定量毛细管电泳法测定虫草类保健品中的核苷及碱基类物质

目的：定量毛细管电泳法因采用了定量阀进样的方式,其进样量为固定值,定量结果更加可靠,且重复性好。本文探讨了采用定量毛细管电泳方法测定市售虫草类保健品中虫草素、腺嘌呤、尿嘧啶、腺苷、尿苷含量的可行性,优化了分析条件,并对其进行含量测定。方法：以浓度为40mM、pH9．5的硼砂为缓冲液,工作电压为-15kV,采用定量毛细管电泳的方测定了虫草类制品中的核苷及碱基类物质的含量。结果：五种核苷及碱基类物质在优化的定量毛细管电泳条件下得到了良好的分离和定量结果．峰面积RSD值小于1．4％,测定了其在虫草菌丝体粉末及虫草王胶囊中的含量。结论：首次利用定量毛细管电泳法对虫草类保健品中的虫草素、腺嘌呤、尿嘧啶、腺苷、尿苷的含量进行了定量测定,不同形式虫草类保健品中核苷、碱基的种类和含量有差异。该方法快速、准确,对虫草类保健品的质量控制有重要意义。     

古尼虫草核苷类成分的高效液相色谱分析*

以已知核苷为标样,并以冬虫夏草做对比,利用反相高效液相色谱法对古尼虫草及其发酵菌丝体的核苷类化学成分进行了分析,结果表明古尼虫草和冬虫夏草主要含有7种标样核苷中的腺苷、胞苷、尿苷,其含量相当,而古尼虫草发酵菌丝体中腺苷和尿苷的含量是古尼虫草子座或僵虫体的2~3倍。     

古尼虫草核苷类成分的高效液相色谱分析

以已知核苷为标样,并以冬虫夏草做对比,利用反相高效液相色谱法对古尼虫草及其发酵菌丝体的核苷类化学成分进行了分析,结果表明古尼虫草和冬虫夏草主要含有7种标样核苷中的腺苷、胞苷、尿苷,其含量相当,而古尼虫草发酵菌丝体中腺苷和尿苷的含量是古尼虫草子座或僵虫体的2～3倍。     

HPLC法同时测定延胡索不同部位中11种核苷类成分

目的：测定并评价不同产地延胡索药材不同部位中11种核苷类成分的含量。方法：采用高效液相色谱法测定核苷类成分的含量，并对测定结果进行聚类分析。结果：不同产地延胡索中核苷类物质含量由高到低为鸟苷>尿苷>腺苷>肌苷>胞苷>尿嘧啶>2’-脱氧肌苷>2’-脱氧胸苷>腺嘌呤>胸腺嘧啶>2’-脱氧腺苷，11种核苷总含量整体表现为叶>茎>块茎，不同产地延胡索核苷类总含量由高到低为浙江磐安>浙江东阳>陕西汉中>重庆万州>重庆开州，块茎中的11种核苷成分含量比无显著性差异，但茎和叶差异较大。结论：此方法可用于延胡索不同部位中核苷类成分的测定，为其综合利用提供思路。     

金蝉花核苷类成分的LC-MS定性分析与HPLC含量测定

本文采用高效液相色谱-质谱联用法定性分析金蝉花中核苷类成分,并建立了高效液相色谱法同时测定6种核苷类成分含量的方法。结果定性分析出金蝉花中13种核苷类成分,腺嘌呤、尿苷、肌苷、鸟苷、腺苷和N6-(2-羟乙基)腺苷在2.34～18.67、4.77～38.13、3.48～27.87、1.13～9.07、4.76～38.04、2.84～22.76μg/mL范围内呈良好的线性关系(r0.999 1),平均加样回收率为97.32%～102.37%,且精密度、重复性、稳定性良好,可用于金蝉花中主要核苷类成分的同时测定;不同产地的金蝉花中核苷类成分含量存在差异,安徽大别山和江苏句容的6种核苷类总量相对较高,浙江天目山、安徽宣城和福建三明相对较低;不同部位的金蝉花样品中,腺嘌呤、尿苷、肌苷、鸟苷、腺苷在子实体中含量均高于菌核,而N6-(2-羟乙基)腺苷则在菌核中含量较高。以上结果可为金蝉花的质量控制及深入开发利用提供参考。     

不同采收期玄参中核苷类成分的含量分析

建立UPLC-QTRAP-MS/MS同时测定玄参中10种核苷类成分含量的方法,分析不同采收期玄参中核苷类成分动态积累变化。采用UPLC-QTRAP-MS/MS技术同时测定玄参样品中10种核苷类成分的含量。玄参核苷类成分中,以鸟苷、尿苷、腺苷、尿嘧啶含量较高;不同采收期玄参核苷类成分含量有所差异,11月份核苷含量相对较高。为探究玄参药材的品质形成机制及确定药材适宜采收期提供基础资料。     

