淫羊藿炮制前后UPLC-PDA-MS 的指纹图谱研究

建立了朝鲜淫羊藿和柔毛淫羊藿药材的超高效液相色谱(UPLC)指纹图谱分析方法, 实验结果表明, 两个品种的指纹图谱差别较大, 朝鲜淫羊藿标出35 个共有峰, 质谱鉴定了26 个峰, 柔毛淫羊藿标出13 个共有峰, 质谱鉴定了9个峰; 研究了朝鲜淫羊藿和柔毛淫羊藿炮制前后UPLC 指纹图谱的变化规律, 指纹图谱发生了五处变化. 该法快速、简便, 重现性好, 从整体上显示淫羊藿药材和炮制品的特征, 为淫羊藿的生品和炮制品的质量控制提供了有效手段.     

朝鲜淫羊藿的色谱指纹图谱及液相色谱/电喷雾串联质谱分析

建立了长白山区朝鲜淫羊藿药材的高效液相色谱指纹图谱的分析方法. 确定了18批朝鲜淫羊藿药材的13个共有峰, 该指纹图谱的精密度、稳定性和重现性的相对标准偏差均低于3.0%. 结合液相色谱/电喷雾串联质谱对特征峰进行了结构确认, 并根据电喷雾串联质谱数据推测了13个特征化合物的结构. 结果表明采用高效液相色谱与质谱联用技术对朝鲜淫羊藿色谱指纹图谱中的特征峰进行结构确认, 使其色谱指纹图谱的特征性更强, 更适合于药材质量的鉴别与评价.     

HPLC法同时测定淫羊藿中朝藿定A、B、C与淫羊藿苷的含量

建立了同时测定淫羊藿中朝藿定A、B、C与淫羊藿苷含量的高效液相色谱方法。淫羊藿样品经超声波提取,以50%(φ)乙醇溶液为溶剂,在温度50℃、料液比1∶60条件下超声波提取30 m in,共提取2次。色谱条件:ZORBAX SB-C8色谱柱(5μm,4.6×250 mm);流动相A为乙腈,B为1%乙酸溶液;梯度洗脱;紫外检测波长为270 nm。上述4种类黄酮的标准曲线在6.6~33.0 mg/L(朝藿定A、淫羊藿苷)或6.6~55.0mg/L(朝藿定B、C)范围内呈良好的线性关系(r>0.99);加标回收率在97%~102%之间;相对标准偏差小于2%(n=5)。测得不同产地心叶淫羊藿叶片中4种类黄酮的质量分数差异较大;朝藿定A广泛分布于淫羊藿属中。本方法在淫羊藿药材质量控制中具有一定的参考价值。     

高效液相色谱法测定箭叶淫羊藿中淫羊藿苷的含量

箭叶淫羊藿用70%乙醇回流提取,取部分提取液直接干燥得淫羊藿提取物,以高效液相色谱法测定箭叶淫羊藿中淫羊藿苷的含量,色谱柱为InertsilODS-3柱,流动相为乙腈-水(27∶73),检测波长为270nm。在此条件下,淫羊藿苷与其它黄酮醇苷的色谱峰分离完全,平均回收率为99.46%。该法可用于淫羊藿药材的质量控制。     

高效液相色谱法测定朝鲜淫羊藿中淫羊藿甙的含量

采用高效液相色谱法测定朝鲜建羊藿中淫羊藿成的含量,色谱柱为Shim-PackCLC-ODS柱,流动相为乙睛-水（30:70）,检测波长270um。在此条件下,淫羊藿式与其它黄酮醇咸的色谱峰分离完全。方法回收率为976％,RSD为1.2％,操作简便,结果可靠,为淫羊藿药材的质量控制提供了新的方法。     

箭叶淫羊藿中5种黄酮类化合物的反相色谱分离制备

用半制备型反相高效液相色谱(RPLC)对箭叶淫羊藿提取液中的5种主要黄酮类化合物进行了分离制备,优化了分离条件。每次进提取液35mL,经一步RPLC便可得到纯度均大于95％的化合物Ⅰ、Ⅱ、Ⅲ和Ⅴ,经过10次制备,共制得93mg Ⅰ、71mgⅡ、296mgⅢ和487mg Ⅴ。而经一步纯化得到的化合物Ⅳ的纯度较低,再经第二步RPLC纯化后可使其纯度大于95％,最后得到64mgⅣ。经波谱分析鉴定所得的5种化合物分别为hexandraside F、朝藿定A、朝藿定B、朝藿定C及淫羊藿甙,均为8-异戊烯基黄酮类化合物,其中hexandraside F是首次从淫羊藿属植物中分离得到的。     

