从蛇足石杉中超声提取石杉碱甲和石杉碱乙

用正交试验确立了超声提取蛇足石杉生物碱的最佳条件。以石杉碱甲和石杉碱乙回收率为指标,考察了溶剂倍数、溶剂浓度、超声时间、超声功率等因素的影响。结果表明,在室温下超声提取的最优条件为：酸浓度O．8％(v／v),液固比例20：1,超声功率600W,超声15min。三次重复实验所得石杉碱甲和石杉碱乙回收率分别是9r7．3％和93．5％,相对标准偏差分别为1．31％和1．40％(n=3)。与传统的回流提取工艺相比,过程时间从2h缩短为15min,回收率提高了10％以上。     

HPLC法检测蛇足石杉内生真菌胶胞炭疽发酵液中石杉碱甲和石杉碱乙的含量

建立了高效液相色谱(HPLC)测定蛇足石杉(Huperzia serrate)内生真菌胶胞炭疽发酵液中石杉碱甲(Huperzina A)和石杉碱乙(Huperzine B)含量的方法,并以此方法检测胶胞炭疽发酵液中石杉碱甲和石杉碱乙含量的积累。内生真菌发酵液经氯仿萃取、甲醇溶解、过滤后进行高效液相检测分析,选用Agilent Eclipse plus-C18色谱柱(250 mm×4.6 mm,5μm),以0.015 mol/L乙酸铵(p H 6.8)和甲醇溶液(70∶30)为流动相进行等度洗脱,流速1 mL/min,检测波长为308 nm,连续检测内生真菌胶胞炭疽发酵液中第6–15天石杉碱甲和石杉碱乙的含量积累。结果表明,发酵提取液中的石杉碱甲和石杉碱乙可在25 min内进行很好的分离和分析,石杉碱甲在1.50-48.00μg/mL范围内线性关系良好(相关系数r为0.999 5),石杉碱乙在0.25-7.50μg/mL范围内线性关系良好(相关系数r为0.999 7),石杉碱甲和石杉碱乙的平均加标回收率分别为106.83%、108.06%,相对标准偏差(RSD)分别为3.34%、3.60%。该方法简便、快速、精密度高、结果准确,适用于内生真菌发酵液中石杉碱甲和石杉碱乙含量检测。在发酵过程中,内生真菌发酵液中石杉碱甲和石杉碱乙的含量呈现先增后减,随后有所增加继而又减少的趋势。石杉碱甲和石杉碱乙的含量分别在内生真菌发酵第14天、第8天达到最高,分别为12.417 0μg/mL、4.660 3μg/mL。该方法学的建立为内生真菌胶胞炭疽合成石杉碱甲与石杉碱乙的机制研究提供了检测手段,从而有利于药物新资源的开发。     

石杉碱甲提取工艺研究

以蛇足石杉为原料,采用生物碱分离的传统方法,通过盐酸浸提,氯仿萃取来制取、纯化石杉碱甲,选出最佳、最经济的生产工艺配方是:料液比为1:10,浸泡10 h,酸解3次,每次为45 min,酸解pH值为3.0,酸解温度为55℃,碱化pH为9.0;纯化过程中,用相当于石杉碱甲溶液体积15%的活性炭在pH为3.0条件下除杂,石杉碱甲的得率大于79.4%。     

武夷山脉石杉科植物石杉碱甲含量的研究

石杉植物因所含Hup A为高效、可逆、高选择性的中枢性乙酰胆碱酯酶抑制剂而受关注。实地采集标本并用改进的HPLC法测定了武夷山脉4种石杉科植物及不同居群间的石杉碱甲(Hup A)含量。结果显示石杉碱甲在长柄石杉、四川石杉、有柄马尾杉和华南马尾杉中均有分布,其中以有柄马尾杉中含量最高,达0.1510%;含量最低的是四川石杉,为0.016%。该地区优势种长柄石杉（千层塔）11个居群的石杉碱甲含量测定显示武平县梁野山和三元区中村乡两个居群含量较高,分别为0.0636%和0.0519%。对石松科部分种进行石杉碱甲测定,认为藤石松等不含石杉碱甲。结果表明武夷山脉地区拥有优良的石杉碱甲植物资源。     

