Optimization of Polysaccharide Extraction from Radix Glehniae Root Bark by Response Surface Methodology and Anti-immunosuppressive Activity Analysis

[ Objective ] This study aimed to establish and optimize the extraction technology of polysaccharides from Radix Glehniae root bark, and to explore the processing methods of Radix Glehniae in producing areas. [ Method ] Based on the single-factor experiment, with polysaccharide content and polysaccharide yield as indices, various factors affecting polysaccharide yield from Radix Glehniae root bark were investigated by central composite design-response surface method. Mo- reover, immune functions of cyclophosphamide-induced blood-deficient mice in various polysaccharide groups were compared to investigate the effects of root bark removal of Radix Glehniae on medicinal quality. [ Result] Ultrasonic extraction was the appropriate extraction technology of polysaccharides from Radix Glehniae root bark. The optimal ultrasonic extraction conditions were ultrasonic time 36 min, ultrasonic power 460 W, solid-liquid ratio 1： 10, ethanol concentration 80%, under which polysaccharide yield reached 16.08%. Polysaccharides extracted from Radix Glehniae root （ALP） and Radix Glehniae root bark （BLP） could effec- tively improve immune function of blood-deficient mice with no significant differences. [ Conclusion] Radix Glehniae could be used without removal of root bark. The optimized extraction process was stable and feasible, which laid a solid foundation for further comprehensive development and utilization of Radix Glehniae root bark.

Cloning and Bioinformatics Analysis of the GlROP6 gene in Glehnia littoralis

Rho-related GTPase from plants(ROP)proteins play an essential role in plant stress resistance.In this study,the full-length GlROP6 gene was cloned based on G.littoralis transcriptome sequencing data acquired in response to salt stress.The protein sequence,conserved domains,secondary structure,three-dimensional structure,phylogenetic relationships,and expression pattern of the GlROP6 gene were systematically analysed.Our results showed that the full-length GlROP6 gene had an open reading frame of 606 bp,which encoded 201 amino acid residues with a relative molecular weight of 22.23463 kDa and a theoretical isoelectric point of 9.06.Amino acid sequence analyses indicated that the structure of the GlROP6 protein was conserved,and included five G-box motifs(G1–G5),an effector binding region,a Rho insert region and a C-terminal hypervariable region.According to our phylogenetic analysis,the GlROP6 protein was closely related to the ROP protein of Daucus carota subsp.Sativus.Our quantitative real-time PCR results revealed that GlROP6 was highly expressed in flower,and GlROP6 expression was significantly upregulated in G.littoralis roots treated with NaCl.This study will facilitate investigations into the function of GlROP genes in response to salt stress in G.littoralis.

Radix Glehniae extract inhibits the migration and invasion abilities of lung cancer cells

Objective: To investigate the effect of Radix Glehniae on the migration and invasion abilities of lung cancer cells, and to explore the underlying mechanism. Method: Normal human bronchial cell line 16 HBE and lung cancer cell line SK-MES-1 were cultured in vitro. Radix Glehniae extract was prepared to treat the two kinds of cells. The proliferation ability of 16 HBE cells was determined by CCK-8 assay. The migration and invasion abilities of SK-MES-1 cells were determined by Transwell and Matrigel assays, respectively. The expression of TIMP2 m RNA in SK-MES-1 cells was detected by fluorescence quantitative PCR(q-PCR). The secretion level of TIMP2 protein by SK-MES-1 cells was measured by ELISA experiment. Results: Radix Glehniae extract caused no toxic effects on 16 HBE cells at the concentrations of 1 mg/ml to 15 mg/ml. Radix Glehniae extract(from 5 mg/ml to 15 mg/ml) significantly inhibited SK-MES-1 cell migration and invasion abilities, as well as upregulated TIMP2 m RNA expression and enhanced TIMP2 secretion by SK-MES-1 cells. Conclusion: Radix Glehniae can inhibit the migration and invasion behaviors of lung cancer cells by upregulating TIMP2 expression and secretion.

Research progress in polysaccharide compositions of Glehnia littoralis

More and more attention has been paid to the polysaccharide components of Glehnia littoralis.It has good immunoregulation,antioxidant,antitumor,hypoglycemic and other biological activities,as well as special health care functions.Therefore,it has a broad application prospect in medical,health care,food and other industries.In this paper,the extraction,purification,content determination and pharmacological activities of polysaccharides from Glehnia littoralis were reviewed,which provided a gauge for the further development and utilization of Glehnia littoralis.

