In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants

Objective:To develop the reproducible in vitro propagation protocols for the medicinally important plants viz.,Achyranthes aspera(A.aspera)L.and Achyranthes bidentata(A.bidentata)Blume using nodal segments as explants.Methods:Young shoots of A.aspera and A.bidentata were harvested and washed with running tap water and treated with 0.1%bavistin and rinsed twice with distilled water.Then the explants were surface sterilized with 0.1%(w/v)HgCl_2 solutions for I min.After rinsing with sterile distilled water for 3-4 times,nodal segments were cut into smaller segments(1 cm)and used as the explants.The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog(MS)medium supplemented with 3%sucrose,0.6%(w/v)agar(HiMedia,Mumbai)and different concentration and combination of 6-benzyl amino purine(BAP),kinetin(Kin),naphthalene acetic acid(NAA)and indole acetic acid(IAA)for direct regeneration.Results:Adventitious proliferation was obtained from A.aspera and A.bidentata nodal segments inoculated on MS basal medium with 3%sucrose and augmented with BAP and Kin with varied frequency.MS medium augmented with 3.0 mg/L of BAP showed the highest percentage(93.60±0.71)of shootlets formation for A.aspera and(94.70±0.53)percentages for A.bidentata.Maximum number of shoots/explants(10.60±0.36)for A.aspera and(9.50±0.56)for A.bidentata was observed in MS medium fortified with 5.0 mg/L of BAP.For A.aspera,maximum mean length(5.50±0.34)of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A.bidentata(5.40±0.61)was observed in the very same concentration.The highest percentage,maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of 1BA.Seventy percentages of plants were successfully established in polycups.Sixty eight percentages of plants were well established in the green house condition.Sixty five percentages of plants were established in the field.Conclusions:The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A.aspera and A.bidentata.The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can he easily adopted for commercial large scale cultivation.

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.

Inhibiting Effects of Achyranthes Bidentata Polysaccharide and Lycium Barbarum Polysaccharide on Nonenzyme Glycation in D-galactose Induced Mouse Aging Model

Objective To investigate the inhibiting effects and mechanism of achyranthes bidentata polysaccharide (ABP) and lycium barbarum polysaccharide (LBP) on nonenzyme glycation in D-galactose induced mouse aging model. Methods Serum AGE levels were determined by AGE-ELISA, MTT method was used to determine lymphocyte proliferation, IL-2 activity was determined by a bioassay method. Spontaneous motor activity was used to detect mouse's neuromuscular movement, latency of step-through method was used to examine learning and memory abilities of mouse, colormetric assay was used to determine hydroxyproline concentration in mouse skin, pyrogallol autoxidation method was used to determine superoxide dismutase (SOD) activity of erythrocytes. Results Decreased levels of serum AGE, hydroxyproline concentration in mouse skin and spontaneous motor activity in D-galactose mouse aging model were detected after treated with ABP or LBP, while lymphocyte proliferation and IL-2 activity, learning and memory abilities, SOD activity of erythrocytes, were enhanced. Conclusions ABP and LBP could inhibit nonenzyme glycation in D-galactose induced mouse aging model in vivo and ABP has a better inhibiting effect than LBP.

Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion.Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion,but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear.Therefore,in the current study,we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons.Hippocampal neurons were treated with Mg^2+-free extracellular solution containing glutamate(300μM)for 3 hours as a model of glutamate-mediated excitotoxicity(glutamate group).In the normal group,hippocampal neurons were incubated in Mg^2+-free extracellular solution.In the Achyranthes bidentata polypeptide group,hippocampal neurons were incubated in Mg^2+-free extracellular solution containing glutamate(300μM)and Achyranthes bidentata polypeptide at different concentrations.At 24 hours after exposure to the agents,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear m'orphology,respectively.Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay,respectively.At various time points after glutamate treatment,reactive oxygen species in cells were detected by H2 DCF-DA,and mitochondrial membrane potential was detected by rhodamine 123 staining.To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors,electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons.Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate.In addition,Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current.Furthermore,the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment.These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway.All animal studies were approved by the Animal Care and Use Committee,Nantong University,China(approval No.20120216-001)on February 16,2012.

