梯度洗脱法同时快速分离测定不同品种不同部位淫羊藿中7种异戊烯基黄酮类成分的研究

建立一种同时快速分离测定不同品种、不同部位淫羊藿中双藿苷A、淫羊藿属苷A、朝藿定C、淫羊藿苷、淫羊藿属苷C、意卡瑞苷A和去多甲基羟基淫羊藿素7种异戊烯基黄酮类成分的梯度洗脱方法,其中双藿苷A、意卡瑞苷A和去多甲基羟基淫羊藿素为首次报道其含量测定。实验比较了三种不同的梯度洗脱条件和三种不同的提取分离条件,选出了实验的最佳条件:采用RP-HPLC法,以美国WATERS SUNFIRETM-C18为色谱柱,乙腈-水为流动相,梯度洗脱(乙腈:0 min,20%;10 min,40%;15 min,45%;20 min,60%),流速为1 mL/min,检测波长270 nm,在此条件下,30 m in内所有组分均得到了良好的分离。实验同时考察比较了巫山淫羊藿植株不同药材部位和其它十种不同品种淫羊藿中7种异戊烯基黄酮类成分的分布。结果表明:不同品种淫羊藿中黄酮类成分的含量差异很大,但大部分品种以朝藿定C为主;同一品种巫山淫羊藿不同部位中则双藿苷A和朝藿定C的含量较大(在巫山淫羊藿根中,两者的含量分别为现行质量控制指标淫羊藿苷的20倍和30倍),可见巫山淫羊藿显著不同于其它品种的淫羊藿,朝藿定C在其药用效果中可能比淫羊藿苷扮演着更重要的角色尤其是在根部作为药用时,这也为民间将巫山淫羊藿根作为小黄连使用提供了一定的实验支持。结论:对于品种较多,分布资源较广的淫羊藿药材,作为药用时,应注意品种和药用部位,考察药材的内在质量,不能一概而论。实验所得到的方法能被用做淫羊藿植株中二次代谢产物分布的快速考察、筛选符合要求的淫羊藿原药材和淫羊藿制备药物中的质量考察。     

淫羊藿苷对大鼠正畸牙齿压力区牙周组织RANKL和Wnt3a表达的影响

目的:观察不同剂量的淫羊藿苷对大鼠正畸牙齿移动时压力区牙周组织中RANKL和Wnt3a表达的影响。方法:将24只健康雄性SD大鼠随机分为4组,根据淫羊藿苷灌胃的剂量分为生理盐水组(对照组)、1 mg/kg淫羊藿苷组、3 mg/kg淫羊藿苷组、5mg/kg淫羊藿苷组(实验组),使用50 g力近中移动左侧上颌第一磨牙。通过免疫组化方法检测压力区牙周组织中RANKL和Wnt3a蛋白的表达。结果:生理盐水组上颌第一磨牙压力区牙根和牙槽骨表面粗糙,牙周膜间隙变窄,可见骨吸收陷窝和破骨细胞,不同剂量淫羊藿苷组牙周膜间隙趋于恢复正常,骨吸收陷窝出现明显减少。RANKL和Wnt3a在生理盐水组和淫羊藿苷组的压力区牙周组织中都有表达。与生理盐水组比较,不同剂量淫羊藿苷组压力区牙周组织中RANKL的表达均显著降低,Wnt3a的表达均明显增加,且RANKL的表达随淫羊藿苷剂量的增加而逐渐减少(P0.05),Wnt3a的表达随淫羊藿苷剂量的增加明显增加(P0.05)。结论:不同剂量淫羊藿苷能减少正畸时牙齿移动过程中压力区牙周组织中RANKL的表达,增加Wnt3a的表达,且作用与其剂量具有一定的相关性。     

