Effect of total flavonoids of epimedium on liver microsomal CYP 1A2,CYP3A4 and CYP2E 1 activities in rats

To assess the potential effect of total flavonoids of epimedium （TFE） on cytochrome P450 and activity of its main isoforms in rat liver microsomes. TFE （300 mg/kg） was administered once daily to male Sprague-Dowley rats by gavage for fifteen days. The total cytochrome P450 content and its main isoforms CYP1A2, CYP3A4 and CYP2E1 activities in rat liver microsomes were detected. The activity of CYP1A2 was measured by fluorometry and the activities of CYP3A4 and CYP2E1 were determined by measuring the amount of methanal and p-aminophenol formed using UV/Vis spectrophotometer, respectively. Administration of TFE significantly increased the total CYP450 content and activities ofCYP1A2, CYP3A4 and CYP2E1 in rat liver microsomes, compared with the control group. Particularly, the activities of CYP1A2 and CYP2E1 were enhanced significantly （P〈0.01）. TFE induced the increase in total CYP450 content and its main isoforms CYP1A2, CYP3A4 and CYP2E1 activities in rat liver microsomes.

Isolation and Extraction of Total Flavonoids from Epimedium Koreanum Nakai by Supercritical Fluid Extraction

In the present paper is reported the method for the isolation and extraction of total flavonoids of Epimedium Koreanum Nakai by means of supercritical fluid extraction(SFE). By examining pressure, temperature, amounts of modifier and extraction time, the optimized condition of SFE is confirmed as 30 MPa and 60 ℃, with 70% ethanol as the modifier. The samples were statically extracted for 30 min, followed by dynamic extraction for 120 min at a flow rate of 6 mL/min. The quantitative analysis of total flavonoids was performed by UV-Vis spectrophotometry. Compared with the conventional method, the SFE method is more efficient, more rapid and more friendly environmentally.

Simultaneous determination of seven flavonoids in Epimedium by liquid chromatography-tandem mass spectrometry method

In this paper, a sensitive and specific liquid chromatography-electrospray ionisation-mass spectrometry （LC-ESI-MS） method has been developed and validated for the identification and determination of seven flavonoids, namely epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2＂-O-rhamnosyl icariside II, and baohuoside I in Epimedium from different sources.

Two new prenylflavonoids from Epimedium sutchuenense

Two new prenylflavonoids, named sutchuenmedin A （1） and sutchuenmedin B （2）, were isolated from the 70% EtOH extract of Epimedium sutchuenense. Their structures were determined from spectral analysis. C 2009 Guang Chen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved,

EF延缓HPAT轴衰老的基因表达谱研究

目的:淫羊藿总黄酮(EF)延缓下丘脑-垂体-肾上腺-胸腺(HPAT)轴衰老的分子机制。方法:利用基因芯片技术,分别检测淫羊藿总黄酮组、右归饮组、桃红四物汤组、老年大鼠对照组及青年大鼠对照组下丘脑、垂体、肾上腺、脾脏淋巴细胞的基因表达谱。结果:老年大鼠和青年大鼠相比,HPAT轴多种神经递质、激素、细胞因子或其受体表达下调;EF组HPAT轴多种神经递质、激素、细胞因子或其受体表达上调;右归饮组及桃红四物汤组未见广泛的调节作用。结论:老年大鼠HPAT轴与生长、发育、衰老相关的基因表达以衰退的表现为主;EF能上调神经递质受体的表达并通过NEI网络的下行通路激活神经内分泌和免疫系统;通过下调促凋亡、抗增殖基因,上调抗凋亡、促增殖基因的表达,重塑淋巴细胞基因表达的平衡,延缓免疫衰老。

EF调控老年大鼠淋巴细胞基因表达谱中凋亡相关信号分子表达的研究

目的揭示EF对老年大鼠淋巴细胞凋亡相关信号分子及通路的调节机制。方法利用基因芯片技术,并比较老年大鼠、青年大鼠、EF干预后的淋巴细胞基因表达。结果老年大鼠与青年大鼠比较,促进细胞凋亡作用的基因表达显著上调,具有抗凋亡作用的基因表达显著下调;具有抗增殖作用的基因表达显著上调,具有促进细胞增殖作用的基因表达显著下调;参与淋巴细胞活化增殖及免疫反应等基本信号通路的组成元件中,多个重要信号分子的表达显著下调。EF组抗淋巴细胞凋亡的几个上游因子如CD28、TGFβ及cJun、cFos、cMyc等癌基因显著上调;促细胞凋亡基因Caspase1、Caspase2、Caspase3、Caspase6、CalpainⅡ、CathepsinS、Dnaseγ、PKCdelta等表达显著下调;CathepsinB、Mtal、NFkappaB、Notch等抗凋亡基因表达显著上调。显著上调促细胞增殖基因PCNA、Araf、CyclinGassociatedkinase等的转录,显著下调抗增殖的Rb等基因的转录。显著上调免疫细胞效应及功能的基本调节因子CD2、CD3、CD5、TCR、IL2、IL2R(CD25)、CD28、CTLA4的表达。结论EF能上调老年大鼠淋巴细胞抗凋亡基因表达的同时,下调抗增殖基因的表达;上调促增殖基因表达的同时,下调抗增殖基因的表达,重塑基因表达良性平衡;还协同调节TCRCD3、CD28、CTLA4、TGFβ、IL2R等介导的?

