HPLC特征图谱结合含量测定的白芍及其炮制品的质量对比研究

建立白芍、炒白芍、酒白芍、硫熏白芍HPLC特征图谱,并结合多成分含量测定,为白芍、炒白芍、酒白芍和硫熏白芍的质量控制提供参考。采用Intersustain C₁₈(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-0.1%醋酸水溶液,流速为每分钟1 mL,梯度洗脱,检测波长为230 nm,柱温为30℃,进样量为10μL。14批白芍、炒白芍、酒白芍和硫熏白芍的特征图谱,标定了6个共有峰,并均被指认,分别为没食子酸、儿茶素、芍药内酯苷、芍药苷、1,2,3,4,6-五没食子酰葡萄糖和苯甲酰芍药苷,而硫熏白芍标定7个共有峰,峰7为白芍硫熏后产生;且各色谱谱峰有较好的分离,但不同炮制品特征图谱存在一定差异;含量测定结果显示,白芍炒制、酒制及硫熏后,6种成分均有不同程度的变化;借助中药色谱指纹图谱相似度评价系统和SIMCA-P13.0软件对14批白芍、炒白芍、酒白芍和硫熏白芍进行相似度和正交偏最小二乘判别(OPLS-DA)分析,所建立的白芍和炮制品及硫熏品的质量评价方法稳定性、重复性好,可用于白芍、炒白芍、酒白芍和硫熏白芍的质量控制和评价。     

高速逆流色谱法从白芍中分离纯化五没食子酰基葡萄糖

本文建立高速逆流色谱(HSCCC)方法,从白芍粗提物中分离纯化五没食子酰基葡萄糖.分别采用正己烷-乙酸乙酯-甲醇-水体积比0.5∶5∶1∶5及0.5∶5∶0.5∶5混合溶剂作为两相溶剂体系,上相为固定相,下相为流动相,转速为800 rpm,流速为2.0 mL/min,用HPLC检测及ESI-MS进行验证.经过两次HSCCC分离纯化,得到五没食子酰基葡萄糖纯度为95.7％.     

Discrimination of geographical origin and adulteration of radix astragali using fourier transform infrared spectroscopy and chemometric methods

Introduction – Radix Astragali, one of most widely used and important traditional Chinese medicines, is cultivated in different geographical regions. Because of varying growing conditions, the qualities of Radix Astragali vary, which can give rise to differences in clinical therapy. Detecting adulteration is a routine requirement in pharmaceutical practice. Objective – To develop a simple and accurate approach to discriminate the geographical origin and potential adulteration of Radix Astragali, derived from the root of Astragalus membranaceus (Fischer) Bunge var. mongholicus (Bunge) Hsiao, using Fourier transform infrared (FT‐IR) spectroscopy and chemometric methods. Methodology – To obtain characteristic IR spectra for accurate discrimination, a one‐solvent extraction method was utilised following a novel evaluation method for selecting appropriate solvents. Samples of Radix Astragali from different geographical origins were discriminated using FT‐IR spectroscopy and discriminant partial least squares (DPLS) methods. FT‐IR spectroscopy combined with Mahalanobis distance was employed to detect adulteration of Radix Astragali. Results – In comparison with other solvents, butanone was more effective at extracting samples. Radix Astragali samples were accurately assigned to their corresponding geographical origins by using FT‐IR spectroscopy and DPLS method. Most adulterated samples were detected accurately by application of FT‐IR spectroscopy combined with Mahalanobis distance. Conclusion – FT‐IR spectroscopy combined with chemometric method was developed and demonstrated to be a useful tool to discriminate geographical origin and adulteration of Radix Astragali. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolite identification strategy of non-targeted metabolomics and its application for the identification of components in Chinese multicomponent medicine Abelmoschus manihot L.

