissociated cells were separated into different groups according to their sizes. The two bioactive alkaloids were mainly found in sponge cells obtained from low-speed centrifugation. Further cell purifications were accomplished by a newly developed multi-step protocol. Four enriched cell fractions (C1, C2, C3, and C4) were obtained and subjected to light and transmission electron microscopy, cytochemical staining, and HPLC quantification. Compared to the low concentrations in other cell fractions, DBH and HD accounted for 10.9% and 6.1%, respectively, of dry weight in the C1 fraction. Using the morphological characteristics and cytochemical staining results, cells in the C1 fraction were speculated to be spherulous cells. This result shows that DBH and HD in Axinella sp. are located in sponge cells and mostly stored in spherulous cells.     

人工栽培山莨菪和野生山莨菪中4种托烷类生物碱含量的比较研究

为了评估人工栽培山莨菪的药用价值,采用高效液相色谱技术对人工栽培和野生山莨菪的地上部分和根中具有生物活性的4种托烷类生物碱：樟柳碱、山莨菪碱、东莨菪碱和阿托品的含量进行了测定。结果表明无论是人工栽培还是野生植物,地上部分中4种生物碱含量均远低于根,这解释了人们为什么用山莨菪的根而不是整株人药。在栽培植物的根中,一年生山莨菪中各生物碱含量均小于二年生山莨菪,其根中4种生物碱总量与野生根相比差异不是很明显;二年生山莨菪根中,4种生物碱总量以及樟柳碱、东莨菪碱和阿托品含量均比野生的高。这说明人工栽培的山莨菪,尤其是二年生山茛菪,同野生山莨菪一样具有一定的药用价值。     

Determination of four pyridine alkaloids from Tripterygium wilfordii Hook. f. in human plasma by high-performance liquid chromatography coupled with mass spectrometry

A novel liquid chromatography-atmospheric-pressure chemical ionization-mass spectrometry (LC-APCI/MS) method was developed and validated for the simultaneous determination of four sesquiterpene pyridine alkaloids (wilfortrine, wilfordine, wilforgine and wilforine) in human plasma. The chromatographic separation was performed on a Shim-pack XR-ODS column using an ammonium acetate buffer solution-acetonitrile in a gradient program. The detection was achieved by an ion trap mass spectrometry in the positive selected ion monitoring (SIM) mode. The method utilized acetonitrile as protein precipitation solvent and followed by solid-phase extraction (SPE). Calibration curves were linear for the four alkaloids over the range of 0.5-100.0 μg/L with the limits of quantification of 0.5 μg/L, while the method exhibited the recovery of 86.5-98.6%, intra- and inter-day RSDs of less than 8.2% and 12.8%, respectively. Methodology was validated in line with the EU requirements (Commission Decision 2002/657/EC). Results of incurred samples demonstrated excellent reproducibility. To our knowledge, this is the first analytical method for simultaneous determination of the four sesquiterpene pyridine alkaloids in plasma. The method was applicable to clinical pharmaceutical research of alkaloids in rheumatoid arthritis volunteer patients after oral administrations.     

Components of Stem Barks of Winchia calophylla A. DC. and Their Bronchodilator Activities

The Dai medicinal plant Winchia calophylla A. DC. (Apocynaceae) has efficacy as an anticough and anti-asthmatic medication. In order to investigate its relative bioactive components, we studied the chemical constituents of this plant. Using repeated column chromatography, 28 compounds, including loganin, six phenolic compounds, 17 indole alkaloids, three pyridine alkaloids, and a quinoline alkaloid,were isolated from the stem barks of W. calophylla. Loganin, paeonol, N (4)-methyl akuammicine, and cantleyine exhibited a moderate relaxation effect on isolated smooth muscles of guinea-pig tracheal spirals and lung strips and may be the bioactive components responsible for the bronchodilation produced by W. calophylla.     

Components of Stem Barks of Winchia calophylla A. DC. and Their Bronchodilator Activities

The Dai medicinal plant Winchia calophylla A. DC. (Apocynaceae) has efficacy as an anticough and anti-asthmatic medication. In order to investigate its relative bioactive components, we studied the chemical constituents of this plant. Using repeated column chromatography, 28 compounds, including loganin, six phenolic compounds, 17 indole alkaloids, three pyridine alkaloids, and a quinoline alkaloid, were isolated from the stem barks of W. calophylla. Loganin, paeonol, N (4)-methyl akuammicine, and cantleyine exhibited a moderate relaxation effect on isolated smooth muscles of guinea-pig tracheal spirals and lung strips and may be the bioactive components responsible for the bronchodilation produced by W. calophylla.     

