Comparative Study on the Effect of Different Fertilizers on the Growth and Development of Halenia ellipitica D.Don

[Objective] The aim was to study the effect of different treatments on development and growth of Halenia ellipitica D.Don.[Method] Three treatments (urea treatment group, diammonium phosphate treatment group and control group) were set in the experiment with 3 replications to detect plant height,root length, leaf number, branch number, flower number and biomass of Halenia ellipitica D.Don. in all experimental groups.[Result] The leaf number,branch number, flower number and output of crude drug of Halenia ellipitica D.Don. in urea treatment group were more than that of Halenia ellipitica D.Don. in diammonium phosphate treatment group and control group,however,the plant height and root length were not significantly different among all groups.[Conclusion] The urea generated better effect on Halenia ellipitica D.Don. cultivation than that produced by diammonium phosphate treatment.

Crystal Structure of 1-Hydroxy-2,3,4,7-tetramethoxyxanthone, a Xanthone Isolated from Halenia Elliptica

In an attempt to identify bioactive compounds from a Tibetan medicinal herb Halenia elliptica, four known compounds were isolated. The crystal structure of the title compound 1-hydroxy-2,3,4,7-tetramethoxyxanthone was characterized by X-ray single-crystal structure analysis. The crystal belongs to monoclinic, space group P21/n with a = 7.155(2), b = 13.232(1), c = 16.037(2) ? b = 95.06(2), V = 1512.4(5) 3, C17H16O7, Mr = 332.30, Z = 4, Dc = 1.459 g/cm3, l(MoKa) = 0.71073 ? m(MoKa) = 1.15 cm-1 and F(000) = 696. It exhibits a characteristic approximate plane structure consisting of 13 carbon atoms (C(1) to C(13)) and all of the seven oxygen atoms (O(1) to O(7)). An intra-hydrogen bond was formed between O(6) and O(5)H(5).

Multiple lines of evidence supports the two varieties of Halenia elliptica(Gentianaceae)as two species

Delimiting species requires multiple sources of evidence.Here,we delimited two varieties of Halenia elliptica(Gentianaceae)using several lines of evidence,including morphological traits and mating system in a sympatric population,phylogenetic relationships based on nrITS and cpDNA(rpl16)data,and complete chloroplast genome sequences.Comparative analysis of 21 morphological traits clearly separates the two varieties of H.elliptica.Examination of the flowering process and pollination treatments indicate that H.elliptica var.grandiflora produces seeds via outcrossing,whereas H.elliptica var.elliptica produces seeds via mixed mating.Furthermore,hand-pollinated hybridization of the two varieties produced no seeds.Observations of pollinators showed that when bees began a pollination bout on H.elliptica var.grandiflora they preferred to continue pollinating this variety;however,when they began a pollination bout on H.elliptica var.elliptica,they showed no preference for either variety.Phylogenetic analysis confirmed the monophyly of H.elliptica,which was further divided into two monophyletic clades corresponding to the two varieties.A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H.elliptica.We recommend that the two varieties of H.elliptica should be revised as two species(H.elliptica and H.grandiflora).Our findings indicate that H.elliptica varieties may have split into two separate species due to a shift in mating system,changes in flowering phenology and/or post-pollination reproductive isolation.

Quality Standard of Haleniae Herba

[Objectives]The research aimed to establish qualitative and quantitative detection methods of Haleniae Herba,and provide basis for improving its quality standard.[Methods]Using thin-layer chromatography(TLC),qualitative identification of Haleniae Herba was conducted.Using high-performance liquid chromatography,luteolin content of Haleniae Herba was determined.[Results]TLC identification method had good specificity,with clear spots and good degree of separation,and reference medicinal material of Haleniae Herba was prepared.Content determination results displayed that luteolin had good linear relationship in the range of 0.088-1.760μg.The RSD values of precision,repeatability,stability and sample recovery of the instrument met the requirements.[Conclusions]The established method is simple and reproducible,and can be used for the quality control of Haleniae Herba.