朝鲜淫羊藿药材HPLC指纹图谱研究

目的:建立朝鲜淫羊藿药材HPLC指纹图谱分析方法。方法:采用Agilent C18(4.6 mm×250 mm,5μm)色谱柱,柱温:25℃,流速:1 mL/min,以乙腈、水为流动相进行梯度洗脱,检测波长为270 nm,以淫羊藿苷、朝藿定A、朝藿定B、朝藿定C为对照品。结果:对同一批次朝鲜淫羊藿药材样品进行测定,标定了4个特征峰,通过"中药色谱指纹图谱相似度评价系统2004A版软件"计算其相似度。结论:该方法建立的朝鲜淫羊藿药材指纹图谱方法精密度高、重现性好,可为更好的控制朝鲜淫羊藿药材的内在质量提供科学依据。     

光照对淫羊藿活性成分生物合成的影响

淫羊藿作为传统的中药材,具有补肾阳、强筋骨、祛风湿等功效.其主要功能活性成分为黄酮醇苷成分淫羊藿苷、朝藿定A、B、C等.本实验在武汉植物园内的药园和园外园两个不同生长环境下,研究两种不同光照对巫山淫羊藿(Epimedium wushanense T.S.Ying)WY品系和箭叶淫羊藿(Epimedium sagittatum( Sieb.& Zucc.)Maxim.) LT1、HN3品系4种主要活性成分生物合成的影响.结果显示,在药园环境生长的淫羊藿的光合速率要高于园外园生长的淫羊藿,药园生长的箭叶淫羊藿LT1、HN3品系的4种活性成分含量均高于园外园生长的同品系,而巫山淫羊藿WY品系则相反.光合速率对4种活性成分含量的积累有一定影响,与朝藿定A、B和淫羊藿苷含量呈显著正相关,与朝藿定C含量呈显著负相关.文中对光合速率与活性成分积累间的关系进行了讨论,还针对不同的淫羊藿品系提出了相应的栽培建议.     

偏斜淫羊藿化学成分研究

采用70%乙醇超声波辅助提取偏斜淫羊藿（Epimedium truncatum H.R.Liang）叶片中的类黄酮化学成分,利用LC-MS联用技术鉴定其化合物组成,利用HPLC方法测定其主要活性成分的含量并与药典规定药材箭叶淫羊藿（Epimedium sagittatum）主要活性成分的含量进行比较。结果从偏斜淫羊藿叶片中鉴定出6个化合物：朝藿定B、朝藿定C、淫羊藿苷、3,5,7-三羟基4’-甲氧基-8-异戊烯基黄酮-3-O-α-L-吡喃鼠李糖基（1→2）-α-L-吡喃鼠李糖苷、槲皮素-3-O-鼠李糖甙、山奈苷。该6个化合物均为首次在该植物中发现。偏斜淫羊藿富含朝藿定C,其含量显著高于箭叶淫羊藿主要活性成分的总含量。结果表明LC-MS可以简单、快速地对淫羊藿化学成分进行定性和定量分析,偏斜淫羊藿富含朝藿定C,具有潜在的药用价值。     

淫羊藿苷对链脲佐菌素致糖尿病大鼠附睾功能的干预作用

目的:研究淫羊藿苷(Icariin)对链脲佐菌素(STZ)致糖尿病雄性大鼠附睾损伤的影响。方法:SD大鼠给予STZ(60mg/kg ip)建立糖尿病模型。动物随机分为6组(n=14):正常组、模型组、淫羊藿苷低、中、高剂量组(剂量分别为20 mg/kg、40 mg/kg、80 mg/kg)、罗格列酮组(3 mg/kg)。12周后,处死大鼠。检测血清中葡萄糖、附睾中乳酸脱氢酶(LDH)、酸性磷酸酶(ACP)、谷氨酰转肽酶(γ-GT)、α-糖苷酶(α-glucosidase)活力及唾液酸(SA)、果糖(fructose)的含量;取附睾以4%多聚甲醛固定,HE染色,于光镜下观察组织学变化。结果:与正常组比较,糖尿病模型组附睾中LDH、ACP及γ-GT活力降低,唾液酸、果糖含量降低。病理学检测可见附睾管中成熟精子数目减少。淫羊藿苷组比模型组大鼠特异性酶类活力提高,并在组织学上有明显的改善。结论:淫羊藿苷对STZ致雄性大鼠附睾功能损伤有明显的改善作用,其机制可能是增强了附睾相关标志酶的活性及改善了内能量代谢。     