Identification of multicomponent in traditional Chinese medicine (TCM) is complex and time-consuming. The inspection of the full-scan mass chromatograms was usually performed manually, which is labor-intensive. It is difficult to distinguish low response signals from complex chemical background. Furthermore, this process is typically based on earlier knowledge of the chemical composition of TCM, and those molecules that have not been characterized earlier were thus ignored. In this paper, a strategy using UPLC-MS combined with pattern recognition analysis was developed to simplify and quicken the identification of multicomponent in Abelmoschus manihot (L.) Medik. First, complex signals obtained by UPLC-MS were processed using automated data mining algorithm and further processed with multivariate chemometric methods. Multicomponent in Abelmoschus manihot L. can be clearly displayed in S- and VIP-plot. Using this method, 320 peaks which present in Abelmoschus manihot L. were detected. In the next step, accurate mass spectra of the characteristic markers acquired by QTOF MS were used to estimate their elemental formulae and enable structure identification. By searching in METLIN database, 41 components were tentatively identified in Abelmoschus manihot L. Our results showed that UPLC-MS based-pattern recognition analysis approach can be used to quickly identify TCM multicomponent and for standardization of herbal preparations.     

Structural characterization and biological activities of two α-glucans from <Emphasis Type="Italic">Radix Paeoniae Alba</Emphasis>

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, cancer, and other diseases. In this study, two polysaccharides RPAPW1 and RPAPW2 were isolated from Radix Paeoniae Alba by DEAE-52 cellulose chromatography and G-25 sephadex. According to physicochemical methods, NMR and methylation analysis, RPAPW1 and RPAPW2 were established to be α-glucans consisting of predominant 4-linked α- Glc residues branched at O-6 and contained trace amount of protein and uronic acid. Immunological tests indicated that RPAPW1, RPAPW2 and could promote splenocyte proliferation and RAW264.7 phagocytic activity. In vitro, RPAPW1 and RPAPW2 elicited a week reducing power, DPPH scavenging activity and could not protect the PC12 cells from H₂O₂ damage. These data implied polysaccharides RPAPW1 and RPAPW2 had the potential to be a natural immunopotentiating and antioxidant supplement for preparing functional foods and nutraceuticals.     

The chemotaxonomic classification of Korean Campanulaceae based on triterpene,sterol, and polyacetylene contents

The taxonomic usefulness of selected marker compounds was investigated by analyzing the chemotaxonomy of 25 taxa in the Korean Campanulaceae. Our data permit discrimination of the source plants of crude drugs listed in the Korean Pharmacopoeia and the Korean Herbal Pharmacopoeia. Chemotaxonomic analysis methods were validated, and quantitative measurements of six marker compounds were made using HPLC. Marker compound similarities among taxa were identified through multivariate statistical analyses (principal component analysis and hierarchical cluster analysis). The chemical analysis method was validated with regard to linearity, lower limit of detection, lower limit of quantitation, precision, accuracy, and recovery. The analysis revealed differences in the marker compound composition of each genus. Eight genera comprising Adenophora, Codonopsis, Asyneuma, Campanula, Hanabusaya, Platycodon, Wahlenbergia, and Peracarpa clustered according to the chemical classification. The results indicated that the six marker compounds used in this analysis were useful in identifying Korean Campanulaceae. These marker compound data were able to successfully discriminate among the three species that are sold as the crude drug Adenophorae Radix.     

濒危高原植物羌活化学成分与生态因子的相关性

羌活（Notopterygii Rhizoma Et Radix） 是伞形科植物羌活（N. incisum）或宽叶羌活（N. franchetii）的干燥根茎和根,为濒危高原药用植物。考察羌活主产地生态气象因子对其主要化学成分含量的影响,探讨气象因子与羌活品质的关系。应用HPLC方法检测羌活醇,异欧前胡素2种主要化学成分含量。采用中国气象科学数据共享服务网平台获得不同产地的平均气压、年降水量、平均风速、日照时数、相对湿度、年平均温度、年最高温度、年最低温度等9个生态因子数据,通过偏最小二乘回归分析（PLS）、冗余分析（RDA）分析了其化学成分含量与生态因子之间的相关性。化学分析表明,羌活中羌活醇的含量高于异欧前胡素,而宽叶羌活中羌活醇含量低于异欧前胡素。经PLS及RDA分析,羌活中羌活醇与日照时数呈强正相关关系,其次是年降水量和海拔。异欧前胡素与年降水量呈强负相关关系,其次是温度。宽叶羌活中影响羌活醇含量的主导因子分别是海拔、年降水量,且都呈正相关。同时2个主导因子对异欧前胡素含量的影响呈负显著相关。因此日照时数、海拔、年降水量是影响羌活化学成分累积的主要因素,日照时数有利于羌活化学成分累积,在一定范围内,海拔越高,年降水量越大,越能促进羌活醇的积累,而不利于异欧前胡素含量富集。基于化学成分含量主成分分析（PCA）表明,羌活和宽叶羌活以四川产为最优。研究结果对保护濒危高原植物羌活及引种栽培奠定基础。     