卵萼花锚STR基因家族的生物信息学分析

【目的】对卵萼花锚(Halenia elliptica)的异胡豆苷合成酶(strictosidine synthase,STR)基因家族成员进行鉴定和生物信息学分析,为进一步研究卵萼花锚STR家族基因的功能奠定基础。【方法】基于卵萼花锚基因组数据,采用生物信息学方法鉴定STR基因,分析其家族的进化关系、理化性质、基因结构和蛋白质保守基序,并与大花花锚(H.grandiflora)、花锚(H.corniculata)、四数獐牙菜(Sinoswertia tetraptera)和达乌里秦艽(Gentiana dahurica)的STR基因进行比较。【结果】卵萼花锚STR基因家族共有10个成员,基因数量与其他龙胆科物种相似。卵萼花锚STR基因与大花花锚具有较高的同源性,将所有HeSTR基因分为6组,同一组成员具有相似的基因结构和蛋白保守基序。HeSTR蛋白的氨基酸数量介于189~388个之间,除HeSTR14.1外,其他均为亲水蛋白。HeSTR基因的外显子数量为3或4个。卵萼花锚STR基因家族成员与四数獐牙菜、花锚和大花花锚的共线性较高。【结论】研究结果为深入探究STR基因功能及进一步解析卵萼花锚单帖吲哚生物碱龙胆苦苷的合成途径提供了理论依据。

Identification of the metabolites of biologically active xanthones isolated from Halenia elliptica D.Don by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry

Metabolism study has been carried out on 1-hydroxy-2,3,5-trimethoxyxanthone （HM-1） and 1-hydroxy-2,3,4,7- tetramethoxyxanthone （HM-2）, which are two biologically active ingredients isolated from the Tibetan herb, Halenia elliptica D. Don., in rat liver microsomes in vitro. A method of high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry （LCMSn-ESI-IT-TOF） was applied to analyze metabolites of HM-1 and HM-2 on line, and five metabolites were identified containing 1,5-dihydroxy-2,3-dimethoxyxanthone （HM-5）, 1,7-dihydroxy-2,3,4-trimethoxyxanthone （HM-9）, 1,4, 7- trihydroxy-2,3-dimethoxyxanthone （HM-10）, 1,4-dihydroxy-2,3,7-trimethoxyxanthone （HM-11） and 1,2-dilaydroxy-3,4,7- trimethoxyxanthone （HM-12）. Among these metabolites, HM-9, HM-11, and HM-12 were isomers mutually. The results indicated that HM-1 and HM-2 occurred Phase I metabolic reaction of demethylation in rat microsomes in vitro.

Chemical constituents of Lomatogonium carinthiacum and Halenia corniculata

Objective:To study the chemical constituents from traditional Chinese(Mongolian)medicine,Lomatogonium carinthiacum and Halenia corniculate.Methods:The chemical constituents were isolated and purified by silicagel column,Sephadex LH-20,ODS and high performance liquid chromategramphy.The structures were identified by NMR and MS analysis technics.Results:Twelve compounds were isolated and identified as isovitexin(1),Luteolin-5-O-β-D-glucoside(2),Isosaponarin(3),Luteolin-7-O-β-D-glucoside(4,7),1,4,8-Trimethoxy-xanthone-6-O-β-D-glucoronyl-(1→6)O-β-Dglucoside(5),friginoside D(6),1-hydroxy-2,3,5-trimethoxyxanthone(8),1-hydroxy-2,3,4,5-tet ramethoxyxanthone(9),1-hydroxy-2,3,4,7-tetramethoxyxanthone(10),1-hydroxy-2,3,4,5,7-pentame thoxyxanthone(11)and usnic acid(12).Conclusion:Compounds 6 and 12 are obtained from L.carinthiacum and H.corniculate for the first time.

Isolation, purification and structural characterization of a water-soluble polysaccharide HM41 from Halenia elliptica D.Don

A water-soluble polysaccharide, HM（41）, was obtained from Halenia elliptica D. Don by acidic ethanol fractionation and gel filtration. Its homogeneity was confirmed by chromatography using multiple systems. HM（41） was composed of rhamnose（Rha）, arabinose（Ara）, xylose（Xyl）, mannose（Man）,galactose（Gal）, glucose（Glc） with a molar ratio of 1.0：5.5：1.8：3.0：9.4：21. The average molecular weight of HM（41） was approximately 1.17 ＊ 10~4. Periodate oxidation, Smith degradation, methylation and GC, IR,NMR, XRD, GC–MS analysis were used for the structural analysis of HM（41）. Its main chain was composed mainly of β-（1→4）Gal, β-（1→4）Glc and β-（1→6）Glc. β-（1 →4）Gal were substituted at 6-O and on average there were 14 branches among 23 main chain residues;（1→ 4）Glc had no branch;（1→6）Glc were substituted at 3-O and on average there were 9 branches among 14 main chain residues. The side chain was composed of（1→3,6）-Rha,（1→4）/（1→5）-Ara,（1 →4）/（1→5）-Xyl,（1→4,6）-Man and（1→2）-Glc. The terminal residue was composed of Ara, Xyl, Man, Gal, and Glc. Then, we demonstrated that HM and HM（41） had strong scavenging activities in vitro hydroxyl. Overall, HM and HM（41） may have potential applications in the antioxidants for medical and food industry.