The Achyranthes bidentata polypeptide k fraction enhances neuronal growth in vitro and promotes peripheral nerve regeneration after crush injury in vivo

We have previously shown that Achyranthes bidentata polypeptides （ABPP）, isolated from Achyranthes bidentata Blume （a medicinal herb）, exhibit neurotrophic and neuroprotective effects on the nervous system. To identify the major active component of ABPP, and thus optimize the use of ABPP, we used reverse-phase high performance liquid chromatography to separate ABPP. We obtained 12 fractions, among which the fraction of ABPPk demonstrated the strongest neuroactivity. Immunocytochemistry and western blot analysis showed that ABPPk promoted neurite growth in cultured dorsal root ganglion explant and dorsal root ganglion neurons, which might be associated with activation of Erk1/2. A combination of behavioral tests, electrophysiological assessment, and histomorphometric analysis indicated that ABPPk enhanced nerve regeneration and function restoration in a mouse model of crushed sciatic nerve. All the results suggest that ABPPk, as the key component of ABPP, can be used for peripheral nerve repair to yield better outcomes than ABPP.

Chemical Components of Achyranthes bidentata Leaves by Ultra High Performance Liquid Chromatography-Mass Spectrometry

[Objectives] To study the chemical components and relative content of Achyranthes bidentata leaves and provide a scientific basis for further development and utilization of A. bidentata leaves.[Methods] The chemical components of A. bidentata leaves were rapidly analyzed using the ultra high performance liquid chromatography-time of flight-high resolution mass spectrometry (UHPLC-TOF-MS).[Results] Thirty eight chemical compounds were identified in samples of A. bidentata leaves collected from Wen County of Henan Province, in which seven chemical compounds had the relative content higher than 5%, linoleic acid reached 25.7% and inokosterone A reached 13.8%.[Conclusions] A. bidentata leaves contain many kinds of chemical compounds. This study is expected to provide a certain basis for further extraction of linoleic acid and inokosterone A.

Protective effects of Achyranthes bidentata polypeptides on retinal ganglion cells post-optic nerve crush in rats

Achyranthes bidentata polypeptides（ABPP） have been reported to inhibit apoptosis of retinal ganglion cells（RGCs）.The present study investigated the protective effects of ABPP on RGCs in a rat model of optic nerve injury.With prolonged injury time,RGC densities were gradually decreased.ABPP（5 μg） significantly increased RGC densities and upregulated growth associated protein 43 expression in rats with optic nerve injury.Results demonstrate that ABPP can protect RGCs and promote axonal growth after optic nerve crush.

Achyranthes bidentata polypeptide protects dopaminergic neurons from apoptosis induced by rotenone and 6-hydroxydopamine

It has been well documented that Achyranthes bidentata polypeptides（ABPPs） are potent neuroprotective agents in several types of neurons. However, whether ABPPs protect dopaminergic neurons from apoptosis induced by neurotoxins is still unknown. This study was designed to observe the effect of ABPPk, a purified fraction of ABPPs, on apoptosis of dopaminergic neurons. SH-5YHY cells and primary dopaminergic neurons were pre-treated with ABPPk（25, 50, or 100 ng/mL） for 12 hours. Cells were then exposed to 6-hydroxydopamine（50 or 150 μM） or rotenone（50 or 200 μM） for 36 hours to induce cell apoptosis. Our results demonstrate that ABPPk markedly increased viability in SH-SY5Y cells and primary dopaminergic neurons, decreased lactate dehydrogenase activity and number of apoptotic dopaminergic neurons, elevated mitochondrial membrane potential, and increased Bcl-2/Bax ratio. These findings suggest that ABPPk protects dopaminergic neurons from apoptosis, and that ABPPk treatment might be an effective intervention for treating dopaminergic neuronal loss associated with disorders such as Parkinson＇s disease.