微量热法研究板蓝根中四种有机酸对微生物生长代谢的影响

微量热法研究传统中药板蓝根中四种有机酸对大肠杆菌、金黄色葡萄球菌和痢疾杆菌生长代谢的影响。得到加药与不加药时大肠杆菌、金黄色葡萄球菌和痢疾杆菌生长代谢的“效-时”曲线, 以生长速率常数(k1, k2)、最大产热功率(Pm)和最大达峰时间(tm)等热力学参数来评价四种有机酸对微生物生长代谢抑制的强度和程度。四种有机酸抗微生物活性作用的顺序为: 丁香酸>邻氨基苯甲酸>水杨酸>苯甲酸, 其中苯甲酸对金黄色葡萄球菌和痢疾杆菌的生长代谢具有促进作用。本研究对板蓝根的进一步研究提供了基础和依据。     

微量热法研究板蓝根中四种有机酸对微生物生长代谢的影响

微量热法研究传统中药板蓝根中四种有机酸对大肠杆菌、金黄色葡萄球菌和痢疾杆菌生长代谢的影响。得到加药与不加药时大肠杆菌、金黄色葡萄球菌和痢疾杆菌生长代谢的“效-时”曲线, 以生长速率常数(k1, k2)、最大产热功率(Pm)和最大达峰时间(tm)等热力学参数来评价四种有机酸对微生物生长代谢抑制的强度和程度。四种有机酸抗微生物活性作用的顺序为: 丁香酸>邻氨基苯甲酸>水杨酸>苯甲酸, 其中苯甲酸对金黄色葡萄球菌和痢疾杆菌的生长代谢具有促进作用。本研究对板蓝根的进一步研究提供了基础和依据。     

Microcalorimetry coupled with chemometric techniques for toxicity evaluation of Radix Aconiti Lateralis Preparata (Fuzi) and its processed products on Escherichia coli

As a widely used traditional Chinese medicine (TCM), Radix Aconiti Lateralis Preparata (Fuzi) is not only efficacious but also poisonous. Its toxicity and processed products should be taken into account and effectively evaluated. In this study, a non-invasive and non-destructive microcalorimetric method was employed to evaluate and compare the toxicity of Fuzi and its three processed products including Yanfupian, Heifupian, and Danfupian on Escherichia coli (E. coli). Some important metabolic information, such as the power–time curves and some quantitative thermokinetic parameters including growth rate constant k, heat output power P, inhibitory ratio I, and half inhibitory concentration IC₅₀, of E. coli growth affected by various concentrations of Fuzi and its processed products were obtained. Combined with chemometric techniques including multivariate analysis of variance and principal component analysis on this information, it could be seen that Fuzi and its processed products could be distinguished according to their toxic effects on E. coli. The IC₅₀ values of 14.6 mg/mL for Fuzi, 59.2 mg/mL for Yanfupian, 118.3 mg/mL for Heifupian, and 182.7 mg/mL for Danfupian illustrated that the sequence of toxicity on E. coli was Fuzi > Yanfupian > Heifupian > Danfupian. This study provided a useful method and idea of the combination of microcalorimetry and chemometrics for studying the toxic effects of TCMs and other substances.     

DNA Extraction Protocol for Biological Ingredient Analysis of Liuwei Dihuang Wan

Traditional Chinese medicine(TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients,which often include some mis-identified herbal materials, adulterants or even some biological contaminants.For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation,Liuwei Dihuang Wan(LDW), as an example to develop a TCM-specific DNA extraction method.An optimized cetyl trimethyl ammonium bromide(CTAB) method(TCM-CTAB) and three commonlyused extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer(ITS2) and the chloroplast genome trnL intron was carried out.The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3–4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting     

Chemometric Methods to Quantify 1D and 2D NMR Spectral Differences Among Similar Protein Therapeutics

