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.

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。

基于叶绿体matK基因序列探讨10种石杉科石杉属植物的系统关系及分子鉴定

目的：利用叶绿体matK基因序列探讨10种石杉属植物的系统发育及分子鉴定。方法：分别从9种石杉属植物中提取总DNA，经扩增纯化后，用PCR产物直接测序。结果：对石杉属Huperzia Bernh．9个种的叶绿体matK基因进行了测序，序列长度为1589bp，并对10个种（亮叶石杉H.lucidula的matK基因序列从GenBank中下载）的marK序列进行比对，其中有35个系统发育信息位点及35个变异位点，以垂穗石松Lycopodiella cernua为外类群，利用MEGA 3．1软件计算种与种之间的遗传距离在1．59％～0．25％，并构建系统树，获得最简约树和邻接树。结论：小杉兰组和蛇足石杉组植物分别聚成一枝，构成姊妹群，其靴带支持率分别为98％和99％。长柄石杉与蛇足石杉分别聚在2个不同进化枝，二者间有19个碱基差异，遗传距离为1．21％。建议应将长柄石杉单独列为一个种。

HPLC法测定6种石杉科植物中石杉碱甲的含量

目的 测定 6种石杉科植物中石杉碱甲的含量。方法 用 2 %酒石酸溶液提取总碱 ,HPL C法测定 ,外标法定量。结果 石杉碱甲在所测定的 5种石杉科植物蛇足石杉、皱边石杉、四川石杉、金丝条马尾杉、柳杉叶马尾杉中均有较高分布 ,分别达到 0 .0 4 81% ,0 .0 5 79% ,0 .0 70 6 % ,0 .0 345 % ,0 .196 1% ,在闽浙马尾杉中含量较少 ,仅为0 .0 0 6 1%。结论 方法简便、准确 。

蛇足石杉中石杉碱甲的酶法提取研究

[目的]筛选蛇足石杉中石杉碱甲的酶法提取的最佳提取条件。[方法]采用纤维素酶酶解细胞壁,从而溶出更多的蛇足石杉细胞中的物质,并设计正交试验选出酶法提取的最佳提取条件。[结果]蛇足石杉中石杉碱甲的酶法提取的最佳工艺条件为纤维素酶用量0.125 g,酶解pH4.5,酶解温度60℃,酶解时间2.5 h;在此条件下,蛇足石杉中石杉碱甲的提取率为0.589‰;各因素中温度对纤维素酶的酶解效果影响最大。与酸提法相比,酶法提取的石杉碱甲的提取率提高了40.3%。[结论]该方法简便快捷、提取率高,可用来提取蛇足石杉中石杉碱甲。

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 。

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。

蛇足石杉内生真菌清除DPPH·与抑制乙酰胆碱酯酶活性研究

为了从蛇足石杉[Huperzia serrata（Thunb.）Trev.]中分离出能产生清除DPPH·和抑制乙酰胆碱酯酶活性物质的内生真菌,采用平板分离蛇足石杉内生真菌,摇瓶发酵并测定其发酵液和菌丝体提取物清除DPPH自由基、抑制乙酰胆碱酯酶活性的能力,形态学和ITS-rDNA序列分析法鉴定获得的部分内生真菌。结果表明,共分离得到94株内生真菌,有31株内生真菌发酵产物显示出乙酰胆碱酯酶抑制活性,其中18株具有显著的抑制活性,其抑制率范围在50.3%～82.78%,分别分离自蛇足石杉茎部（9株）、叶部（8株）和根（1株）。有19株内生真菌发酵产物有清除DPPH·活性,清除率在30%以上。有5株内生真菌的发酵物兼有乙酰胆碱酯酶抑制活性和清除DPPH·活性,以ITS-rDNA序列比对法对5株菌进行了鉴定,其分别属于Arthrinium sp.、Podospora sp.、Trichoderma sp.、Colletotrichum sp.、Colletotrichum sp。表明中药蛇足石杉植株内存在能产生抑制乙酰胆碱酯酶和抗DPPH自由基活性物质的内生真菌。

蛇足石杉内生真菌2F09P03B产石杉碱甲发酵条件的研究

目的研究发酵条件对蛇足石杉支顶孢属内生真菌2F09P03B石杉碱甲生成的影响,并探讨发酵条件的优化。方法通过测定2F09P03B生长曲线、石杉碱甲积累曲线和培养液pH值随时间的变化情况,了解该菌株的培养特性;考察不同碳源和氮源对石杉碱甲积累的影响。在单因素试验的基础上,利用正交试验优化培养基碳源、氮源组成;考察MgSO4、KH2PO4、NaCl、CaSO4对石杉碱甲生成的影响,确定无机盐组成;优化接种量、发酵时间、培养温度、摇床转速等发酵工艺条件。结果菌株2F09P03B的最佳培养基组成为:马铃薯150g/L,玉米粉30g/L,麦芽糖25g/L,酵母膏10g/L,麸皮30g/L,KH2PO41.5g/L,MgSO40.5g/L,NaCl0.3g/L,CaSO40.3g/L,pH自然;最佳发酵工艺条件为:接种量8%,发酵时间10d,培养温度28℃,摇床转数140r/min。在此条件下,发酵液中石杉碱甲含量为8.17μg/L,最高达到8.32μg/L,约为其干重的0.07%左右。结论发酵条件对菌株2F09P03B石杉碱甲产量有明显影响。优化后的发酵条件可使该菌株石杉碱甲产量明显提高,有望成为石杉碱甲新药源。

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。

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。

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.

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。