防控花生白绢病的根际放线菌分离鉴定及防效评价

花生白绢病是由齐整小核菌Sclerotium rolfsii Sacc.引起的发生在花生茎基部的真菌病害,严重制约花生的品质与产量。本研究利用稀释涂布法从怀牛膝根际土壤中分离纯化了116株放线菌,并从中筛选鉴定了能够防控花生白绢病的菌株。通过平板对峙试验筛选得到两株抑菌活性较好且稳定的菌株Soil-1-5和Soil-3-28,它们对齐整小核菌的抑制率分别为80.43%和92.34%。根据形态学观察、生理生化试验及16S rRNA基因序列分析鉴定,两株菌分别被鉴定为疮痂链霉菌Streptomyces scabiei和藤黄灰链霉菌Streptomyces luteogriseus。采用菌丝生长速率法测定了拮抗菌株无菌发酵滤液对植物病原菌的抑制作用。结果表明,菌株Soil-1-5和Soil-3-28的无菌发酵滤液稀释5倍后对齐整小核菌的抑制率分别为73.67%和57.11%,且对禾谷镰孢菌等6种植物病原菌均有不同程度的抑制作用。此外,两菌株的无菌发酵滤液对齐整小核菌菌核萌发和菌核形成也有较好拮抗作用。盆栽试验结果表明,菌株Soil-1-5和Soil-3-28对花生白绢病的防治效果分别为51.92%和31.74%,其中菌株Soil-3-28对花生生长有促进作用。综上,菌株Soil-1-5和Soil-3-28对花生白绢病有较好的防治效果,具有潜在应用价值。

牛膝多糖对雏鸡免疫功能的影响

文章旨在研究牛膝多糖对雏鸡免疫功能的影响。试验将120只1日龄雏鸡随机分为4组,每组3个重复,每个重复10只鸡(公母各半)。在常规饲料中分别添加200、150、100、0 mg/kg牛膝多糖。所用雏鸡连续饲喂至30日龄,每组取10只进行取样。计算动物机体免疫器官指数,MTT检测T细胞增殖活性,Griess检测巨噬细胞释放NO能力,ELISA检测血清总IgG水平。结果表明,在雏鸡饲料中添加中剂量(150 mg/kg)的牛膝多糖能显著提高雏鸡免疫器官指数、T细胞增殖活性、巨噬细胞释放NO能力和血清总IgG水平(P<0.05)。结论:牛膝多糖在一定程度上对雏鸡的免疫功能具有增强作用,从而提高机体抗病能力,饲料中牛膝多糖最适添加量为150 mg/kg。

药对何首乌-牛膝抗骨质疏松的作用机制研究

目的:利用网络药理学研究牛膝何首乌联用抗骨质疏松的作用机制。方法:运用中药系统药理学数据库与分析平台(TCMSP)与文献检索筛选何首乌、牛膝两味中药所含的活性成分。利用Disgent数据库搜索与骨质疏松相关的蛋白靶点;利用Pubchem平台,预测何首乌、牛膝所含活性成分所作用的人类蛋白靶点。分析牛膝何首乌药的交集靶点,并将交集靶点进行蛋白网络分析,构建药物-成分-靶点的网络图。通过微生信平台对关键基因作用的靶向细胞进行富集分析。结果:确定何首乌成分5个,牛膝成分10个,二者共有成分1个,获得药对与疾病交集基因35个;PPI蛋白分析获得10个主要作用成分;绘制GO分析可视图、KEGG分析可视图并对其进行解析,解析结果表明何首乌牛膝主要成分通过抑制骨吸收,诱导骨形成等通路来减轻骨质疏松症的发展。结论:牛膝何首乌药对抗骨质疏松的作用机制可能是通过靶点与共有通路完成的,并且该药对可通过多通路、多靶点、多成分、多层次发挥对骨质疏松症的治疗作用。