NMR spectroscopy is an emerging analytical tool for measuring complex drug product qualities, e.g., protein higher order structure (HOS) or heparin chemical composition. Most drug NMR spectra have been visually analyzed; however, NMR spectra are inherently quantitative and multivariate and thus suitable for chemometric analysis. Therefore, quantitative measurements derived from chemometric comparisons between spectra could be a key step in establishing acceptance criteria for a new generic drug or a new batch after manufacture change. To measure the capability of chemometric methods to differentiate comparator NMR spectra, we calculated inter-spectra difference metrics on 1D/2D spectra of two insulin drugs, Humulin R® and Novolin R®, from different manufacturers. Both insulin drugs have an identical drug substance but differ in formulation. Chemometric methods (i.e., principal component analysis (PCA), 3-way Tucker3 or graph invariant (GI)) were performed to calculate Mahalanobis distance (D _M) between the two brands (inter-brand) and distance ratio (D _R) among the different lots (intra-brand). The PCA on 1D inter-brand spectral comparison yielded a D _M value of 213. In comparing 2D spectra, the Tucker3 analysis yielded the highest differentiability value (D _M = 305) in the comparisons made followed by PCA (D _M = 255) then the GI method (D _M = 40). In conclusion, drug quality comparisons among different lots might benefit from PCA on 1D spectra for rapidly comparing many samples, while higher resolution but more time-consuming 2D-NMR-data-based comparisons using Tucker3 analysis or PCA provide a greater level of assurance for drug structural similarity evaluation between drug brands.     

Application of response surface methodology to optimise ultrasonic-assisted extraction of four chromones in Radix Saposhnikoviae

Introduction – Radix Saposhnikoviae is one of the most famous Chinese herbal medicines with many pharmacological activities towards inflammatory symptoms and antioxidation. Chromones are considered as one of the effective components. It is important to find a reasonable method to extract the chromones in S. divaricata. Objective – To develop an ultrasonic‐assisted extraction (UAE) to extract chromones in Radix Saposhnikoviae and to optimise extraction conditions. Methodology – Four chromones (prim‐O‐glucosylcimifugin, cimifugin, 5‐O‐methylvisammioside and sec‐O‐glucosylhamaudol) were extracted by the UAE method combined with response surface methodology (RSM). Box–Behnken design (BBD) was applied to evaluate the effects of three independent variables (ethanol concentration, extraction time and extraction temperature) on the chromones yield of Radix Saposhnikoviae. Results – Correlation analysis of the mathematical‐regression model indicated that a quadratic polynomial model could be employed to optimise the extraction of chromones by UAE method. The optimal conditions to obtain the highest chromones yield of Radix Saposhnikoviae were a solvent of 75% ethanol, an extraction time of 48 min and an extraction temperature of 67°C. Conclusion – Under these optimal conditions, the experimental values agreed closely with the predicted values. The analysis of variance indicated a high goodness of model fit and the success of RSM method for optimising chromones extraction in Radix Saposhnikoviae. Copyright © 2011 John Wiley & Sons, Ltd.     

DNA barcoding provides distinction between Radix Astragali and its adulterants

Based on variable nuclear and/or organellar DNA sequences among vastly divergent species as well as morphologically indistinguishable species, DNA barcoding is widely applicable in species identification, biodiversity studies, forensic analyses, and authentication of medicinal plants. The roots of Astragalus membranaceus and A. membranaceus var. mongholica are commonly used as Radix Astragali in several Asian countries, including China, Japan, and Korea. However, in addition to the two species recorded in the Chinese Pharmacopoeia, there are twenty-three species from different genera including Astragalus, Oxytropis, Hedysarum, and Glycyrrhiza, which have been used as adulterants not only in trading markets but also by the herbal medicine industry. Therefore, a simple, reliable, and accurate classification method is important for distinguishing authentic Radix Astragali from its adulterants. In this study, we acquired data for 37 samples from four related genera within the family Fabaceae. Then we compared four candidate DNA barcoding markers using ITS, matK, rbcL, and coxI sequences from nuclear, chloroplast, and mitochondrial genomes, all commonly used for plants to identify genetic variations among genera, intraspecies, and interspecies. We observed higher divergences among genera and interspecies for ITS, which have the average Kimura 2-parameter distances of 4.5% and 14.1%, respectively, whereas matK was found to have sufficient divergence at the intraspecific level. Moreover, two indels detected in the matK sequence are useful for PCR studies in distinguishing Radix Astragali from its adulterants. This study suggests that the combined barcoding regions of ITS and matK are superior barcodes for Radix Astragali and further studies should focus on evaluating the applicability and accuracy of such combined markers for a wide range of traditional Chinese herbs.     