工业制备高效液相色谱法制备淫羊藿中的黄酮系列对照品

淫羊藿甙和朝藿定A、B、C是淫羊藿中重要的活性成分,本研究应用工业制备高效液相色谱从淫羊藿粗提物中分离制备了这4个成分。淫羊藿粗提物经大孔吸附树脂粗分离获得相应的组分后,利用工业制备高效液相色谱完成精制纯化。采用自装填Chromatorex C18制备柱(220 mm×77 mm, 10 μm),乙腈-水(26:74或30:70, v/v)为流动相进行洗脱,在35 min内,实现了这4种成分的基线分离及规模制备。从300 g粗提物(总黄酮含量约20%)中获得淫羊藿甙33 g、朝藿定C 4.6 g、朝藿定B 3.7 g和朝藿定A 0.6 g,产品纯度均达到98%以上。此方法通过两步分离即可实现这4种成分的完全分离,具有快速高效、产品纯度高的特点,适于淫羊藿中淫羊藿甙、朝藿定A、B、C系列对照品的规模制备。     

益气通痹胶囊质量标准研究

采用薄层色谱法对益气通痹胶囊中黄芪、何首乌、五味子、赤芍、延胡索进行定性鉴别,采用高效液相色谱法测定了制剂中淫羊藿苷的含量. 所用薄层色谱具有鉴别特征,色谱斑点清晰,专属性强. 淫羊藿苷在0.55~3.30 μg范围内呈良好的线性关系(r =0.99996),平均回收率为98.4%,RSD为0.62 %. 所建立的定性和定量方法,操作简便可靠,专属性强,重现性好,能较全面反映该制剂内在质量,可作为益气通痹胶囊新药研发质量控制标准.     

淫羊藿根与叶活性成分的分析和比较

采用细胞膜色谱法（ＣＭＣ）筛选，并结合离体药理实验确定主要活性成分，利用高效液相色谱法分析比较了淫羊藿根与叶中的活性成分及其差异性．色谱条件为Ｋｒｏｍａｓｉｌ ＯＤＳ 柱（１５０ ｍｍ× ４．６ ｍｍ．Ｉ．Ｄ ）流动相甲醇－水（７０：３０，Ｖ：Ｖ）；检测波长２７０ｎｍ。筛选发现淫羊藿根中的两个有效成分ＹＹＨ－２１４和ＹＹＨ－２１６对血管有较强的舒张作用，表明活性成分在ＣＭＣ模型体系中的保留特性与药理作用之间存在良好的相关性。在此色谱条件下淫羊藿叶未检测到这两种活性成分．     

动态超声辅助萃取与HPLC联用测定淫羊藿中黄酮类化合物

建立了动态超声辅助萃取与高效液相色谱在线联用系统,用于测量淫羊藿中黄酮类化合物。萃取过程在一个循环体系中完成,萃取完成后,通过采样环采集20μL萃取液,萃取液被流动相载入色谱系统进行分离检测。4种黄酮类化合物用HPLC–MS区分,通过比较色谱峰面积选择优化萃取条件。选择50℃的水浴温度,50%的乙醇作为萃取剂,萃取剂流速为1.5 mL/min,萃取时间为8 min,样品量为15 mg。用该法测量淫羊藿样品中4种黄酮类化合物,日内、日间精密度分别为1.05%～2.05%,0.30%～3.63%(n=6)。淫羊藿中的主要化合物淫羊藿苷的加标回收率为95.2%。该方法可用于测量淫羊藿中的黄酮类化合物。     

Geographical classification of Epimedium based on HPLC fingerprint analysis combined with multi‐ingredients quantitative analysis

A reliable and comprehensive method for identifying the origin and assessing the quality of Epimedium has been developed. The method is based on analysis of HPLC fingerprints, combined with similarity analysis, hierarchical cluster analysis (HCA), principal component analysis (PCA) and multi‐ingredient quantitative analysis. Nineteen batches of Epimedium , collected from different areas in the western regions of China, were used to establish the fingerprints and 18 peaks were selected for the analysis. Similarity analysis, HCA and PCA all classified the 19 areas into three groups. Simultaneous quantification of the five major bioactive ingredients in the Epimedium samples was also carried out to confirm the consistency of the quality tests. These methods were successfully used to identify the geographical origin of the Epimedium samples and to evaluate their quality.     

Optimization for quantitative determination of four flavonoids in Epimedium by capillary zone electrophoresis coupled with diode array detection using central composite design

Herba Epimedii (family Berberidaceae), Ying-Yang-Huo in Chinese, is a famous Chinese herbal medicine. Flavonoids are thought to be the major active components in it. A capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of four flavonoids including icariin, epimedin A, epimedin B and epimedin C in Epimedium. The effects of the experimental variables on CZE had been optimized by using central composite design (CCD). The best separation of four flavonoids could be obtained using 50 mM borate buffer (pH 10.0) containing 22% acetontrile as modifier, while separation voltage was 15 kV and temperature was at 25 degrees C. The method developed is accurate, simple and reproducible, which could be used for quality control of Epimedium and its medical preparations.     