石杉碱甲对氧自由基的影响

目的:研究石杉碱甲对氧自由基的影响,探讨石杉碱甲抗老年性痴呆的机制.方法:按50 mg/kg·d给大鼠皮下注射D-半乳糖6周后,按石杉碱甲0.1mg/kg·d、0.2mg/kg·d给药2周,检测血清和脑组织中SOD活力和MDA含量.结果:石杉碱甲能明显提高大鼠血清和脑组织中SOD的活力,同时降低MDA的含量.结论:加快氧自由基的清除、减少自由基损伤是石杉碱甲抗老年性痴呆的机制之一.     

石杉碱甲结构改造的研究进展

石杉碱甲是高效、高选择性的可逆性乙酰胆碱酯酶抑制剂,是治疗早老性痴呆症的一个有前景的药物.本文概述了石杉碱甲的性质、结构和构效关系,从结构简化、C10、吡啶酮环、脂桥环、环外双键和桥头氨基等方面综述了石杉碱甲结构改造的研究进展,并描述了所得的新型石杉碱甲类似物的抗乙酰胆碱酯酶的生物活性.在已合成的大量的石杉碱甲类似物中,部分类似物的活性优于天然石杉碱甲,石杉碱甲结构改造的研究取得了可喜的进展.     

石杉碱甲的研究进展

本文综述了1984年以来对石杉碱甲的药理作用、构效关系、合成及其天然来源等方面的研究进展;详细罗列了巳确认的石杉碱甲天然来源,以及从中分离出的其它生物碱;探讨了石碱杉甲的结构特征与其抑制乙酰胆碱酯酶活性能力之间的关系。     

千层塔中产石杉碱甲内生真菌的研究

从千层塔中分离产石杉碱甲内生真菌。采用平板分离法分离千层塔内生真菌,通过抑制乙酰胆碱酯酶活性和抗氧化活性筛选,结合薄层层析和HPLC测定活性菌株中的石杉碱甲含量,通过形态学ITS-rDNA序列分析鉴定内生真菌。共分离得到94株内生真菌,筛选到S29、L44、S94共3株乙酰胆碱酯酶抑制活性和清除DPPH·自由基活性都在50%以上,筛选到一株S29能产石杉碱甲的菌株。摇瓶发酵产量为每克干菌体的石杉碱甲含量为50.64μg。形态学与ITS-rDNA序列分析法鉴定该内生真菌为Podospora sp.属。药用植物千层塔体内Podospora sp.属的内生真菌可产石杉碱甲。     

石杉碱庚的结构鉴定

从石杉（Ｈｕｐｅｒｚｉａｓｅｒｒａｔａ（Ｔｈｕｎｂ．）Ｔｒｅｖ．）的正丁醇提取部分分离得到一个新的水溶性石杉类生物碱,通过ＩＲ、ＭＳ、１ＤＮＭＲ和２ＤＮＭＲ确定了其化学结构,命名为石杉碱庚（ｈｕｐｅｒｚｉｎｅＧ）。     

湘西蛇足石杉中石杉碱甲、乙和丙含量的测定

采用超声萃取法提取湘西蛇足石杉总生物碱,用高效液相色谱法同时测定其石杉碱甲、乙和丙含量,并分析其在植株不同部位的分布。结果表明:湘西4个样地蛇足石杉中石杉碱甲、乙和丙含量基本一致,分别达到0.5‰、0.3‰和0.04‰;但植株不同部位三种石杉碱含量差异显著,其中石杉碱甲和乙的分布均为叶>茎>根,石杉碱丙则是根大于叶和茎。由此可知,湘西蛇足石杉具有资源优势,石杉碱在植株的分布具有明显的部位选择性;采用HPLC可同时检测石杉碱甲、乙和丙,方法简单快速、准确可靠。     

Uptake Hydrogenase (Hup) in Common Bean (Phaseolus vulgaris) Symbioses

Strains of Rhizobium forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (hup) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the hup structural genes of Bradyrhizobium japonicum was present in 100 of 248 strains examined. EcoRI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal SacI fragment, which contains part of both bradyrhizobial hup structural genes. The DNA with homology to the hup genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the hup structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup⁺ strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains.     

