Two Novel Hydroperoxylated Lycopodium Alkaloids from Huperzia serrata

Two novel hydroperoxylated Lycopodium alkaloids, 11alpha-hydroperoxyphlegmariurine B (1) and 7-hydroperoxyphlegmariurine B (2), along with a known compound, phlegmanurine B (3), were isolated from the total alkaloid fraction of the Chinese medicinal herb Huperzia serrata (Thunb.) Trev. Their structures and relative configurations were elucidated on the basis of spectroscopic analyses.

Huperzine V,A New Lycopodium Alkaloid from Huperzia serrata

Huperzine V, a new Lycopodium alkaloid, was isolated from the whole plant of Huperzia serrata, and the absolute stereochemistry was determined by X-ray crystallographic analysis.

Huperzine W,a Novel 14 Carbons Lycopodium Alkaloid from Huperzia serrata

Huperzine W, a novel 14 carbons Lycopodium alkaloid, was isolated from the whole plant of Huperzia serrata, and its stucture was determined by spectroscopic analysis.

Huperzine H, a New Lycopodium Alkaloid from Huperzia serrata

A new lycopodium alkaloid with a novel skeleton was isolated from Huperzia serrata. Its structure was determined on the basis of spectral evidences.

Lycodine-Type Lycopodium Alkaloids from the Whole Plants of Huperzia serrata

t Three new lycodine-type Lycopodium alkaloids,namely 1-methyllycodine(1),8a-hydroxy-15,16-dehydro-desN-methyl-a-obscurine(2),N-methyl-16-hydroxyhuperzine B(3),and one new natural lycodine-type Lycopodium alkaloid,N-methylhuperzine A(4),along with 11 known analogues(5–15),were isolated from the whole plants of club moss Huperzia serrata.The structures of 1–4 were elucidated on the basis of NMR spectroscopic and mass spectrometry data.Among them,compound 1 was the first lycodine-type alkaloid possessing a methyl group at C-1.In addition,the structure of 5 was confirmed by the single-crystal X-ray crystallography data and its^(13)C NMR was reported for the first time in current study.Compounds 1–5 were tested their BACE1 inhibitory activity.

Tissue Culture and Gemmae Propagation of Shoot Tipof Huperzia serrata

To solve the problems of microbial contamination and no rooting during the tissue culture of Huperzia serrata, wild seedlings of Huperzia serrata were cultured by indoor hydroponics or soil cultivation methods, then the stem tips were used in tissue culture. The above operation significantly reduced microbial contamination during tissue culture process. Different types and concentrations of hormones were added into the basic medium MS to screen the optimal formula for induced plantlet growth and rooting. It was found that 1/2MSCO was the best medium on which stem, leaves and roots of tissue culture plantlets of Huperzia serrata grew quickly. At the same time, sand planting was a convenient and effective approach for gemmae propagation of Huperzia serrata, the survival rate of seedlings germinated from gemmae was nearly 100%.