The bioactive alkaloids identified from Cortex Phellodendri ameliorate benign prostatic hyperplasia via LOX-5/COX-2 pathways

BackgroundThe bioactive alkaloids identified from Cortex Phellodendri (CP) were highly effective in treating rats with benign prostatic hyperplasia (BPH). Specifically, lipoxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) were identified as two primary targets for alleviating inflammation in BPH rats. However, it remains unknown whether the alkaloid components in CP can interact with the two target proteins.PurposeTo further identify bioactive alkaloids targeting LOX/COX pathways.MethodsAn affinity-ultrafiltration mass spectrometry approach was employed to screen dual-target LOX-5/COX-2 ligands from alkaloid extract. The structures of bioactive alkaloids were characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. To understand the molecular mechanisms underlying the effects of bioactive alkaloids, the expression levels of LOX-5 and COX-2 in BPH model rats were investigated at both protein and mRNA levels. The LOX-5/COX-2 enzymes activity experiments and molecular docking analysis were performed to fully evaluate the interactions between bioactive alkaloids and LOX-5/COX-2.ResultsAfter comprehensive analysis, the results showed that bioactive alkaloids could suppress the expression of LOX-5 and COX-2 simultaneously to exert an anti-inflammatory effect on the progression of BPH. In addition, the screened protoberberine, demethyleneberberine was found to exhibit prominent inhibitory activities against both LOX-5 and COX-2 enzymes, palmatine and berberine with moderate inhibitory activities. Molecular docking analysis confirmed that demethyleneberberine could interact well with LOX-5/COX-2.ConclusionThis study is the first to explore the inhibitory effects of bioactive alkaloids from CP on LOX-5 and COX-2 activities in BPH rats. Our findings demonstrate that the bioactive alkaloids from CP can ameliorate BPH via dual LOX-5/COX-2 pathways, which serves as an efficient approach for the discovery of novel drug leads from natural products with reduced side effects.     

Rapid Analysis of Four Alkaloids in <i>Uncaria rhynchophylla</i> by Core-Shell Column HPLC and Quantitative Analysis of Multi-Components by Single Marker (QAMS)

As a traditional herbal medicine, the major alkaloids in Uncaria rhynchophylla have been proven to have blood pressure-lowering and sedative effects. It is essential to develop an effective method for the determination of the major alkaloids in U. rhynchophylla. In this research, a rapid quantitative analysis involving multi-components analysis by a single marker strategy coupled with core-shell column HPLC was adopted to analyse four alkaloids (corynoxeine, isocorynoxeine, isorhynchophylline, rhynchophylline) in U. rhynchophylla. Isorhynchophylline was selected as the internal reference substance, the content of which was determined by the traditional external standard method. Relative correction factors (RCF) between isorhynchophylline and the other three alkaloids were calculated respectively. The results showed that the QAMS method had good robustness under different HPLC instruments. Nineteen batches of U. rhynchophylla were tested. No significant difference was observed between the results by QAMS and EMS (Correlation coefficient > 0.99, p > 0.05). The QAMS method could be employed as a rapid, effective technique for the quality control of U. rhynchophylla.     

Peimisine and peiminine production by endophytic fungus Fusarium sp. isolated from Fritillaria unibracteata var. wabensis

Steroidal alkaloids, as the major biologically active components in Bulbus Fritillariae, possess a variety of toxicological and pharmacological effects on humans. The objective of this work was to determine whether endophytic fungi isolated from fresh bulbs of Fritillaria unibracteata var. wabensis can produce one or more alkaloids like its host plant. Four classical reagents including Wagner's, iodine-potassium iodide, Mayer's and improved Dragendorff's were used for primary screening. Then thin-layer chromatography (TLC) and high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) were employed to identify the fermentation products of the selected strains. The results showed that extract from one stain (WBS007) has positive reactions in process of primary screening. A further TLC scan and HPLC-ELSD showed that strain WBS007 had two components with the same TLC relative front (Rf) value and HPLC retention time (RT) as authentic peimisine and peiminine. In addition, strain WBS007 was identified as Fusarium sp. based on phylogenetic analysis of ITS sequences. Thus, strain WBS007 produced the bioactive ingredient peimisine and peiminine, as does its host plant, and could be used for the production of peimisine and peiminine by fermentation.     