Classification of the uptake hydrogenase-positive (Hup+) bean rhizobia as Rhizobium tropici.

Phenotypic and genetic characterization indicated that Hup+ bean rhizobial strains are type IIA and type IIB Rhizobium tropici. The Hup+ strain USDA 2840, which did not cluster with either of the two types of R. tropici in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences similar to those of three reference strains of R. tropici type IIA. The Hup+ strain USDA 2738, which clustered with the reference strain of R. tropici IIB in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences more closely resembling those of the reference strains of R. tropici type IIA than those of type IIB. DNA amplification with the Y1 and Y2 primers to generate a portion of the 16S rDNA operon was useful to distinguish R. tropici type IIA strains from other bean rhizobial strains. The phylogenetic position of the type IIA strain of R. tropici USDA 2840, determined from the partial 16S rDNA sequence, indicated a more distant relationship with the type IIB strain of R. tropici CIAT899 than with the as yet unnamed rhizobial species of Leucaena leucocephala, TAL 1145. Therefore, we suggest that it may be appropriate either to separate R. tropici types IIA and IIB into two different species or to identify TAL 1145 to the species level as a third type of R. tropici.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within Hup+Rhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H₂-uptake hydrogenase positive (Hup⁺) or negative (Hup⁻) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H₂ oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H₂-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H₂-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup⁺ strains in this symbiotic species.     

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H₂ is an obligate by-product of N-fixation. Recycling of H₂ through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup⁻, with the plants releasing H₂ produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H₂ induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup⁻ symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup⁺ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup⁺ symbiotic relationships with rhizobia. Conversely, Hup⁻ associations could be beneficial to leguminous plants due to H₂-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup⁺, while others were Hup⁻. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.     

Ecology of Hup+ Rhizobium strains of cow pea miscellany: native frequency and competence

Rhizobium strains nodulating summer legumes cow pea [Vigna unguiculata (L.)], green gram [V. radiata (L.) (Wilczek)], black gram [V. mungo (L.) (Hepper)] and cluster bean [Cyamopsis tetragonoloba (L.) (Taub)] and a winter legume chick pea [Cicer arietinum (L.)] were surveyed in the Northern Plains of India and screened for hydrogenase activity to determine distribution of Hup character in the native ecosystem. It was observed that 56% of the Rhizobium strains of summer legumes were Hup⁺ whereas that of the winter legume, chick pea, were all Hup^-. Ex planta acetylene reduction activity was observed in most of the Hup⁺ but not in the Hup^- strains of any of the host species. In summer legume, mixed inoculation of Hup⁺ and Hup^- strains, under sterilized as well as unsterilized soil conditions, showed that the host species were predominantly nodulated with Hup⁺ strain.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Alkaloid fingerprinting resolves Huperzia selago genotypes in Iceland

The club moss family Lycopodiaceae produces a diverse array of bioactive lycopodium alkaloids (LAs). In particular, the alkaloid huperzine A (hupA) has grasped attention since it is a potent acetylcholinesterase inhibitor of medical interest in Alzheimer's disease. Although the structural diversity and bioactivities of LAs have been studied to some extent, their chemotaxonomic value is mostly unexplored, especially to a lower taxonomic unit (e.g. subspecies or genotypes). This study focused on previously reported Icelandic Huperzia selago genotypes, and aimed to evaluate the chemotaxonomic value of LAs in resolving them. Using liquid chromatography-mass spectrometry (LC-MS), alkaloid fingerprints of H. selago taxa were subjected to principal component analysis (PCA). Our results revealed that each genotype tends to have its own alkaloid profile. Genotype 1 and 3 form distinct groups in a PCA plot, where genotype 2 is an intermediate between the other two genotypes. HupA and its derivative, huperzine B, both contribute to the differentiation of genotype 3 from the others. Therefore, our study demonstrated the potential of alkaloid fingerprints in resolving deep taxonomic groups and selecting plant taxa of medicinal importance.