DNA-纳米金探针法检测蛇足石杉内生真菌发酵液中石杉碱甲的含量

本研究将DNA、酶和抗体修饰于纳米金颗粒表面制备纳米金探针,建立了DNA-纳米金探针检测石杉碱甲(HupA)的方法。以磁珠作为载体,将石杉碱甲完全抗原(HupA-OVA)标记在磁珠上,采用竞争免疫分析模式,发现HupA浓度在0.008～16 ng/mL范围内,抑制率(I)与HupA浓度的对数值(lgc)呈良好的线性关系,线性方程为I=0.3006 lgc+0.6142,线性相关系数为0.9952,检测限为19.41 pg/mL。应用该方法对蛇足石杉内生真菌发酵液中HupA进行了检测,加标回收率为99.33%～103.73%,相对标准偏差(RSD,n=3)为1.70%～4.67%。该方法灵敏度高、特异性强、操作简单,可用于蛇足石杉内生真菌发酵液中HupA含量的检测。     

石杉碱O的结构鉴定

蛇足石杉[Huperziaserrata(Thunb.)Trev.]中,R~f与石杉碱甲相近部位分离得到一个新生物碱,经IR,MS,1D和2DNMR确定了其化学结构,为一个有内氢键烯醇型新的lycopodine类生物碱,命名为石杉碱O(HuperzineO)。     

高效毛细管电泳法测定石杉碱甲片中石杉碱甲含量

提出了毛细管电泳法分离测定石杉碱甲片中石杉碱甲含量的方法。以pH为4.6的乙酸盐溶液为电解质溶液,运行电压13kV,于波长310nm处进行紫外检测。石杉碱甲的质量浓度在5.0～60mg.L-1范围内与峰面积呈线性关系。采用该方法对石杉碱甲片样品中石杉碱甲的浓度进行了测定,所得加标回收率在97.8%～98.7%之间,测定值的相对标准偏差(n=5)在0.53%～0.85%之间。     

石杉碱甲-AChE复合物中石杉碱甲的结构特征: 量子化学研究

在运用量子化学从头计算方法(HF/4-31G)结合点电荷模型方法对AChE-HupA复合物活性位点的410个原子和1929个点电荷进行理论计算的基础上, 比较了石杉碱甲分子在形成复合物前后的结构变化特征。发现复合物中石杉碱甲分子构象并非能量最低构象, 它的能量比HF/4-31G全优化得到的构象的能量高91.8kj/mol。和单分子状态相比, 形成复合物后季铵基和内酰胺基的N-H, C=O键的键长变长、键强减弱, 其总原子净电荷也发生了明显的变化。且这些基团的空间取向都有不同程度的改变, C(8)-N(21)键的旋转达20ⅲ。这些信息将有益于设计新的AChE抑制剂。     

高效液相色谱法定量测定中药千层塔提取物中的石杉碱甲

建立了高效液相色谱快速定量测定中药千层塔提取物中石杉碱甲含量的分析方法。千层塔提取物经甲醇/水/甲酸(10/90/0.2, v/v/v)提取并定容后,过滤膜后直接分析。色谱分离选用XCharge C18色谱柱(150 mm×4.6 mm, 5 μm),以水(含0.1%三氟乙酸)和乙腈(含0.09%三氟乙酸)为流动相进行梯度洗脱,流速为2 mL/min,于310 nm波长下检测,可在10 min内完成石杉碱甲的快速分离分析。结果表明,石杉碱甲在2.12～106 mg/L范围内线性关系良好(相关系数为0.9999);平均加标回收率为102.34%,相对标准偏差(RSD)为0.46%;日内及日间精密度均小于2%,满足定量要求。该方法简便、快速,结果可靠,重现性好,可作为千层塔提取物质量评价的依据。     

响应面法优化蒙药瞿麦中总生物碱提取工艺研究

研究了响应面法优化蒙药瞿麦总生物碱的最佳提取工艺。采用紫外-可见分光光度法,以总生物碱含量作为评价指标,用Box-Behnken响应面设计考察各因素对其影响,对结果进行二项式拟合及响应曲面分析得到最佳提取工艺。结果表明:蒙药瞿麦中总生物碱的最佳提取工艺为超声温度53℃,料液比1∶31 g/mL,超声时间41 min,超声功率140 W。四因素影响顺序为料液比超声温度超声功率超声时间。此方法简便、快速、灵敏,结果准确可靠,优化后的提取工艺稳定可行,且具有提取率高、提取时间短等优点,对今后的科研工作有一定参考价值。     

