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.

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。

蛇足石杉仿野生栽培关键技术分析

为保护蛇足石杉野生资源,促进可持续利用,针对蛇足石杉仿野生栽培相关的适宜温湿度、光照强度、肥料效应和不同居群在当地的生长速度等部分关键技术环节展开初步研究,为后续蛇足石杉仿野生栽培体系的创建提供参考。

共生培养法促进石杉碱甲增量富集的研究

为了促进蛇足石杉中石杉碱甲增量富集,本文利用内生真菌与蛇足石杉的共生进化关系,以共培养体系(co-culture system,CCS)为基础,提出了增加石杉碱甲产率的新方法。以蛇足石杉植株存活率为指标,确定共培养体系最佳培养条件;以细胞存活率、愈伤组织诱导率和增殖率为指标,确定最佳培养基条件;以石杉碱甲和麦角甾醇为指标性成分,控制植物细胞和内生真菌比例关系,维持培养体系内植物细胞和内生真菌之间的生长平衡,获得最佳的继代时间。结果表明:CCS的最佳培养条件为光照强度100 Lux,湿度95%,MS液体培养基,蔗糖20.0 g/L,2,4-二氯苯氧基乙酸5.0 mg/L和激动素1.0 mg/L;4 d为最佳继代时间,其石杉碱甲的浓度最高,与1 d时相比增加了49.34%,并且石杉碱甲与麦角甾醇的浓度比值控制在1.5~2.2之间时,内生真菌生长对石杉碱甲的富集有良性促进作用。该研究为高效、低成本增加石杉碱甲产量提供新的探索路径,亦可以有效解决药源不足问题,具有很好的应用前景。

千层塔基原及药效文献研究

千层塔为民间常用中草药,具有清热解毒、燥湿敛疮、止血定痛等功效,现代用于治疗阿尔茨海默病,具有极大的药用价值。通过本草考证和梳理现代研究文献,对千层塔的基原、名称、采收加工、资源利用、毒性、功效主治、民族用药及其有效成分石杉碱甲,治疗阿尔茨海默病的应用,进行系统考证与综述,为进一步开发应用千层塔提供参考依据。

千层塔中脂肪族类化学成分的抗肿瘤活性研究

千层塔中脂肪族类化学成分及其抗肿瘤活性研究。在肿瘤抑制活性引导下,采用AB-8大孔树脂、氧化铝、硅胶、Sephadex LH 20和半制备液相等方法,对千层塔甲醇提取物进行较系统分离纯化,并结合现代波谱手段对分离到的化合物进行结构鉴定。共分离鉴定了9个单体化合物,分别为pentyl-4-oxoicosa-2(Z),6(Z)-dienoate(1)、2-(2-ethoxy-1-hydroxy-2-oxoethyl)-2-hydroxy-4-methylpentanoic acid(2)、ethyl (9Z,12Z)-octadeca-9,12-dienoate(3)、(Z)-nonadec-10-enal(4)、油酸酰胺(5)、反式-9-十八碳烯酸(6)、bacillamidin A(7)、(3Z,6Z,9Z)-3,6,9-二十二碳三烯(8)、oplopandiol(9)。其中,化合物1和化合物2为新化合物,化合物3~9系首次从千层塔中分离。肿瘤抑制活性结果显示,化合物1和3对HepG2细胞具有抑制作用,其IC50分别为41.12、80.54μmol/L。

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。