Determination on Heavy Metals Content of Achyranthes bidentata Blume. through Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

[Objective] The inductively coupled plasma mass spectrometry(ICP-MS)was constructed to determine the contents of lead,cadmium,mercury and arsenic in Archyranthes bidentata Blume.[Method]Under the optimum operation condition of ICP-MS,the samples were digested by microwave.The element 114In was taken as an internal standard element to compensate body effect and ICP-MS method was used to determine the contents of lead,cadmium,mercury and arsenic.[Result]For the determined elements,the correlation coefficient(r)of standard curve was over 0.9995 and recovery rate was from 96.7% to 106.4% while RSD was less than 11.2%.The result of determination showed that the heavy metal content in Archyranthes bidentata Blume.beyond standard was serious.[Conclusion]The constructed ICP-MS method with simple operation,rapid response,accuracy and high sensitivity in this experiment could be used for quality control of Chinese medicinal materials by detecting heavy metal contents in different Chinese medicinal materials from original places.

Effects of Pb on Rhizosphere Soil Enzyme Activity and Chemical Constituents of Achyranthes bidentata Blume

[Objectives]The purpose of this study was to investigate the effects of Pb on rhizosphere soil enzyme activity and chemical constituents of Achyranthes bidentata Blume.[Methods]A.bidentata Blume plants were cultivated in self-made polyvinyl chloride(PVC)pots.The soil was added with different levels of Pb(0,200,400,600,800 and 1000 mg/kg air-dried soil)to investigate the effects of Pb on dry mass,active ingredients(oleanolic acid and ecdysterone)and rhizosphere soil enzyme activity of A.bidentata Blume.[Results]The root dry mass of A.bidentata Blume cultivated in the soil with Pb level above 400 mg/kg significantly reduced.The Pb residues in the A.bidentata Blume.plants growing in the soil with Pb level below 400 mg/kg complied with national standard.The contents of oleanolic acid and ecdysterone in A.bidentata Blume growing in the soil with Pb level above 600 mg/kg declined significantly.At the Pb level of 1000 mg/kg,the activity of urease was inhibited significantly.The activity of phosphatase was inhibited in the presence of Pb in the soil.The activity of sucrase was activated in the soil with Pb level below 400 mg/kg,and was inhibited in the soil with Pb level above 400 mg/kg.[Conclusions]This study has important guiding significance for the reasonable selection of planting base for A.bidentata Blume and the guarantee of its yield and quality.

Deep chemical identification of phytoecdysteroids in Achyranthes bidentata Blume by UHPLC coupled with linear ion trap-Orbitrap mass spectrometry and targeted isolation

Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones.In this work,a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode.As a result,47 phytoecdysteroids were unambiguously or tentatively characterized.Among them,seven known compounds were identified according to the reference standards along with molecular formula,retention time and fragmentation patterns,while others were mostly potential new compounds.Through targeted isolation,the structures of three new compounds were determined by NMR spectra,which were consistent with LC-MS characterization.The present study provides an efficient method to deeply characterize phytoecdysteroids.

怀牛膝多糖的抗氧化性研究

研究怀牛膝多糖的抗氧化活性。采用传统热水浸提法和微波辅助提取法从怀牛膝中提取怀牛膝多糖,经分离纯化获得怀牛膝多糖提取物。分别测定各种多糖提取物和叔丁基对苯二酚对DPPH自由基、羟基自由基、超氧自由基的清除作用,比较和评价怀牛膝多糖的体外抗氧化活性。研究表明,怀牛膝多糖具有一定的抗氧化活性,有望作为天然抗氧剂,为怀牛膝多糖的进一步研究提供一定的理论基础。

中药牛膝中化学成分的研究

目的对牛膝 (AchyranthesbidentataBl.)的化学成分进行分离、鉴定。方法采用硅胶吸附柱色谱 ,分配柱色谱等分离手段 ;利用理化性质、理化反应以及波谱技术 (NMR、MS等 )对化合物进行鉴定。结果得到 5个化合物 :5 羟甲基糠醛 ( 5 hydroxymethylfuraldehyde) ( 1 ) ,人参皂苷Ro( gin senosideRo) ( 2 ) ,竹节参皂苷 1 (PJS 1 ) ( 3) ,polypodineB( 4 ) ,β 蜕皮甾酮 (ecdysterone) ( 5 )。结论化合物 1～ 4为首次从牛膝中分离得到。

