Comparative analysis of twenty-five compounds in different parts of Astragalus membranaceus var.mongholicus and Astragalus membranaceus by UPLC-MS/MS

As a traditional Chinese medicine,the root of Astragalus membranaceus var.mongholicus(AMM) or A.membranaceus(AM) has been widely used in China and other Asian countries for thousands of years.Till now,the flavonoids,phenolic acids and saponins are considered as the main active components contributing to their therapeutic effect in these plants.In order to clarify the distribution and contents of these compounds in different organs of these plants,a rapid and sensitive analytical method for simultaneous determination of 25 active compounds including seven types(i.e.dihydroflavones,isoflavane,isoflavones,flavones.pterocarpans,phenolic acid and saponins) within 10 min was established using ultra-pressure liquid chromatography coupled with tandem mass spectrometry(UPLC-MS/MS).Then,the established method was fully validated and successfully applied to the determination of the contents of these analytes in different parts(root,rhizome,stem,leaf and flower) of AMM and AM.The results indicated that the contents of the same type of compounds in two different species plants were significantly different.Moreover,the obvious differences were also found for the distribution and contents of different type of compounds in five organs of the same species.The present study could provide necessary information for the rational development and utilization of AMM and AM resource.

Study on Tissue Culture and Radiation Mutation of Astragalus membranaceus Bge. var. mongholicus(Bge.) Hsiao

[Objective] The research aimed to study the tissue culture technology and callus induction by radiation mutation of A. membranaceus Bge. [ Method ] With the different parts of Astragalus membranaceus Bge. var. mongholicus （ Bge. ） Hsiao aseptic seedling as explants （ leaves, cotyledons, hypocotyls） induced callus, and cotyledon and hypocotyls taken by the method of radiation mutation were studied. [ Result]The results showed that the three explants had relatively high callus induced rate in the medium which respectively made up of MS ＋6-BA 2.0 mg/L ＋ NAA2.0 mg/L, LS ＋6-BA2.0 mg/L ＋NAA0.1 mg/L, MS ＋ 6-BA2.0 rng/L ＋ NAA2.0 rag/L; the optimum mutation time of hypocotyls and cotyledons was 15 minutes; the growth of the callus induced from hypocotyls would be better as the mutation time increased, but when it reached a certain time the growth would be weaken, the induction rate also would be reduced. [ Conclusion] This study will provide the scientific reference in tissue culture and mutation breeding of A. membranaceus Bge.

Analysis of principal isoflavone glycosides and aglycones in Radix astragali

Two major isoflavone glycosides [calycosin 7-O-β-D-glucopyranoside （1） and ononin （2）] and their aglycones [calycosin （3） and formononetin （4）] were simultaneously quantified with HPLC/DAD method. Two unknown compounds were identified as calycosin 7-O-β-D-glucopyranoside-6＇＂-O-malonate （U1） and formononetin 7-O-β-D-glucopymnoside-6＇＂-O-malonate （U2）, respectively, with LC/MS^n. Raw Radix astragli were shown to have higher contents of isoflavone glycosides （1, 2）, but lower contents of aglycones （3, 4） than the processed herbal materials. After being moistened with water and stored up for 24 h at 35 ℃, the glycosides and their m_alonates were almost completely transformed to their corresponding aglycones. The different contents of the isoflavone glycosides and their aglycones in raw and processed Radix astragali materials might be due to enzymolysis of the glycosides during processing.

Content Determination of Astragaloside Ⅳ in Astragalus from Three Different Regions by HPLC

[Objectives] The content of astragaloside in Astragalus membranaceus(Fisch.) Bge.var.mongholicus(Bge.) Hisao from three different regions was determined.[Methods] Referring to the method recorded in the Chinese Pharmacopoeia(2015 edition),the content of astragaloside IV in A.membranaceus was determined by HPLC.[Results] There were great differences in the astragaloside IV content of A.membranaceus among different regions.The content of astragaloside IV in A.membranaceus cultivated in Inner Mongolia was highest(0.155%),followed by that(0.143%) in A.membranaceus cultivated in Gansu,and the content of astragaloside IV in A.membranaceus cultivated in Shanxi was lowest(0.080%).The contents of astragaloside IV in A.membranaceus from different regions were all in line with the standard(not less than 0.040%) of Chinese Pharmacopoeia(2015 edition).[Conclusions]The content of astragaloside IV in A.membranaceus cultivated in three different regions met the medicinal standards.