基于UPLC-MS/MS法研究淫羊藿属植物品种品质与生长海拔、植物类群间关系

应用UPLC-MS/MS法对10种淫羊藿属植物主成分含量差异进行比较研究,以探讨药材品种品质与生长海拔、植物类群间联系。采用Phenomenex-C18柱(50 mm×2.1 mm,3μm),以乙腈-0.1%甲酸水为流动相梯度洗脱,流速为0.2 m L/min;质谱离子源为ESI,多反应监测正离子模式进行检测。淫羊藿苷、朝藿定A、朝藿定B、朝藿定C分别在0.995~9950.000、0.992~9920.000、1.021~10210.000、1.035~10350.000 ng/m L浓度范围内显示出良好的线性关系(r≥0.9996),平均加样回收率为98.7%~101.2%,RSD为1.7%~2.4%;结果表明,不同品种淫羊藿主成分含量差异较大,且含量有随海拔升高而降低的趋势,小花类群各成分含量均明显高于大花。本研究证明了品种品质与生长海拔、植物类群间具有相关性;该方法准确、快速、灵敏度高,适用于淫羊藿药材的质量控制。     

淫羊藿提取物的抗炎作用及其机制

淫羊藿是一种常用的传统中药。黄酮类成分是淫羊藿的主要有效成分,其中淫羊藿苷是含量最高的单体成分,淫羊藿素是淫羊藿苷的代谢产物。最近十余年来,学者对淫羊藿提取物的药理作用进行了许多研究,表明淫羊藿黄酮,尤其是淫羊藿苷及其衍生物,在骨质疏松症、骨关节炎、神经和精神疾病、动脉粥样硬化、哮喘和肺部疾病、炎症性肠病、肾脏疾病、皮肤病、自身免疫性疾病和癌症等多个与慢性炎症有关的疾病模型中显示了良好的抗炎作用。淫羊藿苷及其衍生物发挥抗炎作用的分子机制主要包括降低炎症细胞因子释放和NF-κB信号通路激活,抑制NLRP3/caspase-1/IL-1β、STAT和MAPK介导的信号传导通路,上调Nrf2/ARE/HO-1信号通路以及糖皮质激素受体和雌激素受体下游信号通路等。本文综述了该领域的近期研究进展,提示淫羊藿及其所含的活性化合物具有治疗多种慢性炎症相关疾病的潜力。     

淫羊藿提取物的抗炎作用及其机制（英文）

淫羊藿是一种常用的传统中药.黄酮类成分是淫羊藿的主要有效成分,其中淫羊藿苷是含量最高的单体成分,淫羊藿素是淫羊藿苷的代谢产物.最近10余年来,学者对淫羊藿提取物的药理作用进行了许多研究,表明淫羊藿黄酮,尤其是淫羊藿苷及其衍生物,在骨质疏松症、骨关节炎、神经和精神疾病、动脉粥样硬化、哮喘和肺部疾病、炎症性肠病、肾脏疾病、皮肤病、自身免疫性疾病和癌症等多个与慢性炎症有关的疾病模型中显示了良好的抗炎作用.淫羊藿苷及其衍生物发挥抗炎作用的分子机制主要包括降低炎症细胞因子释放和NF-κB信号通路激活,抑制NLRP3/caspase-1/IL-1β、STAT和MAPK介导的信号传导通路,上调Nrf2/ARE/HO-1信号通路以及糖皮质激素受体和雌激素受体下游信号通路等.本文综述了该领域的近期研究进展,提示淫羊藿及其所含的活性化合物具有治疗多种慢性炎症相关疾病的潜力.     