牛膝-菟丝子药对防治高血压肾损伤网络药理学机制研究

目的:通过使用网络药理学方法探讨怀牛膝-菟丝子药治疗高血压肾损伤的潜在作用机制。方法:使用TCMSP、Uniprot数据库分别获取怀牛膝、菟丝子的活性成分和靶点基因,高血压肾损伤的疾病相关靶点是通过GenenCards、Omim数据库获取。在Venny在线工具中获取药物与疾病的交集基因,使用STRING数据库、Cytoscape 3.9.1软件构建蛋白质相互作用网络。通过Metascape数据库和微生信网站获取的数据构建GO和KEGG通路富集分析,建立活性成分-靶点-关键通路网络。结果:在TCMSP数据库中共筛选出怀牛膝、菟丝子符合条件的有24种活性药物成分,活性成分靶点及疾病靶点分别为206、1762个。Venny在线工具中获得了药物和疾病的113个共同基因。PPI网络显示怀牛膝、菟丝子药对发挥重要作用的靶点有:丝裂原活化蛋白激酶1、肿瘤抑制蛋白P53、蛋白激酶B1、肿瘤坏死因子、原癌基因、白细胞介素6等。从GO和KEGG通路富集分析结果中可得到,药物和疾病的交集基因与无机物、氧化应激、化学应激、活性氧、金属离子等分子生物进程关系密切,相关信号通路包括有:癌症的途径、脂质与动脉粥样硬化、糖尿病并发症中的AGE信号通路等。结论:怀牛膝-菟丝子治疗高血压肾损伤具有基本药理活性,可能通过AGE-RAGE信号通路、肿瘤坏死因子信号通路等作用于肿瘤抑制蛋白P53、蛋白激酶B1等靶点改善高血压肾损伤。

红花-牛膝治疗口腔黏膜下纤维化作用机制的网络药理学分析

目的联合使用网络药理学和分子对接技术探究红花-牛膝的有效成分作用于口腔黏膜下纤维化(OSF)的治疗靶点。方法通过网络药理学方法获得“红花”“牛膝”的有效成分及作用靶点,以及OSF的疾病靶点。利用STRING平台构建蛋白互作PPI网络图;对关键活性成分与核心靶点进行分子对接分析,并对交集靶点进行了GO和KEGG富集分析。结果发现红花-牛膝作用于OSF的主要活性成分为木犀草素、黄芩苷、黄连素和黄连碱等;主要核心靶点为肿瘤坏死因子(TNF)、基质金属蛋白酶9(MMP9)、丝裂原活化蛋白激酶3(MAPK3)、低氧诱导因子1α(HIF-1α)等;主要涉及癌症中的蛋白多糖、丝裂原活化蛋白激酶(MAPK)、白介素-17(IL-17)、C型凝集素受体等信号通路。分子对接结果亦表明活性分子与靶点基因均具备良好的结合力。结论本研究结果提示红花-牛膝可能通过多活性成分、多靶点以及多种信号通路来治疗OSF。

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。

Tissue-based metabolite profiling and qualitative comparison of two species of Achyranthes roots by use of UHPLC-QTOF MS and laser micro-dissection

Achyranthes bidentata and Achyranthes aspera are saponin and steroid rich medicinal plants, used extensively for therapeutic treatments in Traditional Chinese Medicine(TCM) and Ayurveda. A. bidentata is reported to be one of the rare and extensively exploited medicinal plant species that face the issue of being endangered.Finding qualitative substitute with identical phyto-constituents contributing to similar composition and pharmacological benefits will help in reducing the burden of exploitation of the natural habitats of such plants.In the present study, a comparative metabolite analysis of the whole drug and specific tissues isolated by laser micro-dissection(LMD) was carried out for both the selected species, by use of ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UHPLC-QTOF MS). The results of the study indicate that the cortex and the medullary ray tissues are rich in their content of steroidal and saponin constituents such as(25 S)-inokosterone-20,22-acetonide, ginsenoside Ro, bidentatoside II and achyranthoside B.Metabolite profiling of the whole tissues of both the species indicates presence of identical constituents. Thus,it is inferred that A. bidentata and A. aspera can be used as qualitative substitutes for each other.