青海地区藏药甲地然果(椭圆叶花锚)HPLC指纹图谱和5个主成分含量测定研究

目的:建立椭圆叶花锚HPLC指纹图谱与含量测定方法。方法:采用Inspire C18(4.6 mm×250 mm,5μm)色谱柱进行分离,乙腈-甲醇-水(含0.03%冰乙酸)系统梯度洗脱,检测波长254 nm,流速1.0 mL·min^(-1),进样量10μL,柱温35℃。采用以上色谱条件对22批椭圆叶花锚样品进行指纹图谱分析,标定了25个共有峰,经对照标品比对,指认出其中5个主要成分的色谱峰,并对这5个成分进行了含量测定。结果:5种主要成分依次为去甲氧基花锚苷、花锚苷、獐牙菜苦苷、异荭草苷、1-羟基-3,4,5-三甲氧基口山酮,含量分别为11.2692~20.4006 mg·g^(-1)、5.0018~11.7832 mg·g^(-1)、3.2782~15.3578 mg·g^(-1)、1.1517~4.5183 mg·g^(-1)、1.4075~6.9511 mg·g^(-1)。22批椭圆叶花锚样品指纹图谱和对照指纹图谱相似度在0.908~0.991之间,各批次药材成分种类基本一致。采用SPSS 25.0与SIMCA 14.1软件对椭圆叶花锚样品5种成分含量进行聚类分析、PCA分析和OPLS-DA分析,可将样品分为3类,椭圆叶花锚差异的标志性成分为花锚苷、去甲氧基花锚苷和獐牙菜苦苷。结论:该方法具有简便、专属性强、稳定、可重复的特点,可为椭圆叶花锚药材的鉴别与质量评价提供参考。

花锚治疗肝胆相关疾病网络药理学分析

目的:基于网络药理学与分子对接方法分析花锚治疗肝胆相关疾病的潜在作用机制。方法:通过TCMIP V2.0、PubChem、GeneCards、OMIM数据库预测花锚黄酮类成分的潜在靶标及疾病靶标;应用STRING 11.0数据库构建蛋白互作网络,通过GO和KEGG富集分析,并通过Cytoscape 3.8.1软件构建花锚药材-黄酮成分-关键靶标多维网络;最后采用AutoDockVina分子对接软件对活性成分与关键靶点进行验证。结果:筛选出10个活性成分,4个关键靶点(TP53、AKT1、ESR1、MAPK1);GO和KEGG富集分析结果显示生物学反应包括144个条目,细胞组成涉及30个条目,分子学功能涉及61个条目,KEGG富集分析获得91个通路;分子对接结果表明10个活性成分与4个关键靶点的最低结合能均小于-5.0 kcal/mol,具有较好的结合性。结论:通过网络药理学、分子对接技术揭示了花锚黄酮治疗肝胆相关疾病的作用,为后续的临床试验及药物研究提供了理论参考。