偏最小二乘法和近红外光谱技术快速测定发酵黄芪茎叶中黄酮含量及抗氧化活性

试验测定发酵前、后黄芪茎叶黄酮含量和抗氧化活性,并应用近红外光谱技术(NIR)结合化学计量学法快速预测发酵黄芪茎叶黄酮含量,为定量检测发酵黄芪茎叶黄酮含量提供理论依据以及为黄芪茎叶深加工利用提供技术支持。试验采用硝酸铝-亚硝酸钠比色法测定发酵黄芪茎叶黄酮的含量,测定黄酮对1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除能力和羟基自由基的清除能力,利用偏最小二乘方法构建发酵黄芪茎叶黄酮的快速预测模型。结果表明,发酵黄芪茎叶黄酮抗氧化能力强于发酵前,构建快速预测模型采用了二阶导数(SD)+正态变量变换(SNV)+去趋势算法(Detred)的方法对全谱图进行预处理的效果较好,优化后的模型决定系数(R^(2))、校正均方根误差(RMSEC)、校准标准差(SEC)分别为0.92、0.24、0.24,其相对分析误差(PRD)为3.46,外部验证R2为0.88。研究表明,构建的NIR模型校正和交互验证决定系数均较大,PRD均大于2,说明模型预测性能较好,建立的模型有助于检测发酵黄芪茎叶黄酮含量活性成分。

Influence of Flavonoid of Astragalus membranaceus′s Stem and Leaves on the Function of Cell Mediated Im munity in Mice

Objective: To investigate the immune regulation of flavonoid of Astragalus membranaceus′ s stem and leaves (FAM). Methods: Changes of total T cell count and subsets in mice were determined by monoclonal antibody assay before and after treatment with FAM, and the lymphokine activated killer(LAK) activity was tested simultaneously by isotope technique. Results: FAM could promote the proliferation of lymphocytes induced by ConA, raise the T cell count and regulate the T cell subsets disorder, elevate the LAK activity induced by IL 2. Conclusion: FAM possesses the effect of immune stimulation and immune regulation in treating immunosuppressive mice. This study provides the experimental basis for clinical application of FAM.

基于HPLC结合化学计量学研究黄芪茎叶的最适采收期

目的:建立黄芪茎叶中7种黄酮类和3种皂苷类成分含量的HPLC同步检测方法,研究不同采收期黄芪茎叶中活性成分的动态变化规律,明确黄芪茎叶的最适采收期。方法:对色谱条件进行方法学考察,确定10种活性成分的HPLC同步检测方法,进而测定不同采收期黄芪茎叶中10种活性成分的含量,再结合聚类分析和主成分分析以及熵权TOPSIS法分析上述成分含量的动态变化与分布规律。结果:10种成分随不同采收期均呈显著变化(P<0.05),其中黄芪茎中黄酮总量在8月下旬~9月较高,皂苷总量在8月上旬达到最高;黄芪叶中黄酮总量在8月~9月上旬较高,皂苷总量变化趋势不明显。化学计量学分析结果显示,黄芪茎和叶中的化学成分含量在8月前后均有明显不同,进一步的熵权TOPSIS分析结果表明,8~9月的黄芪茎质量综合评价较高,8月的黄芪叶质量综合评价最高,建议黄芪茎叶适宜的采收期为8月下旬。结论:所建立的HPLC方法可用于黄芪茎叶中黄酮类和皂苷类活性成分的同步检测,所明确的黄芪茎叶最适采收期可为黄芪茎叶的质量控制和精深开发利用提供科学依据。

蒙古黄芪育苗基质的筛选及应用潜力初步评价

为筛选出适宜培育高质量幼苗的育苗基质,促进移栽蒙古黄芪(Astragalus membranaceus var.mongholicus)的生产,以酒糟为主料,牛粪和沼渣为辅料,按不同体积比配制了6种有机复合基质,并对其理化性质,以及碱解氮、速效磷、速效钾的含量进行了测定。通过PLS-DA分析辅料对复合基质性质的影响,采用模糊评价法评估各复合基质肥效,进而筛选出基质F2(酒糟∶牛粪=60∶20)和Z2(酒糟∶沼渣=60∶20),将二者分别与纯土按体积比3∶7混合制成育苗基质后,通过发芽和幼苗生长试验评价应用潜力。结果表明,各有机复合基质理化性质基本符合常规基质要求,分别以牛粪和沼渣为辅料的两组复合基质,F2和Z2的肥效评价系数较高。混合育苗基质“F2+纯土”组的蒙古黄芪种子发芽率最高,且发芽势和发芽指数显著高于“Z2+纯土”组和对照组;同时,该组幼苗的整株长、株高和鲜重等指标也显著优于其他两组。由此可见,有机复合基质F2性能良好,与纯土混合后制成的育苗基质可用于蒙古黄芪优质幼苗的培育,具有较好的应用潜力。