高效液相色谱法测定三种虫草发酵液与菌丝体核苷化合物含量

本项研究测定了三种虫草(江西虫草、古尼虫草、戴氏虫草)发酵液与菌丝体中核苷类化合物的含量,并建立了定量分析的方法体系。其中,采用液体发酵的方法获得虫草菌丝体及其发酵液,应用超声破碎法提取虫草菌丝体中的核苷类化合物,最后应用高效液相色谱法-二极管阵列检测器进行色谱分析,并以保留时间和吸收光谱双指标作为定性依据。实验取得了预期结果:优化了5种核苷化合物:腺苷、虫草素、尿苷、肌苷、鸟苷的色谱分离条件,分析了3种虫草中核苷类化合物的含量,精密度、稳定性、重现性和回收率实验表明这一方法可高效地定量分析虫草中的核苷类化合物。实验表明,HPLC-DAD法对虫草中的核苷类化合物的定量分析是一种有效的方法,该方法可快速准确地检测虫草有效成分的含量,本研究同时测定了胞外部分的化合物含量,系首次对这三种虫草细胞内外成分同时进行测定,对虫草的品质评价体系及其质量控制标准的建立具有重要意义。     

灵芝孢子粉中核苷类成分分析

本文利用高效液相色谱方法（HPLC）同时对灵芝孢子粉中的15种核苷类成分的含量进行测定。采用Ultimate AQ-C₁₈（4.6mm×250mm,5μm）色谱柱,以甲醇和水为流动相进行梯度洗脱,流速1.0mL/min,检测波长259nm,柱温30℃,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,适用于灵芝孢子粉中核苷类成分的测定分析。运用建立的方法对不同破壁时间、不同采收时期龙泉、奉化、大别山、黄山4个产区的灵芝孢子粉中的15种核苷类成分的含量进行测定。结果表明破壁处理对灵芝孢子粉中核苷类成分提取率的影响不大,不同产地的灵芝孢子粉中核苷类成分的组成和含量具有显著差异,且孢子粉中的核苷含量随着产粉时间的延长有所增加。各待测样品中均含有胞嘧啶、尿苷、腺嘌呤、鸟苷、腺苷等成分,其中尿苷、鸟苷、腺苷3种核苷的含量占总量的比例在待测样品中均达到70%以上,为灵芝孢子粉中的主要核苷类成分。     

不同来源的蛹虫草子实体活性成分的比较

通过比较不同来源的蛹虫草子实体的活性成分以探讨蛹虫草品质的差异。对17个蛹虫草菌株栽培得到的子实体及16个市售样品中的多糖、核苷类成分、游离糖醇及小分子糖类的含量进行分析测定,并比较其核苷类成分HPLC指纹图谱。结果表明,因菌株不同蛹虫草子实体的活性成分具有不同程度的差异,菌株对虫草素含量的影响最大,其次是N ⁶-(2-羟乙基)腺苷,因菌株不同虫草素含量可相差14倍,N ⁶-(2-羟乙基)腺苷的含量差异可达6倍以上。市售样品中的核苷类成分分析结果也证明了在测定的几种成分中,虫草素是含量差异最大的活性成分。17个蛹虫草菌株子实体的多糖含量为1.81%-4.92%,甘露醇含量为1.44%-4.47%,海藻糖含量为3.58%-25.43%。16个市售样品的多糖含量为2.84%-5.55%,甘露醇含量为0.96%-3.93%,海藻糖含量为1.04%-19.91%。采用数据归一化法进行子实体品质综合评价研究,结果表明菌株G7a、G10a、G15a综合品质较好,多数成分含量均大于平均值,是生产高品质蛹虫草的合适菌株。     

豆粕营养成分对蝙蝠蛾拟青霉发酵产物的影响

为探索培养基中的豆粕以及制备豆粕的原材料大豆对蝙蝠蛾拟青霉发酵产物的影响,我们比较了不同品种大豆和用其制成的豆粕的主要营养成分含量,包括粗蛋白、粗脂肪、6种微量元素（钙、镁、铜、锌、铁、锰）和总异黄酮（大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素、染料木素）,以及豆粕作为培养基对蝙蝠蛾拟青霉发酵产物的生物量和有效成分含量包括腺苷、腺嘌呤、虫草素和麦角甾醇的影响,结果表明大豆中钙和大豆苷与发酵产物中腺苷含量正相关。豆粕中钙、大豆苷、大豆苷元、染料木素与发酵产物中腺苷含量正相关;豆粕中钙、镁、大豆苷、大豆苷元、染料木素、总异黄酮与发酵产物中腺嘌呤含量负相关;豆粕中铁与菌丝体干重值正相关。结果表明,培养基中的豆粕和原料大豆主要营养成分含量对蝙蝠蛾拟青霉发酵产物品质有显著影响。     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Determination of ribonucleosides,deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.     

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [¹⁴C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[¹⁴C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (K_D) were 1.2 × 10⁻⁶ M and 1.4 × 10⁻⁷ M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10⁻⁶ M and 1.1 × 10⁻⁷ M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.     

Independent blood-brain barrier transport systems for nucleic acid precursors.

The blood-brain barrier permeability to certain 14-C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15s later. The amount of 14-C-labelled base or nucleoside remaining in brain was expressed in relation to 3-H2O (a highly diffusible internal standard) and 113m-In-labelled EDTA (an essentially non-diffusible internal standard). Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine. Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (Km equals 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (Km equals 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [14-D]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.