大孔吸附树脂分离纯化淫羊藿EF5黄酮

目的:采用HPLC法和UV法监测AB-8大孔吸附树脂分离纯化以淫羊藿苷、朝藿定A、朝藿定B、朝藿定C和宝藿苷I这五种黄酮为主要成分的淫羊藿EF5黄酮并筛选出最佳分离纯化工艺参数。方法:采用UV法,以淫羊藿总黄酮洗脱率为指标,考察树脂对淫羊藿总黄酮的吸附容量、解吸附率、吸附动力学,以确定树脂分离纯化淫羊藿EF5黄酮的最佳吸附性能;采用HPLC法,以淫羊藿EF5黄酮中五种黄酮单体的洗脱率为指标,考察洗脱液浓度和洗脱液用量等参数,确定淫羊藿EF5黄酮的最佳洗脱参数。结果:AB-8大孔吸附树脂对淫羊藿总黄酮的吸附容量以湿树脂计为(58.37±2.36)mg·g-1,解吸附率为(86.77±2.32)%,最佳吸附时间为180min,最佳洗脱液为60%乙醇,最佳洗脱液用量为5BV,纯化后真空干燥所得棕褐色淫羊藿EF5黄酮粉末中朝藿定A、朝藿定B、朝藿定C、淫羊藿苷和宝藿苷I的含量分别为3.51%、4.87%、1.22%、33.05%、1.36%,淫羊藿EF5黄酮纯度为44%。结论:AB-8大孔吸附树脂分离纯化得到的淫羊藿EF5黄酮的纯度较高,适合工业生产。

Characteristics of Lymphocyte Nuclear Factor-κB Signal Transduction Kinase Expression in Aging Process and Regulatory Effect of Epimedium Flavonoids

Objective：To study the characteristics of lymphocyte nuclear factor kappa B（NF-κB） signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids（EF） on it.Methods：Sixty SD rats were divided into six groups,according to animals＇ age,i.e.,the 3 days（d） group,the 4 months（m） group,the 10 m group,the 18 m group,the 27 m group,and the 27 m＋EF group.RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip（128 related genes）,the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.Results：The mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats＇ splenic lymphocytes lowered with aging;the highest expression was presented at 3 d after birth,and then,it lowered gradually,with the lowest level at 18 m or 27 m.After EF intervention,the expression level was raised to the 10-18 m level in the aged rats.Conclusion：The changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging,and will also give a hint to the molecular index for assessment of senility retarding researches.

Qualitative and Quantitative Evaluation of Epimedium and Ginseng Contained Combinations Using HPLC

Aim To develop a rapid, effective method for the determination of flavonoidsand ginsenosides in one injection and evaluate the flavonoids and ginsenosides content to controlthe ratio of Epimedium and Ginseng herbs in botanical combinations. Methods The quality evaluationwas determinatted using reversed-phase high performance liquid chromatog-raphy (HPLC), referred bythe major flavonoids from Epimedium, epimedin A, epimedin B, epimedin C, and icariin as thestandards, and the major ginsenosides Rg_1, Re, Rf, Rb_1, Rb_2, and Rd as the standards, includedEpimedium brevicornum Maxim., E. sagittatum (Sieb. et Zucc) Maxim., E. koreanum NaKai, P. ginseng C.A. Meyer, P. quinquefolium L., P. notoginseng and some products containing the above herbs. ResultsThe main flavonoids and ginsenosides could be clearly resolved in the single analysis. ConclusionThe results can be effectively used in evaluating qualitatively and quantitatively the ration ofEpimedium and Ginseng contained products.