牛膝醇提物诱导兔骨髓间充质干细胞软骨分化的蛋白组学分析

背景:前期研究发现牛膝醇提物具有诱导骨髓间充质干细胞定向软骨细胞分化的作用,但具体作用蛋白靶点和网络机制不详。目的:观察牛膝醇提物诱导兔骨髓间充质干细胞软骨分化的蛋白质组学分析及蛋白质相互作用网络构建。方法:采用密度梯度离心联合细胞贴壁法分离培养新西兰大白兔骨髓间充质干细胞,取第3代细胞随机分成5组:空白组、对照组、牛膝醇提物低、中、高剂量组,连续成软骨诱导培养21 d后,用qRT-PCR检测Ⅱ型胶原mRNA表达及甲苯胺蓝染色鉴定软骨细胞形成,应用绝对定量同位素标记(iTRAQ)结合双向液相色谱-串联质谱(2DLC-MS/MS)技术对各组差异表达蛋白质进行鉴定,并对差异蛋白进行GO分析、KEGG分析及蛋白质网络相互作用分析。结果与结论:(1)qRT-PCR结果显示牛膝醇提物高剂量组较其他组Ⅱ型胶原mRNA表达明显增高(P<0.05),牛膝醇提物高剂量组甲苯胺蓝染色阳性,蛋白组学分析共鉴定到1354个差异蛋白点,上调蛋白633个,下调蛋白721个。(2)根据qRT-PCR结果对空白组、牛膝醇提物高剂量组、对照组3组差异表达蛋白进行生物信息分析。GO分析发现这些差异表达蛋白参与代谢、细胞分化、细胞周期与凋亡、炎症反应、免疫调控、氧化应激、磷酸化、泛素化、癌相关等;KEGG分析获得与骨关节病最相关的10个典型信号通路:白细胞介素17信号通路,Toll样受体信号通路,Wnt信号通路,PI3K-Akt信号通路,mTOR信号通路,Jak-STAT信号通路,NF-kappa B信号通路,MAPK信号通路,AMPK信号通路,HIF-1信号通路;根据组间差异蛋白,使用Cytoscape3.6.0软件成功构建蛋白互作网络图。(3)结果表明,牛膝醇提物可以通过调控氧化、细胞周期与凋亡、细胞结构改变、细胞分化、代谢及炎症损伤等环节诱导兔骨髓间充质干细胞成软骨分化,具有多靶点、多中心的网络调控作用,其具体作用机制有待进一步研究。

基于网络药理学和分子对接探究牛膝-当归防治膝骨关节炎的作用机制

运用网络药理学和分子对接的方法从分子层面探讨牛膝-当归防治膝骨关节炎的作用机制.主要通过在中药系统药理学数据库与分析平台(traditional chinese medicine systems pharmacology database and analysis platform,TCMSP)中对“牛膝”“当归”的主要活性成分进行筛选,在GeneCards、OMIM、DrugBank中收集和膝骨关节炎有关的疾病靶点并找出药物与疾病共同作用的靶点.构建蛋白质-蛋白质相互作用网络(protein-protein interaction,PPI)并选出关键靶点,利用R软件进行GO功能与KEGG信号通路富集分析.构建“药物-疾病-靶点”网络,选取重要成分与关键靶点进行分子对接实验.共筛选出二者的有效活性成分19个,得到疾病与药物共同靶点103个.分析PPI网络后共得到15个关键靶点,其中PTGS2、HSP90AA1、CASP3、JUN起主要作用.富集分析结果显示,牛膝-当归药对主要通过调控细胞白介素-17、肿瘤坏死因子、Toll样受体等信号通路来对膝骨关节炎起到防治作用.在分子对接中,主要化学成分槲皮素、β-谷甾醇和豆甾醇与关键靶点的对接效果良好.牛膝-当归药对可通过多个活性成分作用于多个疾病靶点并调控多条信号通路的方式来防治膝骨关节炎.