Bioinformatic Analysis of ARF7 in Huperzia serrata

[Objectives]This paper aims to study the function of the ARF7 gene family of Huperzia serrata.[Methods]Based on the full-length transcriptome sequencing results of H.serrata,the unigenes of 17 H.serrata ARF7 candidate genes were analyzed using biogenetics technology.[Results]The molecular weight of the ARF7 proteins ranges from 11.28 to 608.54 kDa.HsARF7-4,HsARF7-5,HsARF7-8,HsARF7-9,HsARF7-10,HsARF7-13,HsARF7-16 and HsARF7-17 are basic proteins;all the proteins are unstable;except that HsARF7-6 and HsARF7-15 are hydrophilic,the rest are lipophilic proteins;there are 197 types of cis-acting elements,and 17 of the proteins contain as more as 20 acting elements such as GTGANTG10,CBFHV,GT1CONSENSUS,NODCON2GM,CAATBOX1,MYBCORE,GATABOX,WRKY71OS and RAV1AAT;HsARF7-1,HsARF7-2,HsARF7-4,HsARF7-6,HsARF7-8,HsARF7-10,HsARF7-12,HsARF7-14 and HsARF7-16 all have the 6 types of motif predicted;the proteins have domains such as PABP-1234 super family,RRM_SF super famliy,PRK10263 super family,AUX_IAA super family and MFS super family;the proteins all can cross cell membrane and have no signal peptide,and they are all located in the nucleus.On the level of secondary structure,the proportion ofβ-sheet is the smallest.In HsARF7-7 and HsARF7-9,α-helix has the largest proportion,and in the remaining proteins,random coil has the largest proportion;and tertiary structure prediction found that HsARF7-1,HsARF7-2,HsARF7-4,HsARF7-14 and HsARF7-17 have a tertiary structure of polyadenylate-binding protein,and HsARF7-7 has a tertiary structure of auxin-induced protein iaa4.[Conclusions]The above analysis results can provide a certain theoretical basis for further research on the biological function and regulatory mechanism of the ARF gene of H.serrata in the future,and provide a reference for the study of the growth and development of H.serrata.

Comprehensive relative quantitative metabolomics analysis of lycopodium alkaloids in different tissues of Huperzia serrata

Qian ceng Ta,the whole plant of Huperzia serrata,is an important landscape and medicinal herbs and contains abundant bioactive lycopodium alkaloids.Although the structures of more than 100 lycopodium alkaloids in Huperzia serrata have been isolated and identified,the content and distribution of these alkaloids in different tissues are still unclear.In current study,an ultra-performance liquid chromatography-mass spectrometry based comprehensive metabolomics strategy was developed,including the extraction,separation,identification,and statistical analysis.The results showed that different types lycopodium alkaloids could be separated at different time-windows,which was helpful for further metabolite identification.Peak4388 and peak3954 were metabolite biomarkers for the different tissues according to the principle component analysis and partial least squares-discriminant analysis model.A computational tool based in-house database was also built up and used for putative identification.Of the 2354 true peaks after four-step filtration,118 peaks were putatively identified as lycopodium alkaloids by using in-house database,and four of which was identified by authentic standards.Alternatively,another computational software was used to predict the fragmentation pattern,to dereplicate the structure of identified peaks,and identified the peak3585 to N-methylhuperzine A.The integration of both computational tools could be used for more metabolites identification.

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

A new Lycopodium alkaloid together with a phenolic glycoside—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.

基于全长转录组的蛇足石杉ARF基因家族鉴定分析

生长素响应因子(ARF)是介导生长素信号传导并调节多种生物学过程的转录因子家族。为探究蛇足石杉ARF基因家族成员及其在高温和干旱胁迫响应中的作用,该研究利用全长转录组和RNA-seq数据,分析蛇足石杉ARF基因家族成员的系统发育及表达模式,并通过生物信息学分析,对ARF基因家族的理化性质、结构域、保守基序、系统发育、组织表达模式及高温和干旱胁迫下的表达模式进行分析。结果表明:(1)在蛇足石杉全长转录组中共筛选出24个ARF家族成员,均为酸性蛋白且均为亲水蛋白。(2)亚细胞定位预测显示,所有24个HsARF均定位于细胞核。(3)系统发育分析发现,HsARF与被子植物拟南芥和水稻亲缘关系较远,与高等开花植物只有2个共同的ARF祖先。(4)结构域分析发现,除HsARF18/23/24外,大多数HsARF有B3结构域。二级结构分析发现,HsARF蛋白占比最多的为无规卷曲,其次为延伸链和α-螺旋。24个HsARF蛋白中所用的三级结构模型只有4种。(5)RNA-seq分析显示,7个HsARF在所有检测的组织中表达量均较高,10个HsARF在茎中的表达量高于根和叶,而HsARF13和HsARF14在叶中的表达量低于根和茎。(6)HsARF在高温和干旱胁迫下表达量发生显著变化,其中18个HsARF基因不同程度高温胁迫诱导,有7个HsARF基因响应干旱胁迫,其中有3个HsARF基因受干旱诱导,有4个HsARF基因受干旱抑制。该研究结果为蛇足石杉HsARF基因的功能和生物育种提供了理论参考。