淫羊藿苷缓解腹部皮瓣缺血再灌注损伤模型大鼠的作用及机制研究

摘要 目的：研究淫羊藿苷缓解腹部皮瓣缺血再灌注损伤（IRI）模型大鼠的作用及机制。方法：取30只SD级大鼠作为研究对象,将其按照随机数字表法分作假手术组、模型组以及淫羊藿苷组,每组各10只。其中模型组和淫羊藿苷组大鼠均制作大鼠腹部皮瓣IRI模型,假手术组以及模型组大鼠予以生理盐水腹腔注射,淫羊藿苷组大鼠则予以淫羊藿苷腹腔注射。对比各组大鼠皮瓣存活面积及存活率、血清炎症因子以及氧化应激指标水平、皮瓣组织中p38丝裂原活化蛋白激酶（p38 MAPK）信号通路相关蛋白表达情况。结果：模型组、淫羊藿苷组大鼠的皮瓣存活面积及存活率均低于假手术组,但淫羊藿苷组大鼠的皮瓣存活面积及存活率均高于模型组（P＜0.05）。模型组、淫羊藿苷组大鼠血清白细胞介素-10（IL-10）均低于假手术组,但淫羊藿苷组高于模型组;模型组、淫羊藿苷组大鼠血清肿瘤坏死因子-?琢（TNF-?琢）均高于假手术组,但淫羊藿苷组低于模型组（P＜0.05）。模型组、淫羊藿苷组大鼠血清超氧化物歧化酶（SOD）、谷胱甘肽（GSH）水平均低于假手术组,但淫羊藿苷组大鼠血清SOD、GSH水平均高于模型组;模型组、淫羊藿苷组大鼠血清丙二醛（MDA）水平均高于假手术组,但淫羊藿苷组大鼠血清MDA水平低于模型组（P＜0.05）。模型组、淫羊藿苷组大鼠皮瓣组织p38 MAPK、丝裂原活化蛋白激酶磷酸酶-2（MKP-2）相对表达量均高于假手术组,但淫羊藿苷组皮瓣组织p38 MAPK相对表达量低于模型组,而MKP-2相对表达量高于模型组（P＜0.05）。结论：淫羊藿苷可通过调控p38 MAPK信号通路缓解炎症反应及氧化应激,发挥减轻腹部皮瓣IRI的作用。     

Variation of Epimedins A – C and Icariin in Ten Representative Populations of Epimedium brevicornu Maxim., and Implications for Utilization

The concentration variations of main flavonoids, epimedins A–C and icariin, among ten representative populations of Epimedium brevicornu Maxim . were assessed by HPLC. The populations were collected during the flowering stage and included 419 individual samples. Remarkable variations within and among populations were detected. SXXA Population (see Fig. 1) was an outlier due to its significant low concentrations (<1.00–4.46 mg/g). But even without SXXA, significant concentration differences among populations were still observed in epimedin A (2.31–8.42 mg/g), epimedin B (6.67–55.7 mg/g), epimedin C (5.39–23.0 mg/g), icariin (8.50–39.9 mg/g), and their total (29.1–123 mg/g). All populations except SXXA showed much higher concentrations than the recommended standards (i.e. 5 mg/g for icariin and 13 mg/g for the total). A high‐concentration‐population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis, based on Euclidean distances, was observed. Both methods allowed separation of the populations in four groups defined by the concentrations of four main flavonoids. The populations (SXLC and SXQS) located in north of Yellow River were clustered together and characterized by highest concentrations of epimedin B, icariin, and their total. Considering of the high concentrations of main flavonoids and abundant resources, E. brevicornu could be exploited as a good medical resource for Herba Epimedii and would offer a tremendous potential for commercial development, but SXXA population should be paid special attention, and further study is needed.     

Osteoblastic proliferative activity of Epimedium brevicornum Maxim.

The effect of the extracts of Epimedium brevicornum Maxim. was investigated on proliferative activity in vitro. The osteoblast-like UMR106 cells was employed as an osteoblast model. The EtOH extract and the n-butanol fraction from the crude extract were found to show proliferation stimulating activity. Three flavonoid compounds (icariin, epimedin B and epimedin C) were isolated from this fraction by activity-guided assay, and the effects on cell proliferation were studied. Icariin produced the most significant promoting effect on the proliferation in osteoblast-like UMR106 cells. The results suggested that E. brevicornum Maxim. extracts might have potential activity against osteoporosis, and flavonoids such as icariin might be the active constituents stimulating osteoblasts.     