藏药花锚中的酮类成分及其抗氧化活性

目的 研究藏药椭叶花锚中(口山)酮类成分及其抗氧化活性。方法 利用正相硅胶柱色谱、Sephadex LH-20柱色谱、反相高效液相色谱和重结晶等手段进行分离;借助红外光谱、质谱及核磁共振谱等波谱方法进行结构鉴定;以不同浓度化合物对Fe2+-半胱氨酸(Cys)诱导大鼠肝微粒体丙二醛生成的影响评价其抗氧化活性。结果 分离得到8个(口山)酮化合物(化合物Ⅰ～Ⅷ),其结构鉴定为1,7-二羟基-2,3,5-三甲氧基(口山)酮、1-羟基-2,3,4,7-四甲氧基(口山)酮、1,7-二羟基-2,3,4,5-四甲氧基(口山)酮、1,7-二羟基-2,3-二甲氧基(口山)酮、1,5-二羟基-2,3-二甲氧基(口山)酮、1-羟基-2,3,5-三甲氧基(口山)酮、1-羟基-2,3,4,5-四甲氧基(口山)酮、1-羟基-2,3,5,7-四甲氧基口山酮;其中1,7-二羟基-2,3,5-三甲氧基(口山)酮为新化合物;化合物Ⅰ、Ⅲ(10μg/ml、100μg/ml)对Fe2+-Cys诱导大鼠肝微粒体丙二醛生成有显著抑制作用。结论 从椭叶花锚乙醇提取物乙酸乙酯萃取部分分离得到8个(口山)酮化合物,且该类化合物具有一定抗氧化活性。

以体内吸收分布特点指导花锚成分的研究

目的 以体内吸收和分布特点为指导 ,对传统抗肝炎藏药花锚的成分进行研究 ;探索用此方法进行中草药药效成分研究的可行性。方法 利用高效液相色谱二极管阵列检测方法 ,确定能被吸收和分布在大鼠血液、器官和组织中的花锚成分 ,并利用各种色谱分离技术和波谱鉴定其结构。结果 从花锚乙醇提取物中分离鉴定了能被吸收并分布在大鼠血液、器官和组织中的 6个酮苷元类成分。结论 花锚中的酮苷元类成分是能够在大鼠体内检测到的主要被吸收和分布的成分 ,在消化、吸收和代谢过程中它们很快被转化为酮苷元。因此 ,花锚中酮苷类成分的活性主要是通过转化为酮苷元来实现。利用现代灵敏分析检测手段 ,以天然药物成分的吸收分布特点为指导 ,进行天然药物成分的研究是一种可行并且值得进行深入探索的方法。

藏药椭圆叶花锚的资源学研究

目的研究藏药椭圆叶花锚Halenia.elliptica D.Don品种、资源分布、蕴藏量,分析资源可持续利用前景,提出资源保护方案。方法标本查阅与野外踏查相结合分析椭圆叶花锚资源分布与品种情况;生态学方法调查椭圆叶花锚分布生境、生物学特性及生物量、蕴藏情况;通过市场调查、企业调查、医院调查分析椭圆叶花锚使用情况。结果椭圆叶花锚分布区广,适生生境在青藏高原面积广大,种子萌发率高,可形成优势种群,单位面积产量较高,蕴藏量可观,目前药用量有限。结论椭圆叶花锚资源能够满足目前藏医临床与制药产业的需求,栽培过程中应注意控制光照。

椭圆叶花锚的引种栽培

椭圆叶花锚 (HaleniaellipiticaD .Don)是藏族民间用于治疗黄疸型肝胆疾病和病毒性肝炎的一种名贵草药。引种栽培试验研究表明 ,在青海高原自然环境条件下 ,椭圆叶花锚为二年生草本植物 ,完成植物生长发育需要的时间为 10～ 12个月。自然状态下的植物种子萌发率很低 ,种子经过低温条件处理后可显著提高其萌发率。栽培的椭圆叶花锚植株在植株高度、分枝数量、单株生物量等生长状况指标明显高于野生植株 ,其有效化学成分接近野生状态的水平 。

几种药剂对椭圆叶花锚种子发芽力的影响

目的 研究几种药剂对椭圆叶花锚种子的发芽力的影响。方法 用不同浓度的青霉素、KCN、KNO3、PEG-6 0 0 0、IAA、GA3、6 - BA溶液对椭圆叶花锚浸种。结果 青霉素的 5、2 0、4 0 m g/ L,KCN的 5、2 0 mg/ L,5 %的 PEG-6 0 0 0 ,2 0 m g/ L GA3和 6 - BA的 2 5、75 mg/ L可极显著提高椭圆叶花锚种子平均发芽率、平均发芽势 ,其中以 6 - BA以 2 5 m g/ L 处理效果最好 ,可使其种子发芽率、发芽势分别提高 4 6 .0 %、39.6 7% ;IAA溶液和 KNO3对椭圆叶花锚种子的萌发无明显效果 ;而 15 %、2 5 %的 PEG- 6 0 0 0、80 mg/ L IAA、16 0 mg/ L GA3、10 0 mg/ L 6 - BA则明显抑制种子萌发。用 5 mg/ L 青霉素、2 5 mg/ L 6 - BA分别处理椭圆叶花锚种子后进行的室内土壤播种 ,对种子的萌发无明显影响。结论 不同药剂及同一药剂的不同浓度对椭圆叶花锚种子发芽力的影响不同。