牛膝总皂苷对IL-1β诱导髓核细胞的凋亡及炎性损伤的影响

目的通过观察髓核细胞(nucleus pulposus cell,NPC)的凋亡、炎症及细胞外基质(extracellular matrix,ECM)代谢研究不同浓度牛膝总皂苷(achyranthes bidentata saponin,ABS)对白细胞介素-1β(interleukin-1β,IL-1β)诱导人源NPC损伤的保护作用。方法通过IL-1β诱导建立NPC退变模型,随机将细胞分为正常组、模型组(10 ng/mL IL-1β)、ABS低剂量组(10 ng/mL IL-1β+3μg/mL ABS)、ABS中剂量组(10 ng/mL IL-1β+10μg/mL ABS)及ABS高剂量组(10 ng/mL IL-1β+30μg/mL ABS),干预24 h。CCK-8法检测细胞活力,采用Tunel法和流式细胞术检测细胞凋亡,Western blot检测B细胞淋巴瘤-2相关X蛋白(B-cell lymphoma-2-associated X protein,Bax)、天冬氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase-3,Caspase-3)、B细胞淋巴瘤-xl(B-cell lymphoma-xl,Bcl-xl)、环氧合酶-2(cyclooxygenase-2,COX-2)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)和血小板结合蛋白基序的解聚蛋白样金属蛋白酶-5(recombinant a disintegrin and metalloproteinase with thrombospondin-5,ADAMTS-5)蛋白表达情况。结果与正常组比较,模型组NPC活力显著下降(P<0.01),凋亡率增加(P<0.01),Bax、Caspase-3、COX-2、MMP-3和ADAMTS-5蛋白表达升高(P<0.05),Bcl-xl蛋白表达降低(P<0.05);与模型组比较,不同浓度的ABS处理后,NPC活力显著上升(P<0.05),凋亡率减少(P<0.05),Bax、Caspase-3、COX-2、MMP-3和ADAMTS-5蛋白表达降低(P<0.05),Bcl-xl蛋白表达升高(P<0.05)。结论ABS可以通过抑制由IL-1β诱导引起的NPC凋亡、炎症反应、ECM降解,促进细胞增殖和活性,对NPC产生保护作用,其机制可能与抑制Bax、Caspase-3、ADAMTS-5、MMP-3及COX-2表达,促进Bcl-xl表达相关。

土牛膝的不同种质来源的差异性比较研究

目的从植物形态学、药材性状特征、薄层色谱、有效成分含量、指纹图谱等方面对3种不同种质来源的土牛膝的差异性进行比较,为土牛膝质量标准提升及合理应用提供科学依据。方法在植物分类学鉴定的基础上,对采集的样本进行植物形态学观察;采用传统药材性状鉴别方法对药材性状特征进行比较;通过TLC法比较不同药材粉末薄层色谱图;采用HPLC法测定药材中β-蜕皮甾酮的含量;通过指纹图谱比较不同来源土牛膝药材相似度差异。结果3种不同种质来源土牛膝的植物形态、药材性状特征、薄层色谱图均存在一定差别;土牛膝指纹图谱标定了11个共有峰,不同来源土牛膝药材中β-蜕皮甾酮含量及指纹图谱存在显著差异。结论3种不同种质来源土牛膝在植物形态学、药材性状特征、薄层色谱、有效成分含量及指纹图谱方面均存在较大差异,应加以甄别利用。

牛膝多糖降血糖活性成分的分离鉴定

研究牛膝多糖与降血糖活性的关系。牛膝粉碎后先经乙醇提取,再用热水提取,水提液减压浓缩后经醇沉得到牛膝水提醇沉物。采用链脲佐菌素(streptozocin,STZ)诱导方法建立小鼠糖尿病模型,每日给药牛膝各分离组分,连续给药8周,通过测定小鼠血糖、糖化血红蛋白,检测牛膝中具有降血糖活性的组分。采用DEAE、HW-55F、Sephacryl S-400等凝胶色谱柱对具有降血糖活性的牛膝多糖组分进一步分离。运用Sephadex G-100凝胶色谱、飞行时间质谱(time of flight mass spectrometer,TOF-MS)、气相色谱(gas chromatography,GC)、核磁共振(nuclear magnetic resonance,NMR)等手段鉴定多糖纯度、测定相对分子质量、分析结构。结果表明,牛膝的水提醇沉物能显著降低糖尿病小鼠的空腹血糖(fasting plasma glucose,FPG)和糖化血红蛋白(HbA1c)浓度(P<0.05)。将水提醇沉部分进一步分离得到具有降血糖活性的牛膝多糖,并鉴定其纯度为均一多糖,相对分子质量为1801.5,该多糖由1分子葡萄糖和10分子果糖组成,以葡萄糖分子为起点,与果糖C_(2)相连,果糖之间以β-1,2糖苷键连接形成主链,支链连接在果糖的C6上,该研究明确了牛膝中具有降血糖活性的多糖分子结构。