聚焦微波辅助提取-酸性染料分光光度法测定农吉利中总生物碱

采用聚焦微波辅助提取(FMAE)结合溴麝香草酚蓝分光光度法测定农吉利中总生物碱。粒径为0.180 mm的样品,以甲醇为溶剂,料液比为1比25,在微波功率中火条件下提取50min。在pH 7.2的磷酸二氢钾-氢氧化钠缓冲溶液中,提取得到的生物碱与溴麝香草酚蓝酸性染料形成缔合物,用三氯甲烷萃取后,在最大吸收波长417nm处测量其吸光度。总生物碱的质量浓度在0.25~250mg·L-1范围内呈线性,方法的检出限(3S/N)为1.5mg·L-1。应用该方法测定农吉利药材中总生物碱,加标回收率为105%,测定值的相对标准偏差(n=6)小于3.0%。     

响应面法优化苦槛蓝总生物碱提取工艺研究

结合单因素试验和响应面法优化苦槛蓝总生物碱的最佳提取工艺条件,并对其抑菌活性进行研究。以苦参碱为对照品,采用酸性染料比色法,以总生物碱含量为指标,分别考察乙醇体积分数、提取温度、提取时间和料液比4个因素对醇提法提取苦槛蓝总生物碱的影响,在此基础上通过Box-Behnken设计-响应面法确定最佳提取工艺,并进行验证。并检测其大肠杆菌和金黄色葡萄球菌的抑菌活性。苦槛蓝总生物碱的最佳提取工艺为:乙醇体积分数86.18%、提取温度75.42℃、提取时间2.622h、料液比0.080。验证实验下总生物碱提取率为(0.400 6±0.009 2)%,与模型结果接近。苦槛蓝提取物对大肠杆菌的抑菌直径达到33.17±0.53mm,金黄色葡萄球菌抑菌直径达到32.62±0.19mm。Box-Behnken设计响应面法能够有效地优化苦槛蓝总生物碱的提取工艺,并且苦槛蓝总生物碱提取物对大肠杆菌和金黄色葡萄球菌有明显的抑菌效果,这为苦槛蓝生物碱类化学成分的分离及其药理活性的深入研究提供了实验依据。     

金银花中绿原酸的微波预处理提取及检测

采用微波预处理法对金银花中绿原酸的提取进行优化,用三波长法测定了绿原酸的含量.设计正交试验,以绿原酸的含量为指标,考察乙醇用量、乙醇浓度和微波处理时间对金银花中绿原酸提取工艺的影响.三波长法检测波长为λ1=280 nm,λ2=325 nm,λ3=380 nm,其回归方程为ΔA=0.0426C 0.0017,相关系数为R=0.998.结果表明在气化剂用量20 mL,乙醇浓度为70(,微波辐射70 s时绿原酸得率最高.与常用的提取和测定方法相比,此方法对设备要求不高,具有更高的准确性与精密度.     

几个石松类生物碱的质谱分析

采用电子轰击电离高分辨质谱及B/E联动扫描(LinkedScan)法,研究了HuperzineE,F,G三个lycopodine类新生物碱的质谱行为。并应用这些结果确定了另一个新生物碱HuperzineO的结构。     

A new alkaloid and arbutin from the whole plant of Huperzia serrata

A new Lycopodium alkaloid together with a phenolic glyco-side-arbutin, were isolated from the whole plant of Huperzia serrata. The new compound was assigned the trivial name of huperzine I. The structures of these two compounds were determined on the basis of spectral evidence.     