藏药蒂达原植物在四川的地理分布与植物特征

目的研究藏药蒂达原植物藏茵陈在四川地区的资源分布。方法在野外考察的基础上,结合文献资料,总结了四川地区用作藏茵陈的植物种类,并研究其地理分布。结果实地考察分析发现,川西獐牙菜和椭圆叶花锚作为蒂达原植物在四川地区分布广泛。结论川西獐芽菜和椭圆叶花锚作为藏药蒂达原植物,应加快引种栽培。

椭圆叶花锚简化基因组的SSR信息分析及SSR引物开发

基于RAD-seq简化基因组测序技术获得椭圆叶花锚(Halenia ellipitica D.Don)简化基因组水平上的序列信息并开发SSR分子标记。利用SR search软件检测而得到双端各有至少100 bp的五种类型的SSR(二、三、四、五、六核苷酸)位点共6 201个,并成功设计其中3 865个SSR引物。在能成功设计引物的SSR位点中,三核苷酸SSR位点最多;重复序列长度包括17种(12~36 bp);重复序列的基序共达316种,其中五核苷酸基序种类最多(91种)。从中挑选65对可对应五种SSR类型的引物,经梯度PCR检验后,利用椭圆叶花锚的4个居群32个个对可扩增的引物进行PCR和聚丙烯酰氨凝胶电泳检测,其中14对引物能在绝大多数个体中扩增,且13对具多态性。13个多态性位点的等位基因数量均值为5.462,多态性较高且不连锁(P<0.05);其中10个位点在多数居群中偏离哈迪温伯格平衡(P<0.01)且存在较高的纯合子数量(观测杂合度Ho均值0.226);近交系数在-0.443~1,均值为0.656;基因流N_m为0.474。

藏药花锚中新化学成分的鉴定

目的 分离并鉴定高原植物椭圆叶花锚 H alenia elliptica全草中的化学成分。方法 采用元素分析 (EA)、核磁共振波谱 (NMR)、质谱 (MS)、红外光谱 (IR)、紫外光谱 (U V)、差示扫描量热 (DSC)等分析方法 ,测定其理化常数和波谱数据 ,鉴定其化学结构。结果 确证从椭圆叶花锚中所得到的两种针状结晶化合物 ,分别为 1-羟基 - 3,7,8-三甲氧基酮 (1- hydroxy- 3,7,8- trimethoxyxanthone)和 1,7-二羟基 - 3,8-二甲氧基酮 (1,7- dihydroxy- 3,8-dimethoxyxanthone)。结论 上述两种酮衍生物为首次从藏药花锚中分离得到。

不同肥料对椭圆叶花锚生长发育的比较研究

[目的]研究不同处理对椭圆叶花锚生长发育的影响。[方法]设尿素处理组、磷酸二铵处理组、对照组3个处理,3次重复,测定各组椭圆叶花锚的植株高度、根势、叶片数、分枝数、开花数以及生物量。[结果]尿素处理组的叶片数、分枝数、开花数以及药材产量(地上、全株生物量)均大于磷酸二铵组与对照组,而各组株高与根势生长情况的差别较小。[结论]尿素对椭圆叶花锚的栽培效果优于磷酸二铵。

藏药椭圆叶花锚中抗肝炎有效成分的含量测定

运用RP-HPLC建立了花锚中青兰苷、去甲氧基花锚苷和花锚苷的含量分析方法,为栽培花锚替代野生花锚入药提供一定的科学依据。花锚苷、去甲氧基花锚苷和青兰苷分别在0.68～3.40μg(r=0.9998)、0.36～1.80μg(r=0.9990)和0.80～4.00μg(r=0.9979)有良好线性关系,平均回收率分别为100.39%(RSD=2.60%)、99.79%(RSD=3.55%)和100.71%(RSD=2.19%)。栽培花锚中花锚苷和去甲氧基花锚苷的含量(抗肝炎活性成分)和在野生花锚中的含量相比无明显差别,可以初步证明栽培花锚可以替代野生花锚入药。