A review of research on coumarins from Radix Glehniae

Radix Glehniae,with a wide range of chemical components of various pharmacological effects,is widely used in the prevention and treatment of various diseases in clinical practice.It has a broad prospect of development and application.Coumarin,as one of its major active components,also displays good biological activities for analgesia and sedation,antibacteria,antivirus,anti HIV,anti-inflammation,anti-tumor,anti mutation,tyrosinase inhibition,vasodilation and blood pressure reduction.The aim of the present review is to discuss the studies on coumarin components,content determination,pharmacological activities,processing methods and provide a guide for the further development and utilization of Radix Glehniae.

New lignan glycosides from Glehnia littoralis

从 Glehnia littoralis，二新 lignan glycosides，命名 glehlinoside E (1 ) 和 F (2 ) 的根乙醇摘录，被获得。他们的结构决心是(?)-secoisolariciresinol 4-O--d-(6-O-feruloyl ) glucopyranoside (1 ) 和(?)-secoisolariciresinol 4-O--d-(6-O-caffeloyl ) 由分析的 glucopyranoside (2 ) 他们的光谱数据分别地。

珊瑚菜果实形成与内源激素含量的相关性分析

为明确珊瑚菜果实形成时期及其4种内源激素含量的动态变化,分析果实形成与内源激素含量间的关系,采用高效液相色谱法测定珊瑚菜果实发育过程中内源生长素(indole-3-acetic acid,IAA)、玉米素(zeatin,ZT)、赤霉素(gibberellin a3,GA_(3))、脱落酸(abscisic acid,ABA)的含量。结果表明,珊瑚菜果实形成过程可划分为乳熟期、中熟期和完熟期;其纵径和横径均呈现出快-慢-快的增长趋势;含水量表现为先升高后降低;百粒重显著增加,至完熟期达2.66 g。在果实发育过程中,ZT和IAA含量均呈升-降-升-降的变化趋势;GA_(3)含量呈降-升-降的变化,其中在果实完熟末期ABA含量达46.02 ng·g^(-1)。相关性分析表明,ZT含量与果实横径呈显著正相关,与果形指数呈极显著负相关;其他性状间相关性不显著。上述结果初步揭示了珊瑚菜果实形成中各指标变化及其与4种内源激素的关系,为激素调控果实形成和提高粒重提供了理论依据。

Glehnia fittoralis Extract Promotes Neurogenesis in the Hippocampal Dentate Gyrus of the Adult Mouse through Increasing Expressions of Brain-Derived Neurotrophic Factor and Tropomyosin-Related Kinase B

Background： Glehnia littoralis has been used for traditional Asian medicine, which has diverse therapeutic activities. However, studies regarding neurogenic effects of G. littoralis have not yet been considered. Therefore, in this study, we examined effects of G. littoralis extract on cell proliferation, neuroblast differentiation, and the maturation of newborn neurons in the hippocampus of adult mice. Methods： A total of 39 male ICR mice （12 weeks old） were randomly assigned to vehicle-treated and 100 and 200 mg/kg G. littoralis extract-treated groups （n = 13 in each group）. Vehicle and G. littoralis extract were orally administrated for 28 days. To examine neurogenic effects ofG. litmralis extract, we performed immunohistochemistry tbr 5-bromo-2-deoxyuridine （BrdU, an indicator for cell proliferation） and doublecortin （DCX, an immature neuronal marker） and double immunofluorescence staining for BrdU and neuronal nuclear antigen （NeuN, a mature neuronal marker）. In addition, we examined expressional changes of brain-derived neurotrophic factor （BDNF） and its major receptor tropomyosin-related kinase B （TrkB） using Western blotting analysis. Results： Treatment with 200 mg/kg, not 100 mg/kg, significantly increased number of BrdU-immunoreactive （＋） and DCX＋ cells （48.0 ±3.1and 72.0 ± 3.8 cells/section, respectively） in the subgranular zone （SGZ） of the dentate gyrus （DG） and BrdU＊/NeuN＋ cells （17.0 ±1.5 cells/section） in the granule cell layer as well as in the SGZ. In addition, protein levels of BDNF and YrkB （about 232% and 244% of the vehicle-treated group, respectively） were significantly increased in the DG of the mice treated with 200 mg/kg ofG. littoralis extract. Conclusion： G. littoralis extract promots cell proliferation, neuroblast differentiation, and neuronal maturation in the hippocampal DG, and neurogenic effects might be closely related to increases ofBDN F and TrkB proteins by G. littoralis extract treatment.