用超临界流体色谱法测定怀牛膝及其制剂中齐墩果酸的含量

采用超临界流体色谱法(SFC)分别测定了不同产地的怀牛膝药材及三种含怀牛膝的制剂中齐墩果酸的含量。方法简单、快速。不需经特殊的纯化处理。各产地药材齐墩果酸含量波动较小(1.75～2.19％,x±SD=1.94±0.17％),可以作为药材与制剂质量评价的指标,药材的加样回收率为97.42％(n=18,RSD=2.80％);制剂天麻丸的加样回收率为94.82％(n=9,RSD=2.51％)。

牛膝的rDNAITS序列分析

目的 探讨不同产地牛膝的 ITS的序列变异 ,为鉴别牛膝提供 DNA分子标记。方法 利用特异性 PCR技术对牛膝的 r DNA ITS区碱基序列进行测定 ,报道了牛膝的 r DNA ITS区的碱基序列。结果 得到核糖体 DNA中的 ITS及 5 .8S r DNA完全序列 ,18S和 2 6 S r DNA部分序列 ,共约 6 5 0 bp。牛膝与川牛膝及土牛膝碱基序列有明显差异 ,不同产地、不同栽培品种牛膝碱基序列无差异。结论 此法可用于牛膝种间及真伪品鉴别。

反相高效液相色谱法测定怀牛膝中羟基促蜕皮甾酮的含量

AIM: To set up an assay method for the determination of ecdysterone (EDS) in the root of Achyranthes bidentata Bl. by RP HPLC. METHODS: The EDS was separated on a Hypersil ODS column (200 mm×4 6 mm ID, 5 μm), using acetonitrile — water (1 0∶5 4) as the mobile phase and detected at 243 nm. The flow rate was 1 0 mL·min -1 . Phenol was used as an internal standard. RESULTS: The calibration curve was in good linearity over the range of 28 04～280 40 μg (γ=0 9997). The average recovery was 95 2%. CONCLUSION: The method is simple, fast, sensitive and is suitable for quantitative analysis of EDS in the root of Achyranthes bidentata Bl.

怀牛膝中甾酮类成分的分离与鉴定

为了阐明怀牛膝中具有促进成骨细胞增殖作用的甾酮类化学成分,采用硅胶柱色谱、ODS反相柱色谱和制备型高效液相色谱等方法分离,从怀牛膝大孔树脂50%乙醇洗脱组分中分离得到了5个甾酮类化合物,并通过UV、NMR和MS等波谱确定了其化学结构分别为β-蜕皮甾酮(β-ecdysterone)(1)、25S-牛膝甾酮(25S-inokosterone)(2)、25R-牛膝甾酮(25R-inokosterone)(3)、旌节花甾酮C(stachysterone C)(4)和旌节花甾酮D(stachysterone D)(5),其中化合物旌节花甾酮C为首次从牛膝中分离得到。

牛膝中脱皮甾酮的含量测定及促成骨样细胞增殖活性

AIM To study the activity of ecdysterone from Achyranthes bidentata Bl. (AB) promoting proliferation of osteoblast like (OB like) UMR106 cells and to determine its content in AB by HPLC method. METHODS Ecdysterone isolated from AB was cultured with OB like cells UMR106 together in vitro and the proliferation of OB like cells was determined by MTT assay. The chromatographic conditions for determining ecdysterone included an ODS column (250 mm×4 6 mm, 5 μm), a mobile phase consisting of a mixture of water acetontrile tetrahydrofuran (86∶11∶3), detection wavelength of 243 nm, and column temperature of 27℃. Phenacetin was used as the internal standard. RESULTS The ecdysterone from AB had significant activity promoting proliferation of OB like cells, the proliferation was promoted by 41% ( n =3). The average recovery of ecdysterone was 96 2% (RSD=2 1%), the calibration was linear in the range of 30～300 μg·mL -1 (γ=0 9998). CONCLUSION Ecdysterone was screened quickly by cultivating with OB like cells together in vitro . The HPLC method is accurate, fast and reproducible for the determination of ecdysterone in AB.