毛细管电泳法测定淫羊藿甙的pK_a值及其在中药淫羊藿中的含量

 利用毛细管电泳中不同 pH条件下有效淌度 (μeff)与缓冲溶液中氢离子的浓度 ([H+])的非线性关系以及1/ μeff与缓冲溶液中 [H+]的线性关系 ,测定了淫羊藿甙的离子淌度 (μA- )和pKa 值 ;考察了在有机改性剂存在的情况下 ,随缓冲溶液中有机改性剂浓度的增加 ,淫羊藿甙的 pKa 值的变化情况。在缓冲溶液为V(2 4mmol/L磷酸盐 )∶V(乙醇 ) =70∶30时 ,对中药淫羊藿中淫羊藿甙的含量进行了定量测定 ,结果发现校正峰面积 (Y)与淫羊藿甙的质量浓度 (X ,g/L)的线性相关方程为Y =6 96× 10 -3 + 17 0X ,线性范围为 0 0 32g/L～ 0 35 4g/L。     

大孔吸附树脂法去除淫羊藿多糖中蛋白的研究

从4种大孔吸附树脂中筛选出ADS-7, 考察了其对淫羊藿多糖中蛋白的去除作用, 并讨论了pH值、 鞣酸、 上样量等对树脂去蛋白效率的影响. 结果表明, 该方法对淫羊藿粗多糖中的蛋白具有较高的去除效率, 淫羊藿粗多糖中的蛋白含量由1.2%下降到0.035%.     

Simultaneous determination of seven flavonoids in Epimedium using pressurized liquid extraction and capillary electrochromatography

Herba Epimedii (known as Yinyanghuo in China) is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a CEC method was developed for the simultaneous determination of seven flavonoids, including hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, icariin, epimedin A, B, and C, in Epimedium using baicalein as internal standard (IS). The influence of relevant parameters such as buffer concentration, pH, and proportion of ACN was investigated and optimized. Baseline separation was obtained using a Hypersil C18 capillary (3 microm, 100 microm/25 cm) with a mixture of 20 mM phosphate buffer (pH 4.0)/ACN (70:30 v/v) as mobile phase running at 30 kV and 25 degrees C in 20 min. All calibration curves showed good linearity (r2 >0.9992) within test ranges. The LOD and LOQ were lower than 8.6 and 42.8 microg/mL, respectively. The RSDs of intra- and interday for relative peak areas of seven analytes were less than 3.1 and 4.4%, and the recoveries were 95.2-103.3%. Samples of different Epimedium species were analyzed using the validated method, which is useful for quality control of Epimedium and its medical preparations.     

Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2'-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.     

Application of HPLC Fingerprint Combined with Chemical Pattern Recognition and Multi-Component Determination in Quality Evaluation of Echinacea purpurea (L.) Moench

Echinacea purpurea (EP) is a common medicinal material for extracting anti-RSV components. However, up to now, there has been no effective and simple method to comprehensively reflect the quality of EP. In our current study, the quality of Echinacea purpurea (L.) Moench samples from six different cultivation locations in China was evaluated by establishing a high-performance liquid chromatography (HPLC) fingerprint, combining chemical pattern recognition and multi-component determination. In this study, the chemical fingerprints of 15 common peaks were obtained using the similarity evaluation system of the chromatographic fingerprints of traditional Chinese medicine (2012A Edition). Among the 15 components, three phenolic acids (caftaric acid, chlorogenic acid and cichoric acid) were identified and determined. The similarity of fingerprints of 16 batches of Echinacea purpurea (L.) Moench samples ranged from 0.905 to 0.998. The similarity between fingerprints of five batches of commercially available Echinacea pupurea (L.) Moench and the standard fingerprint ”R” ranged from 0.980 to 0.997, which proved the successful establishment of the fingerprint. PCA and HCA were performed with the relative peak areas of 15 common peaks (peak 3 as the reference peak) as variables. Anhui and Shaanxi can be successfully distinguished from the other four cultivation areas. In addition, the index components of caftaric acid, chlorogenic acid and cichoric acid were in the range of 1.77–8.60 mg/g, 0.02–0.20 mg/g and 2.27–15.87 mg/g. The results of multi-component index content determination show that the contents of the Shandong cultivation area were higher, followed by Gansu, Henan and Hebei, and the lowest were Anhui and Shaanxi. The results are consistent with PCA and HCA, which proved that the quality of Echinacea purpurea (L.) Moench from different origins was different. HPLC fingerprint combined with chemical pattern recognition and multi-component content determination was a reliable, comprehensive and prospective method for evaluating the quality of Echinacea purpurea (L.) Moench. This method provides a scientific basis for the quality control and evaluation of Echinacea purpurea (L.) Moench.     

Comparison between HPLC and HPTLC densitometry for the determination of icariin from Epimedium koreanum extracts

Dry extracts of the aerial parts of Epimedium koreanum were quantified by HPLC and high performance TLC (HPTLC). A gradient HPLC method was used for the quantification of the prenylflavone glycoside icariin at 270 nm. A direct HPTLC assay was developed for the determination of icariin at 270 nm. The UV detection of both analytical assays were used to examine the purity of icariin peaks and compared with the standards. The assays provide good accuracy, reproducibility, and selectivity for the quantitative analysis of icariin. The icariin contents of five different dry extracts were compared by HPLC and HPTLC densitometry. The quantitative results of both analytical methods did not show any statistically significant differences between them, although a trend to slightly lower mean values could be found for the HPLC method.