Systematic Quality Evaluation of Epimedium wushanense T. S. Ying Based on Two Quality Control Standards: Total Flavonoid Glycosides and Epimedin C

Two quality control standards, total flavonoid glycosides of Epimedii Folium and epimedin C of Epimedii Wushanensis Folium, were used to systematically evaluate the quality of Epimedium wushanense T. S. Ying, so as to provide reference for its germplasm screening and resource utilization. Seven representative populations of E. wushanense covering its main distribution areas were uniformly sampled during the flowering period. There were significant quality differences among the populations of E. wushanense. According to the quality standard of total flavonoid glycosides, all populations were superior to the quality standard of the Chinese Pharmacopoeia for Epimedii Folium, with more than 1.5 % total flavonoid glycosides. The variation ranges of epimedin A, epimedin B, epimedin C, icariin and total flavonoid glycosides were 0.40–0.76 %, 0.51–0.83 %, 1.70–9.31 %, 0.40–1.23 % and 3.05–10.61 %, respectively. According to the quality standard of epimedin C, all populations were better than the quality standard of the Chinese Pharmacopoeia for Epimedii Wushanensis Folium, with more than 1.0 % epimedin C. The variation range of epimedin C was 2.22–10.06 %. When comparing the results of the two methods, a trend of slightly lower mean values was found for total flavonoid glycosides, except for the HBXW population. The quality of E. wushanense was superior to both the quality standard of Epimedii Folium and Epimedii Wushanense Folium in the Chinese Pharmacopoeia. Epimedin C was the most abundant component. Among the investigated populations, HBXW and HBGK exhibited the highest quality, and may provide excellent genetic resources for standardized cultivation. In addition, the habitat of these populations can also serve a reference for cultivation conditions.     

High-performance liquid chromatography-based multivariate analysis to predict the estrogenic activity of an Epimedium koreanum extract

This study characterizes the correlation between the chemical fingerprint and estrogenic activity of an Epimedium koreanum extract. The estrogenic activity of 31 E. koreanum extract samples was evaluated by a luciferase reporter gene assay, and the samples were classified into 3 groups based on their bioactivity. A chemical fingerprint analysis was performed on each sample by high-performance liquid chromatography (HPLC), and 44 common peaks were selected from the chromatogram and used as a dataset for a pattern recognition analysis. A canonical discriminant analysis performed on this dataset determined a distinct distribution of the samples according to their estrogenic activity on the scoring plot. The classification results showed that 90.3% of the original grouped cases had been correctly classified. The total content of the 4 major extract compounds, epimedin A, epimedin B, epimedin C, and icariin, exhibited good correlation (r=0.784) with the estrogenic activities of the respective extracts. This chromatographic fingerprint-chemometric analysis system could be useful for predicting the E. koreanum pharmacological activity and consequent biological activity-relevant quality control assessment.     

游泳对大鼠阳虚状态的影响及数理分析

目的考察一次性游泳对氢化可的松粉针剂诱导大鼠阳虚状态指标的影响,以期暴露模型本身内隐的隐性测评指标并确定特征指标。方法将SD雄性大鼠随机分为正常组、正常游泳组、阳虚组、阳虚游泳组,阳虚组／阳虚游泳组肌注氢化可的松粉针剂20ms／kg,正常组／正常游泳组注射等量的0．9％氯化钠溶液,注射14d后,阳虚游泳组／正常游泳组辅以一次性游泳后,各组麻醉,取血,测定相关指标。结果阳虚游泳组C4、cGMP、TC、L阳虚隐性指标暴露,BUN、17-OHCS、T、T／E2、cAMP／cGMP阳虚评定指标凸显。结论非破坏性一次游泳可以使氢化可的松粉针剂诱导的大鼠阳虚证候模型的隐性指标凸显、部分显性指标放大,建议今后在建立、模拟阳虚证候模型时辅以游泳手段,以便客观、全面、真实的反映模型状态。     

Effects of total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on the proliferation and differentiation of primary osteoblasts.