北沙参种子内源抑制物质活性研究

研究北沙参种子和果皮各分离相对白菜种子萌发和幼苗的影响,探讨北沙参种子内源抑制物的活性。对北沙参种子不同部位的浸提液通过系统溶剂法进行分离,分离出石油醚相、二氯甲烷相、乙酸乙酯相、正丁醇相、甲醇相和水相,并以各分离相对白菜种子进行萌发实验,测定北沙参种子内源抑制物生物活性。结果显示,①北沙参种子、果皮各分离相对白菜种子萌发均有一定的抑制作用,其中抑制作用最强的是甲醇相,其次是乙酸乙酯相;与对照组相比,各处理组的发芽指数均有一定程度的下降;甲醇相的平均发芽天数显著长于其他处理。②北沙参种子、果皮各分离相在抑制白菜幼苗根长和芽长方面表现出明显的差异,其中,甲醇相的抑制作用最显著,其次为乙酸乙酯相。与萌发指标结果一致。北沙参种子不同部位分离相对白菜种子萌发及幼苗存在不同程度的抑制作用,表明北沙参种子、果皮均含有高活性抑制物质,主要存在于甲醇相和乙酸乙酯相,因此,北沙参种子中的内源抑制物是导致其休眠的主要原因。

干旱胁迫对栽培种及野生种北沙参叶片解剖结构及生理特性的影响

为探究干旱胁迫对北沙参生长发育的影响,以栽培种和野生种北沙参为试验材料,测定不同浓度PEG模拟干旱胁迫下北沙参的解剖结构、生理指标及叶绿素荧光参数。结果表明:随着干旱程度的增加,叶片、上下表皮、栅栏组织及海绵组织厚度减小,其中30%PEG胁迫对两种北沙参影响最明显;在PEG胁迫增加的过程中,栽培种较野生种叶绿素含量变化较小,可溶性蛋白含量较高,且较对照呈极显著提高;两种北沙参叶片F_(v)/F_(m)值、光化学荧光淬灭系数(q_(p))和PSⅡ的实际光化学效率(Y(Ⅱ))与对照相比均呈下降的趋势,栽培种比野生种降幅小。这些结果说明栽培种比野生种北沙参对干旱胁迫有较强的耐受能力。

珊瑚菜叶绿体基因组密码子使用偏性分析

为探究珊瑚菜(Glehnia littoralis F.Schmidt ex Miq.)叶绿体基因组密码子使用偏性及其影响因子,本研究基于珊瑚菜叶绿体基因组测序数据筛选到51个蛋白质编码基因,并对其密码子使用模式进行分析。结果显示:珊瑚菜叶绿体基因组密码子总GC含量(G_(Call))为38.8%,密码子第3位碱基的GC含量(GC3)最低,仅为29.2%,3个位置碱基的GC含量均小于50%,表明珊瑚菜叶绿体基因组密码子偏好使用A或U。珊瑚菜叶绿体基因组有效密码子数(ENC)为34.6~54.5,密码子适应指数(CAI)为0.100~0.270,密码子偏好指数(CBI)为-0.190~0.226,最优密码子使用频率(FOP)为0.276~0.550,表明珊瑚菜叶绿体基因组密码子使用偏性较弱;相关性分析结果表明珊瑚菜叶绿体基因组密码子第3位碱基的组成对基因表达有很大影响。中性绘图分析、PR2-plot分析、ENC-plot分析和对应性分析结果表明珊瑚菜叶绿体基因组密码子使用偏性受自然选择和突变的共同影响,以自然选择为主;基于相对同义密码子使用度(RSCU)和高、低表达组的RSCU差值(ΔRSCU)共获得16个最优密码子,且大部分以A或U结尾。综合分析结果表明:珊瑚菜叶绿体基因组密码子使用偏性较弱,且主要受自然选择的影响。