发酵黄芪茎叶饲喂肉羊应用效果试验

为考察发酵黄芪茎叶饲喂肉羊的应用效果,将挑选的黄芪茎叶揉碎与黄豆粉、麦麸、玉米面、解淀粉芽孢杆菌等混合发酵,替代50%的粗饲料饲喂肉羊,粗饲料成本从360元/t降为340元/t,粗饲料成本减少了20元/t,降低了5.56%;通过对356只小尾寒羊90 d的饲喂,试验组羊平均日增重比对照组羊增加了0.02 kg,提高了8.7%,试验组与对照组相比差异极显著(P<0.01);整个试验期间,试验组羊发病率比对照组羊减少了6.74%;试验组羊净收入比对照组羊多36.55元/只,增加经济效益10.53%。说明发酵黄芪茎叶饲喂肉羊可以降低肉羊饲料成本,提高肉羊饲料利用率,增加肉羊增重,增强肉羊机体免疫力。

A reference-grade genome assembly for Astragalus mongholicus and insights into the biosynthesis and high accumulation of triterpenoids and flavonoids in its roots

Astragalus membranaceus var.mongholicus(AMM),a member of the Leguminosae,is one of the most important medicinal plants worldwide.The dried roots of AMM have a wide range of pharmacological effects and are a traditional Chinese medicine.Here,we report the first chromosome-level reference genome of AMM,comprising nine pseudochromosomes with a total size of 1.47 Gb and 27868 protein-encoding genes.Comparative genomic analysis reveals that AMM has not experienced an independent wholegenome duplication(WGD)event after the WGD event shared by the Papilionoideae species.Analysis of long terminal repeat retrotransposons suggests a recent burst of these elements at approximately 0.13 million years ago,which may explain the large size of the AMM genome.Multiple gene families involved in the biosynthesis of triterpenoids and flavonoids were expanded,and our data indicate that tandemduplication has been the main driver for expansion of these families.Among the expanded families,the phenylalanine ammonia-lyase gene family was primarily expressed in the roots of AMM,suggesting their roles in the biosynthesis of phenylpropanoid compounds.The functional versatility of 2,3-oxidosqualene cyclase genes in cluster Ⅲ may play a critical role in the diversification of triterpenoids in AMM.Our findings provide novel insights into triterpenoid and flavonoid biosynthesis and can facilitate future research on the genetics and medical applications of AMM.

移栽期对蒙古黄芪(Astragalus membranaceus var.mongholicus)药材产量和质量的影响

采用初冬和春季移栽对比试验,定期测定土壤含水量、生长发育指标、药材产量和质量指标,旨在探寻地道产区蒙古黄芪(Astragalus membranaceus var.mongholicus)最佳移栽季节。结果表明:与春栽田比较,冬栽田20cm土层内土壤含水量在苗栽返青期显著增高,而40cm以下土层土壤含水量在生长后期显著降低;苗栽返青和开花分别提早10d和13d,返青出苗势、出苗率和出苗指数分别提高32.6%、4.4%和0.2497,返青株成活率提高5.0%,有效延长了黄芪生长天数,为药材根的生长和有效成分积累奠定了良好的营养生长基础。初冬栽和春栽田成药根数分别为201 087根·hm-2和181 250根·hm-2,一等品出成率分别为24.02%和19.69%,药材鲜产量分别为5 400kg·hm-2和5 057kg·hm-2;黄芪甲苷含量分别为0.121%和0.098%,毛蕊异黄酮葡萄糖苷含量分别为0.080%和0.052%,水溶性浸出物含量分别为27.62%和25.54%。初冬移栽黄芪成效更为显著,建议规范化生产中合理调整移栽季节,提倡初冬移栽。

黄芪茎叶总皂苷的响应面提取工艺优化及抗氧化活性研究

采用超声法提取黄芪茎叶中的总皂苷,利用单因素结合响应面法优化其提取工艺,并通过测定提取物对DPPH·、ABTS+·、·OH、O_(2)^(-)·和H2O2的清除能力评价其抗氧化活性。结果表明黄芪茎叶总皂苷的最佳提取工艺为超声功率200 W,乙醇浓度60%,液料比25∶1 mL/g,提取时间61 min,提取温度57℃,在该工艺条件下黄芪茎叶总皂苷提取量为8.44 mg/g,HPLC法测得此时黄芪甲苷转移率达70%以上。黄芪茎叶总皂苷对DPPH·、ABTS+·、·OH、O_(2)^(-)·和H2O2的清除能力与浓度呈正相关,1.0 mg/mL的总皂苷提取物对DPPH·和ABTS+·的清除率分别为84.38%和96.19%,是同浓度下阳性对照Vc活性的87.21%和96.97%,但该浓度提取物对H2O2的清除能力稍弱,仅为59.39%。对于·OH和O_(2)^(-)·,当提取物浓度达到5.0 mg/mL时才表现出较佳的清除能力,清除率分别为79.73%和63.28%。最优工艺条件下所得的黄芪茎叶总皂苷具有一定的抗氧化活性,为黄芪茎叶资源在抗氧化产品中的开发应用提供了理论依据。