In a bioassay-guided drug screening for anti-osteoporosis activity, eight flavonol glycosides were isolated from Epimedium koreanum Nakai, which is traditionally widely used in China for the treatment of impotence and osteoporosis. The effects of total flavonoids and flavonol glycosides on the proliferation and differentiation of rat calvarial osteoblast-like cells were evaluated by the MTT method and measuring the activity of alkaline phosphatase (ALP activity). Total flavonoids (1.2 x10(-2) to 6.0 x10(-7) mg/ml) and flavonol glycosides (2.0 x10(-5) to 1.0 x10(-9) mol/l) exhibited a strong inhibition on the proliferation of primary osteoblasts at most concentrations. However, the total flavonoids and icariin significantly promoted the differentiation of primary osteoblasts. The results suggested that flavonoids from E. koreanum Nakai may improve the development of osteoblasts by promoting the ALP activity; and icariin might be one of the active constituents facilitating the differentiation of osteoblasts.     

不同群落类型柔毛淫羊藿总黄酮和淫羊藿苷含量及其与土壤因子的关系

2009年8月,采用高效液相色谱法和紫外分光光度法测定了唐家河自然保护区次生落叶阔叶林红桦群落(群落Ⅰ)、常绿落叶阔叶混交林细叶青冈群落(群落Ⅱ)和常绿阔叶林油樟群落(群落Ⅲ)下生长的柔毛淫羊藿各器官的总黄酮和淫羊藿苷含量,分析其与土壤因子的关系结果表明：柔毛淫羊藿叶片中总黄酮和淫羊藿苷含量最高、茎中最低;群落Ⅰ的柔毛淫羊藿总黄酮和淫羊藿苷含量［(5.32±0.23)%和(0.47±0.05)%］均显著高于群落Ⅱ［(4.06±0.03)%和(0.32±0.01)%］和群落Ⅲ［(4.15±0.07)%和(0.28±0.09)%］;土壤氮含量和pH值对柔毛淫羊藿的总黄酮和淫羊藿苷含量影响较大,柔毛淫羊藿总黄酮和淫羊藿苷含量与土壤全氮和碱解氮呈显著负相关(P<0.05),与土壤pH值呈极显著正相关(P<0.01).土壤较低的氮供应水平和较高的土壤酸度可能使群落Ⅰ柔毛淫羊藿的总黄酮和淫羊藿苷含量增加.     

葛根素对2型糖尿病大鼠的治疗作用*

目的:观察葛根素对2型糖尿病(T2DM)大鼠的治疗作用。方法:采用高糖高脂饲料喂养加一次性腹腔注射60 mg/kg链脲佐菌素的方法建立T2DM 大鼠模型,随机分为正常组,模型组,二甲双胍(40 mg/kg)组,葛根素低、中、高剂量(40,80,160 mg/kg)组,每组10只大鼠;造模成功后,灌胃给药4周,每周测量大鼠体重和空腹血糖(FBG),末次给药24 h后取血,收集血清,检测各组大鼠的血糖、血清甘油三酯(TG)、总胆固醇(TC) 、低密度脂蛋白-胆固醇(LDL-C)水平、高密度脂蛋白-胆固醇(HDL-C),血清天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)活性,血清尿素氮(BUN)、肌酐(SCr)、尿酸(UA)水平。结果:干预4周后,与正常组比较,模型组大鼠体重显著降低(P＜0.01),FBG,TC,TG,LDL-C,ALT,AST,BUN,SCr,UA均显著升高(P＜0.01),而HDL-C 显著降低(P＜0.01);与模型组比较,二甲双胍组和葛根素各剂量组大鼠体重均显著增加(P＜0.01),FBG,TC,TG,LDL-C,ALT,AST,BUN,SCr,UA均显著降低(P＜0.01),而HDL-C显著升高(P＜0.01)。结论:葛根素能够减少T2DM大鼠体重降低幅度,降低血脂、血糖水平,可用于T2DM的治疗。