沙藏对北沙参种子萌发的影响

以未沙藏、沙藏后萌发与未萌发的北沙参种子为实验材料,运用数字基因表达谱(DGE)技术,对种子的转录本进行功能注释和差异分析。通过分析沙藏和未沙藏以及沙藏后萌发与未萌发的北沙参种子基因表达差异,探究沙藏打破北沙参种子休眠的机理和种子萌发的分子机制。结果表明,测序后共得到218387701条原始序列,有7.85%的Unigene注释到7个数据库中。差异基因的通路分析中,沙藏与未沙藏的种子基因差别在核糖体,萌发与未萌发的种子主要区别在苯丙烷生物合成途径。北沙参种子沙藏后可以引起核糖体基因的表达,打破种子休眠。香豆素作为苯丙烷合成的重要产物,其基因的差异表达,可能与种子的萌发有密切关系。

不同种源北沙参香豆素含量及关键酶基因表达量差异研究

目的:探究北沙参中香豆素含量及IPT基因相对表达量的差异,为北沙参品质评定及进一步选优育种提供理论依据。方法:采用HPLC法测定北沙参中9个香豆素的含量,实时荧光定量PCR检测异戊烯基转移酶基因(IPT)的相对表达量。结果:栽培品种北沙参莱阳、赤峰及野生品种八仙岛、金石滩、海阳中香豆素含量较高;赤峰和海阳种源中北沙参IPT基因相对表达量较高;IPT基因表达量与补骨脂素、异茴芹内酯含量呈极显著正相关(P<0.01);主成分分析得出莱阳与海阳为优质北沙参种源。结论:该实验探究了8个种源北沙参中香豆素的含量、IPT基因表达量的差异及二者间的相关性,可为筛选具有高应用价值的北沙参种源提供参考,有助于北沙参的进一步开发利用。

北沙参补骨脂素合成酶基因 GlPS1 原核表达载体的构建和蛋白诱导表达

北沙参(Glehniae Radix)为伞形科植物珊瑚菜(Glehnia littoralis)的干燥根,具有良好的药用价值和较高的经济价值。补骨脂素(psoralen)是北沙参香豆素类化合物主要的活性成分之一,对白血病细胞有较强的杀伤作用,对支气管平滑肌有舒张作用。补骨脂素合成酶(GlPS)是北沙参补骨脂素生物合成途径的最后一步关键酶。本试验通过PCR扩增获得对北沙参补骨脂素合成酶基因GlPS1,通过酶切、连接、转化、平板培养和提取质粒构建原核表达载体;将构建好的表达载体转入大肠杆菌BL21(DE3)中,实现GlPS1基因的IPTG诱导原核表达。该研究表明被诱导表达的GlPS1蛋白存在于菌体沉淀中,不溶于上清液。研究结果为验证GlPS1基因的功能及使用基因工程方法调控GlPS1蛋白表达奠了基础。

北沙参茎叶和根中挥发油的提取与抗真菌活性研究

目的探究北沙参茎叶和根中挥发油的提取工艺,解析北沙参茎叶和根中挥发油所含化学成分,并初步评价其体外抗真菌活性。方法以水蒸气蒸馏法为基础,通过单因素与正交实验优化北沙参茎叶和根中挥发油提取方法,利用气相色谱-质谱联用(GC-MS)技术鉴定北沙参茎叶和根中挥发油所含化学成分并计算其相对百分含量。采用纸片扩散法测定北沙参茎叶和根中挥发油对茄病镰刀菌、半裸镰刀菌、尖孢镰刀菌、寄生曲霉和黄曲霉的抗真菌活性。结果北沙参最佳提取工艺为料液比1∶15,蒸馏时间5 h,KCl浓度15%。从北沙参茎叶挥发油中鉴定出11种成分,根挥发油中共鉴定出8种成分。人参炔醇为北沙参茎叶挥发油和根挥发油的共有成分,其在茎叶中的含量为38.21%,根中的含量为74.02%。北沙参茎叶挥发油和根挥发油对茄病镰刀菌、半裸镰刀菌、尖孢镰刀菌、寄生曲霉和黄曲霉均有一定的抑制作用,以茎叶挥发油较强。结论北沙参茎叶挥发油和根挥发油的化学成分差异明显,但二者均具有一定的体外抗真菌活性。