Huperzine A production by Paecilomyces tenuis YS-13, an endophytic fungus isolated from Huperzia serrata

Huperzine A (HupA), a naturally occurring alkaloid in the plant family Huperziaceae, has drawn great interest for its potential application in Alzheimer disease therapy. Our primary objective was to identify alkaloid- and HupA-producing fungi from the Chinese folk herb, Huperzia serrata. We established a rapid and efficient model for screening HupA-producing endophytic fungal strains. The presence of HupA in Paecilomyces tenuis YS-13 was analysed by thin-layer chromatography, high-performance liquid chromatography and mass spectrometry. The fermentation yield of HupA was 21.0 μg/L, and the IC₅₀ of the crude extract of YS-13 fermentation broth was 1.27 ± 0.04 mg/mL. This is the first report of P. tenuis as a HupA-producing endophyte isolated from Huperziaceae.     

Endophytic Fungal Community of Huperzia serrata: Diversity and Relevance to the Production of Huperzine A by the Plant Host

As the population ages globally, there seem to be more people with Alzheimer’s disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer’s disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.     

Validation of a microscale extraction and high-throughput UHPLC-QTOF-MS analysis method for huperzine A in Huperzia

Herein we report on an improved method for the microscale extraction of huperzine A (HupA), an acetylcholinesterase‐inhibiting alkaloid, from as little as 3 mg of tissue homogenate from the clubmoss Huperzia squarrosa (G. Forst.) Trevis with 99.95% recovery. We also validated a novel UHPLC‐QTOF‐MS method for the high‐throughput analysis of H. squarrosa extracts in only 6 min, which, in combination with the very low limit of detection (20 pg on column) and the wide linear range for quantification (20–10,000 pg on column), allow for a highly efficient screening of extracts containing varying amounts of HupA. Utilization of this methodology has the potential to conserve valuable plant resources. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of absolute configuration of Pandanus alkaloid, pandamarilactonine-A, by first asymmetric total synthesis

The absolute configuration of pandamarilactonine-A, a pyrrolidine alkaloid, was established on the basis of total synthesis starting from l-prolinol. An insight into the mechanism of the low enantiomeric purity of natural pandamarilactonine-A is discussed.     

川佛手中多酚类物质的微波和超声波联合提取工艺优化

采用微波和超声波提取技术,实现了川佛手中多酚类物质的有效提取.在微波功率800 W和微波提取时间40 min的条件下,通过单因素实验条件优化,考察了超声功率、乙醇浓度、提取温度、超声时间和提取料液比对提取效率的影响;通过正交试验设计,确定了最佳提取工艺条件参数,即超声功率800 W、乙醇体积分数60%、提取温度55℃、超声时间1.0 h和提取料液比1∶20 g/m L;通过验证实验,得到川佛手多酚的提取率约为20.79 mg/g.     

何首乌中蒽醌类物质的超声波提取工艺优化

采用超声波辅助法提取何首乌中蒽醌类物质.讨论了甲醇体积分数、提取时间、提取温度、料液比和提取功率5个因素对提取效率的影响;通过正交试验确定了何首乌中蒽醌类物质的最佳提取工艺条件,甲醇体积分数为80%,提取时间为1.0h,提取温度为45℃,料液比为1∶15(g∶mL),提取功率为700W.此条件下蒽醌类物质的最大提取率为1.472 9%.     

The first enantiospecific total synthesis of a C-quaternary voachalotine alkaloid, (+)-dehydrovoachalotine

The first enantiospecific total synthesis of the indole alkaloid (+)-dehydrovoachalotine (1) has been achieved from d-(+)-tryptophan methyl ester in 28% overall yield. The formation of the prochiral quaternary carbon center at C-16 in the key intermediate (12) was realized via a Tollens reaction from N_a-methylvellosimine (13) in 95% yield. This approach could also be applied to the synthesis of many other indole alkaloids that contain a quaternary carbon center at C(16).     

A new morphinandienone alkaloid from the stems of Fissistigma tungfangense

A new morphinandienone alkaloid, fissistigmine A (1), together with three known alkaloids (2?4), were isolated and identified from the stems of Fissistigma tungfangense. Among them, fissistigmine A (1) represents the first example of a novel naturally occurring morphinandienone alkaloid with a unique cleavage of the C-9?N-17 bond. All isolated compounds were evaluated for their anti-rheumatoid arthritis activities via examining their anti-proliferative effects on synoviocytes in vitro. Compound 1 exhibited inhibitory effect on the proliferation of synoviocytes with an IC₅₀ value of 114.6 ± 2.2 μM.