Cell types, microsymbionts, and pyridoacridine distribution in the tunic of three color morphs of the genus Cystodytes (Ascidiacea, Polycitoridae)

Abstract. The tunic of colonial ascidians of the genus Cystodytes is a dynamic and complex system where a variety of cell types and microsymbionts are found. The tunic is also the site where pyridoacridine alkaloids involved in chemical defense are found. We wanted to explore the composition of symbionts and tunic cell types and their relationship with localization of alkaloids in three color morphs (usually attributed to the species Cystodytes dellechiajei ). Tunic morphology was studied by means of transmission electron microscopy, and energy-dispersive X-ray microanalysis was performed for indirect localization of the bioactive alkaloids produced by these morphotypes. The main cell types identified are bladder cells, pigment cells, amebocytes, phagocytes, and morula cells. Amebocytes include several subtypes that may correspond to a sequence of ontogenetic stages; these cells also seem to give rise to other cell types. In the three morphotypes, the morphology of the tunic and tunic cells is basically the same. The alkaloids are localized in the pigment cells. At least three types of bacteria are present in the tunic, but they are scarce and do not store the targeted bioactive alkaloids. Our results indicate that, although pyridoacridine alkaloids are present in these ascidians, as in a variety of animal phyla, their wide taxonomic range is not necessarily the result of production by common microsymbionts, but rather of the convergent evolution of a successful biosynthetic pathway.     

“COSY”谱用于昆明山海棠生物碱的鉴定

昆明山海棠(Tripterygium hypoglaucum)的生物碱可从甲醇中结晶析出,是多元混合物。高压液相色谱分析出现5个峰,用制备性烷化硅胶RP-18薄层层析板分离纯化,5个生物碱逐一分开。用化学和光谱法证明为:雷公藤次碱、雷公藤碱乙、雷公藤碱丁、雷公藤碱及卫矛碱(1—5)。高压液相色谱分析指出,前二者为其中的主要成分。用高分辨核磁共振谱及COSY谱指定了它们的质子及其在分子中的取向,说明了它们之间结构和构型的关系。     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

Phytochemical Variation in <Emphasis Type="Italic">Fritillaria cirrhosa</Emphasis> D. Don (<Emphasis Type="Italic">Chuan Bei Mu</Emphasis>) in Relation to Plant Reproductive Stage and Timing of Harvest<Superscript>1</Superscript>

Phytochemical Variation in Fritillaria cirrhosa D. Don ( Chuan Bei Mu ) in Relation to Plant Reproductive Stage and Timing of Harvest. Economic development in southwest China and the increasing use of traditional Chinese medicine (TCM) worldwide have led to intensified collection of native medicinal plants. Fritillaria cirrhosa D. Don (Chuan Bei Mu), commonly used for the treatment of cough in TCM, is endemic to the Hengduan Mountain region of southwest China and is under increasing pressure from over-collection and decreasing suitable alpine habitat. The bioactive compounds in F. cirrhosa bulbs, isosteroidal alkaloids, are greatly influenced by environmental conditions and fluctuate in content and concentration with plant age and reproductive stage. Aiming at obtaining useful information for the sustainable management of wild F. cirrhosa populations, we evaluated how the phytochemical composition of F. cirrhosa bulbs varies at various stages of plant reproductive development. Using chemical methods and high-performance liquid chromatography, we extracted and analyzed two major bioactive alkaloids from F. cirrhosa bulb samples collected throughout the Hengduan Mountain region. Plant reproductive stage was found to affect the concentration of bioactive alkaloids in F. cirrhosa bulbs. Bulb alkaloid concentration was highest during the early stages of fruit development and decreased significantly with fruit maturation. These results lend biochemical support to the practice of harvesting F. cirrhosa during the early stages of plant senescence (i.e., early fruit development).     

Anticholinesterase activity in an alkaloid extract of Huperzia saururus.