基于网络药理学和分子对接探讨北沙参-麦冬药对治疗肺癌的作用机制

运用网络药理学和分子对接的方法,探究“北沙参-麦冬”药对治疗肺癌的相关作用机制.运用中药系统药理学数据库与分析平台(TCMSP),获得北沙参的有效活性成分及作用靶点,应用BATMAN-TCM数据库获得麦冬的有效活性成分及作用靶点,应用GeneCards及DisGeNET数据库获得肺癌相关靶点,绘制韦恩图获取药物疾病共同靶点,将其输入Cytoscape 3.9.0软件绘制“药物-成分-疾病-靶点”网络.运用STRING数据库及Cytoscape 3.9.0软件构建蛋白相互作用PPI网络.通过DAVID数据库进行基因本体(GO)、京都基因与基因组百科全书(KEGG)富集分析.运用AutoDock软件进行分子对接验证,Pymol软件进行可视化处理.“北沙参-麦冬”药对通过25个有效成分作用于137个肺癌治疗靶点.筛选出4种核心有效成分,12个核心靶点包括TP53、AKT1、MYC、ESR1、EGFR、VEGFA、CASP3、JUN、TNF、CCND1、HIF1A、IL6,GO富集分析主要涉及基因表达调控、凋亡调控、蛋白结合、转录因子结合、胞外、线粒体等,KEGG富集分析主要涉及TNF、p53、HIF-1、细胞凋亡等信号通路.4种核心有效成分与主要核心靶点TP53、MYC、EGFR、VEGFA、CASP3均具有一定的结合活性.结果可知,“北沙参-麦冬”药对治疗肺癌的机制,可能是通过多成分、多靶点、多通路,诱导肺癌细胞凋亡、抑制其增殖及侵袭转移.

辽宁省珊瑚菜野生资源调查

珊瑚菜(Glehnia littoralis Fr.Schmidt ex Miq.)又名北沙参,为伞形科珊瑚菜属的单种属植物,主要生长在沿海地区的海滨沙滩上,根可入药。由于城市的扩张、旅游景点的设立及港口的建设等原因,珊瑚菜的生存环境遭到破坏,加之人为的掠夺性采挖,其分布及资源逐渐减少,已被列为国家Ⅱ级重点保护野生植物。在辽宁省主要分布区内展开了对珊瑚菜野生资源的调查,结果显示:仅在大连长海一处面积约100 m^(2)的海滩发现一个珊瑚菜极小种群,该种群植株数量不足100株,呈聚集性分布,且以幼年植株为主,能开花结实的数量有限;在绥中的珊瑚菜原生境保护点中未发现珊瑚菜植株。根据调查结果得出结论:野生珊瑚菜目前在辽宁已经极度濒危,应加强保护。

不同轮作方式对北沙参生长及药材质量的影响

目的:探讨不同轮作方式对北沙参生长、质量及其根腐病发病率的影响,为有效缓解北沙参连作障碍及制订合理栽培制度提供理论依据。方法:于内蒙古赤峰市喀喇沁旗牛家营子镇进行三年大田试验,设置4种轮作处理,前茬作物分别为:玉米、牛膝、桔梗和防风。比较各处理北沙参生物量、根腐病发病率、水溶性和醇溶性浸出物含量,并利用UPLC测定5种香豆素(补骨脂素、花椒毒素、佛手柑内酯、欧前胡素和异欧前胡素)含量。结果:与玉米或牛膝轮作后,北沙参长势较好,根腐病发病率下降,根中浸出物和5种香豆素含量显著提高;而与防风轮作后,北沙参根部长势不佳,有效成分含量较低,根腐病发病情况较严重。结论:与玉米或牛膝轮作后,北沙参的生长及药材质量较好,且根腐病的发生较轻;而与防风轮作后,北沙参的长势和质量较差,表明防风不宜作为北沙参的前茬作物。

北沙参及其6种混淆品的鉴别

[目的]对北沙参与其混淆品明党参、田贡蒿、川明参、迷果芹、硬阿魏、蝇子草进行鉴别比较研究。[方法]采用性状鉴别、显微鉴别和薄层层析鉴别等方法对北沙参与其混淆品进行比较研究。[结果]明党参、田贡蒿、川明参、迷果芹、硬阿魏、蝇子草与药材北沙参在性状、显微、薄层层析等方面均有明显差别,可以区分。[结论]混淆品的功效与北沙参不同,不能作药材北沙参使用。