基于不同炮制方法的淫羊藿黄酮类成分含量研究

试验旨在寻求羊脂油炮制淫羊藿的内在机理,以甘肃产淫羊藿为研究对象,用羊脂油、牛脂油、猪脂油进行炮制,分别提取有效成分,利用紫外分光光度法和高效液相色谱法进行有效成分含量测定,比较不同炮制品和生品总黄酮、淫羊藿苷、宝藿苷Ⅰ和朝藿定C的含量。结果显示:各炮制品总黄酮含量均显著低于生品(6.84%),其中羊脂油炙含量(5.69%)显著高于其他炮制品;生品和羊、牛脂油炙炮制品中淫羊藿苷含量无显著差异,羊脂油炙炮制品为0.77%,牛脂油炙炮制品为0.70%,生品为0.70%,猪脂油炙炮制品中淫羊藿苷含量为0.50%,较生品和其他炮制品显著降低;羊脂油炙、牛脂油炙淫羊藿中宝藿苷Ⅰ含量无显著性差异,分别为0.08%和0.07%,但显著高于生品(0.05%)和猪脂油炙淫羊藿(0.04%);羊、牛、猪脂油炙炮制品中朝藿定C含量分别为0.29%、0.27%和0.24%,均显著低于生品(0.57%)。得出结论:淫羊藿最佳炮制辅料为羊脂油,其次为牛脂油。

淫羊藿总黄酮对去势大鼠抗酒石酸酸性磷酸酶5b的影响及与骨密度的相关性

目的:探讨淫羊藿总黄酮(TFE)对去势大鼠抗酒石酸酸性磷酸酶5b(TRACP-5b)及骨密度的影响。方法:10月龄雌性SD大鼠去卵巢法建立大鼠模型,随机数字表法分为空白对照组(N)、模型对照组(O)、TFE低(T_(L))、中(T_(M))、高(T_(H))剂量组,β-雌二醇(E)组各10只,灌胃干预90d。用抗酒石酸酸性磷酸酶试剂盒测定TRACP-5b含量,用小动物分析软件测定活体全身骨密度。结果:(1)TRACP-5b水平O组明显高于N组,具有显著性差异(P<0.01),T_(L)、T_(M)、T_(H)和E组TRACP-5b水平明显低于O组,具有显著性差异(P<0.01)。(2)与N组比较,O、T_(L)、T_(M)、T_(H)和E组腰椎骨密度均下降,唯有O组呈显著性差异(P<0.05);与O组比较,T_(M)、T_(H)组腰椎骨密度升高、呈显著性差异(P<0.05),E组腰椎骨密度升高、具有显著性差异(P<0.01)。(3)与N组比较,O、T_(L)、T_(M)、T_(H)和E组全身骨密度均下降,唯有O组差异有统计学意义(P<0.05);与O组比较,T_(L)、T_(M)、T_(H)和E组全身骨密度均升高,唯有E组呈显著性差异(P<0.05)。结论:TFE可以降低去势大鼠血清中TRACP-5b的含量,并与去势大鼠腰椎骨密度、全身骨密度呈一定的量效关系。

淫羊藿中异戊烯基黄酮的鉴别和含量测定的综合实验设计

为加深学生对传统中药淫羊藿的药效成分异戊烯基黄酮的认识,设计了综合化学实验.将淫羊藿粉末经乙醇热回流提取,蒸馏浓缩得到总浸膏,再将总浸膏溶于水,依次用石油醚、乙酸乙酯和正丁醇萃取,得到石油醚相、乙酸乙酯相和正丁醇相.将三相的有机物分别用薄层色谱展开,在紫外灯下观察254 nm和365 nm荧光颜色和强度,同时用硫酸、氯化铁显色,表明各相均含有黄酮.以淫羊藿苷为标品,通过作标准曲线结合紫外光谱对其各相的异戊烯基黄酮含量进行平行测定,同时考察了方法的重复性和样品的稳定性.

巫山淫羊藿资源、人工种植及活性成分转化应用研究进展

巫山淫羊藿是《中华人民共和国药典》规定的淫羊藿属植物5个品种之一,其用药历史悠久、药效显著、活性成分含量高、种植面积大,是当前主流品种之一。但现代分析发现巫山淫羊藿的淫羊藿苷含量低,达不到《中华人民共和国药典》规定量,而被单列,致使其开发应用受限、种植面积有所缩减。综述我国巫山淫羊藿资源状况、形态与生长特征、人工种植、主要活性物质种类与功效及其转化、加工制备等新近研究文献,为促进巫山淫羊藿人工种植、加大其加工制备及其产品开发应用产业化提供参考。