干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响

以山西道地药材黄芪一年生幼苗为试验材料,设置常规水分条件(CK)、轻度干旱胁迫(A1)、中度干旱胁迫(A2)、重度干旱胁迫(A3)4个不同处理,研究土壤干旱胁迫对黄芪生长及生理的影响。结果表明:黄芪茎叶快速生长集中在出苗后80—120d,以后生长减缓;当茎叶枯萎时,根中生物量短期快速积累。与常规水分条件相比,干旱胁迫处理显著降低了黄芪苗高及茎叶生物量,但对抗氧化能力、根系生长及次生代谢物积累产生了不同的影响。轻度干旱胁迫下SOD、POD、CAT 3种抗氧化酶活性升高,丙二醛(MDA)含量和细胞膜透性降低,同时根长与根生物量增加、多糖与皂苷两种次生代谢物积累增多,黄芪药材的质量得到显著提高(P<0.05);胁迫上升到中度、重度时,SOD酶活性逐渐降低,重度胁迫下低于对照,而POD及CAT酶活性、MDA含量、细胞膜透性均随胁迫增强而升高,相反,根长、根生物量、多糖与皂苷含量降低,导致黄芪药材的质量显著降低(P<0.01)。综上表明,干旱胁迫下,SOD酶表现较为敏感,可能在清除活性氧中起主要作用;轻度水分胁迫能有效启动黄芪体内抗氧化酶系统和次生代谢,它们相互协作共同对抗胁迫对细胞产生的伤害,通过降低地上部分的生长,将营养物质优先运往根部,促进根产量及药材质量的提高。这一结论,可在黄芪多糖和皂苷次生代谢物定向培育的水分管理中加以利用。

膜荚黄芪茎叶总黄酮对小鼠细胞免疫功能的影响

目的：研究黄芪茎叶总黄酮（ＦＡＭ）的免疫调节作用。方法：用单克隆抗体检测实验小鼠药物处理前后Ｔ细胞总数及亚群变化；用核素标记法测定ＬＡＫ细胞活性的变化。结果：ＦＭＡ能促进ＣｏｎＡ诱导的淋巴细胞增殖，增加Ｔ细胞总数并调整Ｔ细胞亚群紊乱；提高ｒＩＬ２所诱导的ＬＡＫ细胞活性。结论：ＦＡＭ对免疫功能低下小鼠具有免疫刺激和免疫调整作用，为临床应用提供了实验依据。

干旱胁迫对黄芪植株生长中黄酮类成分积累的影响

以3年生蒙古黄芪植株为材料,采用盆栽法模拟干旱处理,研究水分胁迫对黄芪生理状态以及其根、茎、叶中毛蕊异黄酮、毛蕊异黄酮葡萄糖苷、芒柄花素和芒柄花苷4种黄酮类化合物含量的影响。结果显示:(1)毛蕊异黄酮和毛蕊异黄酮葡萄糖苷在黄芪根、茎、叶均有分布,不同器官的毛蕊异黄酮葡萄糖苷含量表现为根>叶>茎,而毛蕊异黄酮含量则是叶>茎>根。(2)干旱胁迫下,黄芪根中毛蕊异黄酮葡萄糖苷和芒柄花苷在胁迫第8~12天有一定的积累;叶中的毛蕊异黄酮则在干旱第4~8天有明显的积累,而毛蕊异黄酮葡萄糖苷则没有明显变化。研究发现,4种黄酮类化合物在黄芪各器官中分布和含量不尽相同,同一器官中不同黄酮类化合物对干旱胁迫的响应规律也存在差异;适度的干旱胁迫能够促进黄芪毛蕊异黄酮葡萄糖苷的积累,但过度干旱胁迫则不利于其积累。