An efficient strategy based on MAE, HPLC-DAD-ESI-MS/MS and 2D-prep-HPLC-DAD for the rapid extraction, separation, identification and purification of five active coumarin components from Radix Angelicae Dahuricae

Introduction – Further studies of active coumarin components in Radix Angelicae Dahuricae (AE) are absolutely essential to provide data on pharmacology, toxicology and quality for innovative drug candidates. Thus, the preparation of active component standards and the administration of coumarin monomers should be carried out. The isolation of the low‐level active components from complex Traditional Chinese Medicine (TCM) samples necessitates the development of rapid, simple and economical modern extraction, separation, identification and purification methods. Objective – To develop an efficient strategy for the rapid extraction, separation, identification and purification of coumarins from AE. Methodology – First, active coumarins in AE were extracted with microwave‐assisted extraction (MAE) after the extraction conditions were optimised. Second, gradient extraction methods with MAE were used to partially purify AE. Third, a high‐performance liquid chromatography–diode array detection‐electrospray ionisation tandem mass spectrometry (HPLC‐DAD‐ESI‐MS/MS) method was applied for the preliminary on‐line identification and screening of the main coumarins in AE extract. Finally, a two‐dimensional preparative high‐performance liquid chromatography–diode array detection (2D‐prep‐HPLC‐DAD) system was developed for further preparative separation of those target components. Results – Altogether 10 coumarins have been identified and five of them including xanthotoxol, osthenol, oxypeucedanin hydrate, byakangelicin and imperatorin were deemed as target components for the preparative isolation. All of the five isolated coumarins were at high purities of over 99% and the production rate was much higher than the traditional methods. Conclusion – The present paper demonstrates that these consecutive approaches are very useful for to isolate chemical constituents from TCM.     

Characterization of the cold and hot natures of raw and processed Rehmanniae Radix by integrated metabolomics and network pharmacology

BackgroundThe processing of Chinese materia medica (CMM) is one of the characteristics and advantages of traditional Chinese medicine (TCM). Occasionally, the processing of CMM might reverse the cold/hot nature of CMM. For example, the nature of raw Rehmanniae Radix (RR) is cool, while the processed Rehmanniae Radix (PR) by steaming is hot. Because the cold/hot nature of CMM is defined by the body's response to CMMs, a metabolomics approach, allowing the monitoring of the fluctuation of endogenous metabolites related to an exogenous stimulus, might be an ideal tool to uncover the cold/hot nature of different forms of Rehmanniae Radix.PurposeAn integrated strategy combining metabolomics and network pharmacology was applied to illuminate the different natures of raw and processed Rehmanniae Radix.Study designMice were orally administered RR and PR once daily for ten days. The entire metabolic changes in the plasma of mice were profiled by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS). Furthermore, network pharmacology analysis was performed to identify the underlying targets related to iridoids that significantly changed during the processing.ResultsThe metabolomics analysis results demonstrated a clear separation of the metabolic phenotypes among the control, RR and two PR groups in both the positive and negative modes. Nine lysophosphatidylcholines (LysoPCs), LysoPC (16:0), LysoPC (18:2), LysoPC (18:1), LysoPC (22:6), LysoPC (20:2), LysoPC (18:0), LysoPC (16:1), LysoPC (20:4) and LysoPC (20:5), that decreased in the RR-treated group, but increased in the PR-treated group, were identified to be potential biomarkers related to the natures of RR and PR. The network pharmacology results indicated that four iridoids in Rehmanniae Radix, 8-epiloganic acid, 6-O-p-coumaroyl ajugol, 6-O-p-hydroxybenzoyl ajugol and ajugol, might play important roles in the different natures of raw and processed Rehmanniae Radix.ConclusionsThere might be a strong connection between the cold/hot nature of different forms of Rehmanniae Radix and LysoPC metabolism. This study offers new insight into the cold/hot nature of Rehmanniae Radix.