微生物发酵转化黄酮类化合物研究进展

黄酮类化合物是自然界中存在的天然有机化合物,具有抗氧化、抗癌、预防骨质疏松和增强免疫调节等多种生物活性。通过微生物发酵技术对黄酮类化合物进行结构修饰,可以产生更多结构新颖的衍生物,提高生物利用度。常用的微生物有肠道菌群、内源性微生物和一些转化效果良好的菌株,转化反应类型有糖基化、甲基化、羟基化、氢化和水解反应等。黄酮类化合物的微生物转化以黄酮苷和苷元的相互转化为主,转化后可实现苷元类成分的大量富集。本文以大豆、陈皮、黄芩、淫羊藿中的黄酮为例,对黄酮类化合物的微生物转化现状进行总结分析,可为黄酮类化合物的转化研究和新药研发提供参考。

淫羊藿总黄酮联合黄芪多糖对骨质疏松性骨折模型大鼠骨折愈合的影响

目的探讨淫羊藿总黄酮联合黄芪多糖对骨质疏松骨折模型大鼠骨折愈合、骨痂形成及塑形情况的影响。方法将60只SD大鼠随机分为全处理组(A组)、后处理组(B组)、对照组(C组),各20只。其中,A组和B组分别灌胃淫羊藿总黄酮提取物与黄芪多糖混悬液(1∶1,m/m)690 g/(kg·d)和等量生理盐水,4周后采用双侧卵巢切除术复制大鼠骨质疏松骨折模型,继续灌胃6周;C组大鼠灌胃等量生理盐水4周后造模,连续灌胃等量生理盐水6周。于造模后第10,17,24,30天拍摄患肢股骨正侧位X线摄片,计算矿化骨组织体积(BV)、骨痂总体积(TV)、骨体积分数(BV/TV)、骨矿密度(BMD)。灌胃6周后,处死所有大鼠,每组随机选取10只进行患肢Micro-CT扫描;于腹主动脉取血,测定Ⅰ型胶原N端前肽(PⅠNP)、碱性磷酸酶(AKP)、骨钙素(BGP)水平。结果与本组造模后第10天比较,3组大鼠造模后第17,24,30天的正侧位X线摄片Lane-Sandhu评分均显著升高(P<0.05);与C组比较,A组大鼠造模后第10,30天的正侧位X线摄片Lane-Sandhu评分均显著升高(P<0.05)。与C组比较,A组大鼠的AKP,BGP,PⅠNP,BV,BV/TV,BMD均显著升高(P<0.05),B组大鼠的AKP和BGP均显著升高(P<0.05);与B组比较,A组大鼠的BGP,PⅠNP,BV/TV均显著升高(P<0.05)。结论淫羊藿总黄酮联合黄芪多糖可有效改善骨质疏松性骨折模型大鼠的骨折愈合情况,促进骨痂形成。

基于网络药理学探讨淫羊藿总黄酮防治阿尔兹海默病的机制研究

目的:采用网络药理学的分析方法研究淫羊藿总黄酮治疗阿尔兹海默病的机制。借助网络药理学数据库与分析平台(TCMSP)得到淫羊藿中的有效活性成分,并从中筛选出黄酮类成分。通过Pubchem和Swiss Target Prediction数据库,找到各黄酮类成分的相关靶点;采用Gene Cards数据库获取阿尔兹海默病的相关基因;淫羊藿“黄酮类活性成分-靶点-阿尔兹海默病”相互作用网络利用Cytoscape 3.9.1软件建立;采用Venny2.1.0平台获得总黄酮类成分与阿尔兹海默病的交集靶点,再将交集靶点输入STRING 11.0数据库获取蛋白质之间相互作用的网络图;采用Metascape数据库对GO功能以及KEGG通路进行分析。结果:得到淫羊藿治疗阿尔兹海默病黄酮类的核心成分12个,核心靶点为AKT1、TNF、VEGFA、SRC等10个,包括癌症途径、松弛素信号通路、EGFR酪氨酸激酶抑制剂耐药性等信号通路。结论:构建的“成分-靶点-通路”网络结构图能够揭示淫羊藿黄酮类成分的多成分、多靶点、多通路协同抗阿尔兹海默病的作用,为进一步阐明淫羊藿抗阿尔兹海默病的作用机制提供理论依据。