蒙古黄芪中黄酮类成分的研究

目的研究蒙古黄芪的化学成分,为该中药的开发利用和质量评价提供依据。方法利用多种色谱方法分离纯化,通过理化常数测定和波谱分析鉴定其化学结构。结果从蒙古黄芪中分离鉴定了9个黄酮类化合物,分别为芒柄花素(formononetin,Ⅰ)、(3R)-8,2′-二羟基-7,4′-二甲氧基异黄烷[(3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavane,Ⅱ]、毛蕊异黄酮(calycosin,Ⅲ)、(6aR,11aR)9,10-二甲氧基紫檀烷-3-O-β-D-葡萄糖苷[(6aR,11aR)9,10-dimethoxypterocarpan-3-O-β-D-glucoside,Ⅳ]、7,2′-二羟基-3′,4′-二甲氧基异黄烷-7-O-β-D-葡萄糖苷(7,2′-di-hydroxy-3′,4′-dimethoxy-isoflavane-7-O-β-D-glucoside,)、芒柄花素-7-O-β-D-葡萄糖苷(formononetin-7-O-β-D-glucoside,Ⅴ)、毛蕊异黄酮-7-O-β-D-葡萄糖苷(calycosin-7-O-β-D-glucoside,Ⅵ)、红车轴草异黄酮-7-O-β-D-葡萄糖苷(pratensein-7-O-β-D-glucoside,Ⅶ)和染料木苷(genistin,(Ⅸ))。结论化合物为首次从黄芪属植物中分得,化合物为首次从该种植物中获得,化合物～具有促进细胞增殖的活性。-

高速逆流色谱法分离纯化黄芪中的芒柄花素和毛蕊异黄酮

采用高速逆流色谱法(HSCCC),以正己烷氯仿甲醇水组成二相系统作为固定相与流动相,对黄芪的乙酸乙酯粗提物进行了分离纯化。结果发现:以正己烷氯仿甲醇水(体积比为1.5∶3∶3∶2)组成的系统可以从黄芪的乙酸乙酯粗提物中分离出毛蕊异黄酮,纯度可达95%以上,并可以初步纯化芒柄花素;接着用正己烷氯仿甲醇水(体积比为4∶4∶5∶4)组成的系统进一步纯化芒柄花素,其纯度达95%以上。利用该方法,可以对中药黄芪中的异黄酮进行快速的分离和纯化。

播种量和方式对甘肃渭源蒙古黄芪育苗质量和产量的影响

以陇芪2号为试验材料,采用不同播种量(90,150,210kg/hm^2)和播种方式(条播,撒播,覆膜穴播)两因素育苗,育苗结束后秋季测定种苗质量指标和产量,旨在揭示不同播种量和播种方式互作对蒙古黄芪种苗质量和产量的影响,为其规范化栽培提供依据。结果表明,不同播种量和播种方式交互条件下育苗,蒙古黄芪种苗个体质量和群体质量均存在显著差异。在90kg/hm^2最低播种量基础上,随着播种量的增加,种苗单根鲜重、根长和根粗等个体质量指标均呈降低趋势,种苗产量先极显著提高后下降,产苗数持续增加,优质种苗率显著下降。在同一播量条件下覆膜穴播方式育成种苗个体质量最优,优质种苗率均比条播和撒播高,并随播种量递增,苗产量较撒播和条播呈先增加后下降,而产苗数呈下降趋势。播种量150kg/hm^2条件下采用覆膜穴播育成单位面积优质种苗数最多。说明在保证种苗个体质量和单位面积产苗数的前提下,渭源蒙古黄芪育苗采用覆膜穴播方式,种子播种量为150kg/hm^2最佳。

黄芪多倍体的诱导与鉴定

本试验采用改良琼脂涂抹法(0.2％的秋水仙碱与0.1％的琼脂混合成半固体)处理黄芪Astragalusmembranaceus var.mongholicus幼苗顶芽,得到的诱导株,经生物学性状鉴定和染色体鉴定为四倍体。

砷胁迫对药用植物黄芪中抗氧化酶活性的影响

[目的]探讨砷胁迫对药用植物黄芪中抗氧化酶活性的影响,以期为揭示砷毒害植物机理提供理论依据。[方法]以内蒙药用植物黄芪为材料,分别采用高锰酸钾滴定法、愈创木酚法、邻苯三酚自氧化法以及硫代巴比妥酸法研究不同浓度砷胁迫下对药用植物黄芪中过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)的活性影响以和丙二醛(MDA)的浓度变化。[结果]随着砷浓度升高,黄芪叶片中这3种酶的活性能够维持,特别是POD活性显著增加。从时间动态看,随处理时间的延长,黄芪叶片中的CAT活性先升高再降低,POD活性增加,SOD活性变化不显著;MDA含量先升后降,第8天达到最大值。[结论]该研究可为中药材重金属含量控制和药理活性鉴定提供理论依据。