蛇足石杉COBRA基因家族的分子生物信息学及表达分析

为探明蛇足石杉COBRA基因家族成员分子生物信息学特征及组织表达规律,该文基于蛇足石杉的全长转录组数据,通过生物信息学技术对该家族成员(HsCOBRAs)的理化性质、结构域、保守基序、顺式作用元件、基因表达量等进行分析。结果表明:(1)在蛇足石杉全长转录组中共筛选出24个HsCOBRAs家族成员,其中酸性蛋白9个,稳定蛋白11个,疏水性蛋白5个,具有跨膜结构的蛋白7个,具有信号肽的蛋白3个。(2)亚细胞定位在细胞壁、叶绿体、细胞核、细胞膜上。(3)结构分析发现HsCOBRAs有7种结构域和6种保守基序,部分成员具有高度保守的CCVS结构。(4)HsCOBRAs具有CAAT-box、TATA-box等45种顺式作用元件。(5)HsCOBRA2在叶、孢子、茎、芽胞中的表达量均最高。该研究结果可为HsCOBRAs的进一步研究及生物学功能验证等提供理论依据。

HPLC法测定蛇足石杉中石杉碱甲含量

目的 考察蛇足石杉不同产地、不同部位中石杉碱甲 (Hup A)和总碱含量 ;比较不同提取方法对石杉碱甲含量的影响。方法 采用 RP- HPL C法测定 Hup A的含量。结果 Hup A在根、茎、叶中的含量为茎、叶 >根 ;贵州、广东和安徽产地的 Hup A含量分别为 0 .0 18% ,0 .0 2 1% ,0 .0 2 0 % ,提取方法之间差异不显著。结论 Hup A含量在蛇足石杉中的分布为地上部分大于地下部分。不同产地 Hup A含量有一定差异。

中国蛇足石杉资源调查与评估

通过对我国蛇足石杉主要分布区35个野外样点和总样方量71300m2的调查,结合国内12个标本馆470余号蛇足石杉馆藏标本及现有文献的研究表明:蛇足石杉的主要分布区位于我国中亚热带和南亚热带所在的地理区域,幅员面积260×104km2,实际分布生境总面积约39×104km2;性喜阴湿,所适应的生态环境为中亚热带常绿阔叶林和南亚热带季风常绿阔叶林及其次生生态系统;野外生物量的调查得到了蛇足石杉在不同的地理区域的生物量分布格局,并由此结合专家系统对直接取样结果和间接推导数据的权重分析及环境参数的评估,初步确定我国蛇足石杉天然生物量为4656.6t;并以此为基础,在进一步考虑人的采集能力的前提下,判定我国蛇足石杉的理论可采量为1604t;同时指出,在目前收购价格条件下(30～40元/kg),我国蛇足石杉商业可采量大约不会超过200t。由于蛇足石杉资源分散,而且许多相对集中的产地均位于自然保护区和风景区范围内,因此,不可大规模开采,解决资源短缺的问题还有赖于寻求其他更加科学合理的途径。另外,论文为小型植物的生物量和可采量评估提供了一些新思路和方法。

千层塔内生真菌分离鉴定的初步研究

目的从治疗老年痴呆药用植物千层塔中分离内生真菌,并进行初步鉴定。方法用PDA平板培养基分离菌株,对照真菌鉴定手册,根据菌株的菌落形态、大小、颜色、生长速率、质地、生长培养基颜色变化以及菌丝体和孢子的形态特征进行鉴定。结果从千层塔的茎中分离出4株内生真菌,分别属于顶孢霉属、单轴霉属、酵母和青霉属。结论首次从千层塔中分离和鉴定出4株内生真菌。