怀牛膝对未孕大鼠子宫平滑肌峰电活动的影响

在 Wistar未孕大鼠子宫角浆膜面上埋植一对 Ag- Ag Cl双极电极 ,腹腔注射不同剂量怀牛膝水煎剂后观察大鼠在体子宫平滑肌峰电活动的改变 .结果显示 :怀牛膝水煎剂显著增加未孕大鼠子宫肌电爆发波的最大振幅和峰面积 (P <0 .0 5 ) ,可延长爆发波持续时间和增加爆发波动作电位个数 (P <0 .0 5 ) ;怀牛膝水煎剂明显增大未孕大鼠子宫肌电单波的正波最大振幅、负波最大振幅和峰面积 (P <0 .0 5 ) .怀牛膝显著兴奋在体子宫平滑肌峰电活动的效应类似催产素 .

牛膝“泛糖”机制研究

目的 对牛膝“泛糖”机制进行研究。方法 采用正交试验研究牛膝“泛糖”的主要影响因素。结果 环境温度是牛膝“泛糖”的主要因素 ,其次是相对湿度和药材水分。最佳贮藏条件是环境温度 2 5℃ ,相对湿度 6 0 % ,药材水分 11%。结论 牛膝药材在环境温度 35℃以下 ,相对湿度 70 %以下 ,药材水分 9%～ 13%条件下 ,可安全贮存。

牛膝提取物对成骨样细胞增殖的作用

考察了牛膝 (AchyranthesbidentataBl.)的不同提取部位对成骨样细胞UMR10 6的作用 .通过牛膝的醇提液及醇提液的不同提取部位和UMR10 6成骨样细胞共同体外培养 ,用MTT方法检测了各部位对细胞增殖的作用 ;同时考察了MTT法的实验条件 .结果表明 :牛膝的醇提液及其石油醚和乙酸乙酯萃取的混和物对细胞具有较强的促进增殖作用 ,而正丁醇层和水层对细胞增殖无影响 ;在MTT法中 ,铺板细胞浓度、检测波长和加入MTT后的孵育时间均影响吸光度值的测定 .

怀牛膝多糖的超声波辅助提取工艺及其抗氧化性活性

研究怀牛膝多糖的超声波提取工艺和抗氧化性。在单因素实验基础上采用正交实验得出超声波提取怀牛膝多糖的适宜工艺条件,即超声波功率1 000 W、提取温度60℃、提取时间60 min、料液比(g∶mL)1∶30。在此提取工艺条件下,粗多糖得率为6.1%。体外抗氧化性实验表明,所提取怀牛膝多糖具有较好的抗氧化性,对H2O2、O2-.和.OH的清除率分别可达到75.1%、90.3%和61.3%。

牛膝营养器官中蜕皮甾酮的积累动态研究

目的揭示蜕皮甾酮在牛膝不同营养器官中的积累规律。方法高效液相色谱法。结果在牛膝根、茎和叶3种营养器官中都含有蜕皮甾酮,但不同器官中蜕皮甾酮的量有显著差异。幼嫩的器官中蜕皮甾酮的量较高,随着营养器官发育成熟,其蜕皮甾酮的量随物候期的变化差异显著。至采收期(11月)时,牛膝营养器官中蜕皮甾酮的量为:叶>根>茎。结论牛膝根中蜕皮甾酮的量高峰与其产量高峰一致,都在11月初地上部分枯萎时,因此,此时应为牛膝的适宜采收期,与传统的牛膝采收期相吻合。但此时叶和茎中也含有较高量的蜕皮甾酮,因此,建议对牛膝地上部分进行综合开发利用。