Huperzia saururus (Lam.) Trevis. (Lycopodiaceae) is used widely in Argentinian traditional medicine as an aphrodisiac and for memory improvement. An aqueous extract from the aerial parts was obtained by decoction, revealing the presence of alkaloids, among other constituents. By partition with organic solvent in alkaline media, alkaloids were extracted and then purified by gel permeation. We studied the anticholinesterase activity in vitro of the alkaloid extract using erythrocyte membranes and human serum as sources of acetylcholinesterase and pseudocholinesterase, respectively. The results show a marked inhibition of true acetylcholinesterase with an IC50 value of 0.58 microg/ml. Low inhibition of pseudocholinesterase was observed (IC50 value = 191 microg/ml). This shows a selectivity of the extract for the true acetylcholinesterase. Furthermore, chemical study of the bioactive extract was performed by GC-MS, revealing the presence of seven Lycopodium alkaloids, including some not identified previously: sauroxine, 6-hydroxylycopodine, N-acetyllycodine, lycopodine, lycodine, N-methyllycodine, and clavolonine. Further investigations will be undertaken in order to discover which compound/s are responsible for the aqueous extract's acetylcholinesterase activity.     

Study of Sesquiterpene Alkaloids from Tripterygium wilfordii (IV)

Two sesquiterpene alkaloids,wilforine and wilfordsuine were isolated from Tripterygium wilfordii Hook. f. Wilfordsuine was a new alkaloid. The structures of wilforine and wilfordsuine were elucidated on the basis of spectral data,especially 2D-NMR techniques　(H'-'H COSY,H'-13C COSY, NOESY, COLOC methods).     

Advances in study of ginsenoside biosynthesis pathway in <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Panax ginseng C. A. Meyer is one of the important nutraceutical and medicinal plants, which is used worldwide. Until now, ginseng has been reported to contain saponins, antioxidants, peptides, polysaccharides, fatty acids, vitamins, alkaloids, lignans, and flavonoids. The saponins, known as ginsenosides, are widely believed to be the major bioactive compounds of ginseng. In this article, ginsenoside biosynthesis pathway and key enzymes regulation are also described. This review provides a reference for improving ginsenoside contents through regulation of ginsenoside biosynthesis pathway.     

Development and validation of a rapid capillary zone electrophoresis method for the determination of aconite alkaloids in aconite roots

Introduction – Aconites, with aconite alkaloids as the major therapeutic and toxic components, are used for the treatment of analgesic, antirheumatic and neurological symptoms. Quantification of the aconite alkaloids is important for the quality control of aconite‐containing drugs. Objective – To establish a validated capillary zone electrophoresis (CZE) method for the simultaneous determination of six major alkaloids, namely aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine, in crude and processed aconite roots. Methodology – The CZE method was optimised and validated using a stability‐indicating method. The optimised running buffer was a mixture of 200 mm Tris, 150 mm perchloric acid and 40% 1,4‐dioxane (pH 7.8) with the capillary thermostated at 25°C. Results – Using the optimised method, six aconite alkaloids were well separated. The established method showed good precision, accuracy and recovery. Contents of these alkaloids in crude and processed aconites were determined and it was observed that the levels of individual alkaloids varied between samples. Conclusion – The developed CZE method was reliable for the quality control of aconites contained in herbal medicines. The method could also be used as an approach for toxicological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

低磷胁迫对雷公藤幼苗叶片生理生化特性的影响

为研究雷公藤的耐磷胁迫性,对雷公藤（Tripterygium wilfordii Hook.f.）一年生和三年生同一无性系扦插苗采用土培的方法进行了控制条件下的低磷胁迫实验,以KH2PO4为磷源,土壤中P2O5浓度为0、5、10、15、20、25（CK）mg/kg,低磷胁迫3个月和6个月后取叶片测定其丙二醛（MDA）、脯氨酸（Pro）含量以及超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性和酸性磷酸酶（APA）活性的变化。实验结果表明：低磷胁迫下一年生和三年生雷公藤叶片SOD、APA活性升高,MDA、Pro含量增加,并呈现随低磷胁迫加重和胁迫时间延长而上升趋势,低磷胁迫下各处理的一年生和三年生雷公藤叶片CAT、POD活性普遍低于对照,并随着低磷胁迫程度的加重而呈下降趋势。低磷胁迫下雷公藤幼苗叶片的几种保护酶活性、MDA、Pro含量以及APA活性响应都较为灵敏,保护酶系统在15、20 mg/kg处理下能够起到较好保护作用,表明雷公藤能通过自身生理调节来适应中度和轻度缺磷环境;综合各项生理指标,三年生雷公藤相对于一年生雷公藤显示了较强的抗氧化能力和渗透调节能力,具有较强的耐低磷能力,对磷较为缺乏的林地下套种雷公藤的苗龄选择有参考价值。