草药蛇足石杉的研究进展

对蛇足石杉的药理作用及其临床应用方面的研究作较全面综述 。

一株拮抗真菌的蛇足石杉内生细菌分离鉴定及培养条件优化

从蛇足石杉(Huperzia serrata)叶片中分离到一株对小麦赤霉(Fusarium graminearum)等多种植物病原真菌有强拮抗作用的内生细菌H-6。通过形态和培养特性观察、生理生化实验及16SrDNA序列分析,初步鉴定该菌属于伯克霍尔德属,命名为Burkholderia sp.H-6。同时还对该菌培养条件进行了优化,得出马铃薯浸出液中添加2.5%的甘露醇和0.1%NaNO3有利于细胞生长和抑菌活性的产生,最适培养温度为28℃,最佳初始pH4.0。

生态环境因子对千层塔生长发育的影响研究

目的：通过生态因子对千层塔生长发育的影响研究，建立和优化野生药材千层塔的栽培体系。方法：采用自然栽培法进行定点、定时观察，研究光照、温度、相对湿度、肥料等因子对千层塔生长的影响。结果：结果表明在温度为18．9～26．3℃、相对湿度81％～90％和光照强度1330～3000lx时，千层塔生长良好，初步筛选出利用山区毛竹林种植千层塔的技术方案。结论：提出了“光照强度和环境相对湿度是制约千层塔生长的两大生态环境因子”的观点，同时说明千层塔野生变家种是可行的。

蛇足石杉外植体表面消毒及内生菌消除方法

为了获得蛇足石杉的无菌材料,使用双氧水、酒精、升汞等消毒剂对外植体进行表面消毒,使用混合抗菌溶液及不同温度的热水浴处理外植体以消除内生菌.结果表明:茎段经预处理后,用47℃热水浴处理1 h,再经70%酒精处理30 s,0.1%升汞处理7 min后,能达到较好的表面消毒及内生菌消除的效果;孢子囊和茎尖经预处理后,再经过70%酒精处理30 s,0.1%升汞处理4 min即可达到较好的表面消毒效果;消毒剂结合温度处理能降低内生菌的污染率;孢子囊和茎尖几乎不含内生菌,是可用于组织培养的较好的外植体.

浙江及邻近地区蛇足石杉依存环境的初步研究

对浙江及邻近地区产的蛇足石杉(Huperzia serrata)7个种群的自然环境进行野外观察,测定了土壤含水量、电导率、有机质含量、pH值,植株和土壤中K、Ca、Mg、Fe、Zn、Cu、Mn、Na、Al、Pb、Cd11种元素的含量,以及植株中石杉碱甲(HupA)含量。结果表明,蛇足石杉种群多分布于海拔350-1700m的山地密林下或沟谷阴湿土中,郁闭度、年均降雨量、空气相对湿度均较大;环境中土壤含水量为10%-30%,pH值4.57-5.31,电导率0.061-0.385mscm-1,有机质含量6.18%-9.75%;蛇足石杉对K、Ca、Zn和Na的需要程度较高,对Pb、Cu、Cd3种重金属元素的富集能力较强,在人工栽培中应注意协调各元素的合理配给;蛇足石杉较适宜生长的环境条件为:土壤电导率以及pH值相对较低;郁闭度高,年均降雨量和空气相对湿度较大。基于土壤理化性质,对7个种群分布点进行除趋势对应分析,表明石杉碱甲含量高的磐安种群分布点具有特殊性,提示环境条件对蛇足石杉中HupA含量有较大的影响。

千层塔黄酮类物质的微波提取工艺

对千层塔茎黄酮类物质的微波提取工艺进行了探讨。结果表明,微波提取的影响因素顺序为乙醇浓度>微波功率>辐射时间>料液比。千层塔茎黄酮类物质的最佳提取条件为辐射时间30s,微波功率650W,乙醇浓度90%,料液比1:50(W/V)。此条件下总黄酮提取率为16.651mg/g。