怀牛膝成分的分离与鉴定

目的进一步研究怀牛膝(AchyranthesbidentataBl.)的化学成分。方法反复利用正相和反相硅胶色谱进行分离和纯化,通过理化及波谱分析鉴定化合物结构。结果从正丁醇可溶部分中分离得到6个化合物,分别鉴定为齐墩果酸-3-O-β-D-葡萄糖醛酸苷(oleanolicacid-3-O-β-Dglucuronopyranoside,1)、竹节参苷Ⅳa(chikusetsusaponinⅣa,2)、竹节参苷Ⅳa甲酯(chikusetsusaponinⅣamethylester,3)、竹节参苷Ⅳa丁酯(chikusetsusaponinⅣabutylester,4)、28去葡萄糖牛膝皂苷D甲酯(28deglucosylachyranthosideDmethylester,5)和牛膝甾酮(inokosterone,6)。结论化合物1～5为首次从该植物中分离得到。

怀牛膝愈伤组织诱导及分化的研究

对怀牛膝叶片和茎段愈伤组织的诱导及分化进行了研究.结果表明:KT 0.5 mg/L+2,4-D 1.5 mg/L对叶片愈伤组织的诱导效果最好,KT 0.5 mg/L+NAA 1.5 mg/L对茎段愈伤组织的诱导效果最好,出愈率均达100%;KT 0.5 mg/L+NAA 1.5 mg/L有利于叶片形成的愈伤组织分化为根,6-BA 0.5 mg/L+NAA 1.5 mg/L则有利于茎段形成的愈伤组织分化为芽.

怀牛膝细胞悬浮培养及多糖含量变化的研究

采用正交实验设计研究基本培养基、碳源、2,4-D、6-BA、CH及接种量对怀牛膝悬浮培养细胞生长和细胞中多糖含量的影响。结果表明,(1)6-BA是影响怀牛膝细胞生长的关键因子,其影响效应依次为6-BA>CH>接种量>基本培养基>2,4-D>碳源,细胞生长的适宜培养基为MS+30 g/L葡萄糖+2 mg/L 2,4-D+1 mg/L 6-BA+0.5 g/L CH,适宜接种量为30 g/L。(2)碳源是影响细胞中多糖含量的关键因子,其影响效应依次为:碳源>2,4-D>6-BA>CH>接种量>基本培养基,利于细胞中多糖含量提高的适宜培养基为:LS+30 g/L蔗糖+0.5 mg/L 6-BA+0.5 g/L CH,适宜接种量为30 g/L。

牛膝提取物神经再生素促小鼠坐骨神经再生的实验研究

目的观察中药牛膝提取物神经再生素(NRF)对小鼠损伤坐骨神经的修复作用。方法ICR小鼠50只,雌雄各半,行坐骨神经夹伤术,随机分成5组:NRF高、中、低剂量组,弥可保组(阳性对照),生理盐水组(空白对照)。术后每日腹腔注射给药。采用小鼠坐骨神经功能指数(sc iatic function index,SFI)、组织形态学及电镜检查,观察NRF对损伤坐骨神经的影响。结果与空白对照组相比,NRF可显著改善小鼠的坐骨神经功能。超微结构观察表明,实验组有髓神经纤维的髓鞘形态、厚度、成熟度均优于对照组,而变性纤维的数目少于对照组。结论NRF能促进小鼠坐骨神经损伤后的修复及其功能的恢复。

中药牛膝化学成分的分离与鉴定

目的 对牛膝（Achyranthes bidentataB1．）的化学成分进行分离、鉴定。方法 采用硅胶吸附柱色谱，HPLC等分离手段对牛膝进行分离；利用理化性质、理化反应以及NMR、MS等波谱技术对化合物进行鉴定。结果分离得到5个化合物，分别鉴定为环（酪氨酸-亮氨酸）[cyclo-（-Tyr-Leu），1]、环（亮氨酸-异亮氨酸）[cyclo-（-Leu-Ile），2]、杜鹃花酸（nonanedioic acid，3）、琥珀酸（succinicacid，4）、正丁基-β-D-吡喃果糖苷（n-butyl-β-D-fructopyranoside，5）。结论 化合物1～3为首次从牛膝中分离得到。首次对化合物1和2进行了碳谱数据归属。