箭叶淫羊藿叶片不同发育时期黄酮类成分形成与元素含量关系研究

目的探究箭叶淫羊藿Epimedium sagittatum(Sieb.et Zucc.)Maxim.叶片不同发育时期黄酮类成分变化与元素积累的规律,揭示其黄酮类成分的形成机制。方法以箭叶淫羊藿叶片为研究对象,将其按照不同发育时期分为t1~t6共6个时期,高效液相色谱(HPLC)测定4种黄酮类成分含量,紫外分光光度法测定总黄酮醇苷含量;实时荧光定量法(RT-qPCR)测定17个与黄酮类成分合成关键酶基因的相对表达量;电感耦合等离子体质谱法(ICP-MS)测定25种元素含量;皮尔逊(Pearson)相关性分析黄酮类成分与元素的关系。结果淫羊藿苷含量呈现先上升后下降的趋势,朝霍定A、朝霍定B、朝霍定C、总黄酮醇苷的含量显著降低,但是5种黄酮类成分总量却显著升高,在t4和t5时期大量积累。大部分黄酮类成分合成关键酶基因表达量在t4和t5时期显著上调,在t2时期显著下调,不同基因的表达量随叶片发育而变化;叶片中元素Mg、Ca、Ti、Mn、Fe、Sr和Ba元素含量显著升高,元素K和Co含量显著降低;元素P、K、V、Cu的含量与箭叶淫羊藿中黄酮类成分含量呈显著性正相关。结论箭叶淫羊藿叶片中黄酮类成分形成过程受到叶片发育程度及相关基因表达的影响,黄酮类成分在叶片发育后期大量积累。

New prenylated flavonoid glycosides derived from Epimedium wushanense byβ-glucosidase hydrolysis and their testosterone production-promoting effects

Six new prenylated flavonoid glycosides,including four new furan-flavonoid glycosides wushepimedoside A–D(1–4)and two new prenyl flavonoid derivatives wushepimedoside E–F(5–6),and one know analog epimedkoreside B(7)were isolated from biotransformation products of the aerial parts of Epimedium wushanense.Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data,and the absolute configurations were assigned using experimental and calculated electronic circular dichroism(ECD)data.The regulatory activity of compounds 1–7 on the production of testosterone in primary rat Leydig cells were investigated,and 4 and 5 exhibited testosterone production-promoting activities.Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3β-HSD and 4 also had good affinity with Cyp17A1,which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Epimedium-Derived Flavonoids Modulate the Balance between Osteogenic Differentiation and Adipogenic Differentiation in Bone Marrow Stromal Cells of Ovariectomized Rats via Wnt/β-Catenin Signal Pathway Activation

Objective： To observe the function of wnt/β-catenin signal pathway on the process that epimedium-derived flavonoids （EFs） regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal ceils of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopausal osteoporosis. Methods： Bone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive medium or liquid medium for 15 days. Low- （1 μg/mL）, medium- （10 μg/mL） and high- （100 μg/mL） dose EFs were administrated correspondingly. Alkaline phosphatase （ALP） staining, ALP activity determination, oil red O staining and real- time polymerese chain reaction （RT-PCR） were used to determine the effect of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, Dickkopf-related protein 1 （DKK1） was used in the medium group. Enzyme linked immunosorbent assay （ELISA） and RT-PCR were used to determine mRNA levels of 13-catenin, low density lipoprotein receptor-related protein 5 （LRP5） and T cell factor （TCF） protein, known as wnt/β-catenin signal pathway related factors. Results： EFs increased mRNA expression levels of ALP and early osteoblast differentiation factors, such as runt-related transcription factor 2 （Runx2）, osteocaicin and collagen I, and decreased mRNA expression levels of fat generation factors, such as peroxisome proliferator activated receptor gamma 2 （PPAR r/-2） and CCAAT enhancer-binding protein-α （C/EBP α） in a dose-dependent manner. While osteobiast differentiation factors were down-regulated, fat generation factors were up-regulated when DKK1 was applied. Also EFs up-regulated mRNA expression levels of β-catenin, LRP5 and TCF protein which could be blocked by DKK1. Conclusion： EFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/13-catenin signal pathway, which may be an important molecular mechanism of EFs on treatinq DostmenoDausal osteoDorosis.

气相色谱法测定葛根和淫羊藿总黄酮提取物中大孔树脂有机残留物

目的:建立葛根和淫羊藿总黄酮提取物中大孔吸附树脂7种有机残留物的含量测定方法。方法:采用气相色谱法,DB-1毛细管柱(柱长为60 m,内径为0.25 mm,膜厚度为1.0μm),柱温为程序升温,起始温度为40℃,再以4℃/min升温至175℃,保持12 min,用氢火焰离子化检测器检测,检测器温度250℃,进样口温度200℃;载气为氮气,流速为0.8 mL/min,分流比为20∶1,顶空进样,顶空瓶平衡温度为90℃,平衡时间为50 min。结果:7种有机溶剂残留物的峰面积与浓度呈良好线性关系,正己烷、苯、甲苯、二甲苯、苯乙烯、二乙烯苯、1,2-二乙基苯的平均回收率分别为101%、90%、87%、85%、84%、76%、79%。结论:该方法测定准确度高、精密度重复性好。