HPLC fingerprinting and LC-TOF-MS analysis of the extract of Pseudostellaria heterophylla (Miq.) Pax root

High-performance liquid chromatographic (HPLC) was developed for fingerprint analysis of Pseudostellaria heterophylla (Miq.) Pax. Liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-TOF-MS) technique was first employed to identify the components of the fingerprint. Twelve major peaks in chromatographic fingerprint were analyzed by on-line LC-TOF-MS analysis; one cyclic peptide was unequivocally identified and five cyclic peptides were tentatively assigned based on their MS data. These cyclic peptides served as the marker peaks in the HPLC fingerprints. The chromatographic fingerprints have been analyzed by similarity index calculations and hierarchical clustering analysis (HCA). The result showed that the HPLC fingerprints could be used to determine the optimal harvest time for P. heterophylla (Miq.) Pax and to authenticate the species of the herb.     

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC-MS

High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C18 column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.     

Fingerprint recognition with identical twin fingerprints

Fingerprint recognition with identical twins is a challenging task due to the closest genetics-based relationship existing in the identical twins. Several pioneers have analyzed the similarity between twins' fingerprints. In this work we continue to investigate the topic of the similarity of identical twin fingerprints. Our study was tested based on a large identical twin fingerprint database that contains 83 twin pairs, 4 fingers per individual and six impressions per finger: 3984 (83*2*4*6) images. Compared to the previous work, our contributions are summarized as follows: (1) Two state-of-the-art fingerprint identification methods: P071 and VeriFinger 6.1 were used, rather than one fingerprint identification method in previous studies. (2) Six impressions per finger were captured, rather than just one impression, which makes the genuine distribution of matching scores more realistic. (3) A larger sample (83 pairs) was collected. (4) A novel statistical analysis, which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type, has been conducted. (5) A novel analysis, which aims at showing which finger from identical twins has higher probability of having same fingerprint type, has been conducted. Our results showed that: (a) A state-of-the-art automatic fingerprint verification system can distinguish identical twins without drastic degradation in performance. (b) The chance that the fingerprints have the same type from identical twins is 0.7440, comparing to 0.3215 from non-identical twins. (c) For the corresponding fingers of identical twins which have same fingerprint type, the probability distribution of five major fingerprint types is similar to the probability distribution for all the fingers' fingerprint type. (d) For each of four fingers of identical twins, the probability of having same fingerprint type is similar.     

Monitoring and Evaluating the Quality Consistency of Compound Bismuth Aluminate Tablets by a Simple Quantified Ratio Fingerprint Method Combined with Simultaneous Determination of Five Compounds and Correlated with Antioxidant Activities

A combination method of multi-wavelength fingerprinting and multi-component quantification by high performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was developed and validated to monitor and evaluate the quality consistency of herbal medicines (HM) in the classical preparation Compound Bismuth Aluminate tablets (CBAT). The validation results demonstrated that our method met the requirements of fingerprint analysis and quantification analysis with suitable linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ). In the fingerprint assessments, rather than using conventional qualitative “Similarity” as a criterion, the simple quantified ratio fingerprint method (SQRFM) was recommended, which has an important quantified fingerprint advantage over the “Similarity” approach. SQRFM qualitatively and quantitatively offers the scientific criteria for traditional Chinese medicines (TCM)/HM quality pyramid and warning gate in terms of three parameters. In order to combine the comprehensive characterization of multi-wavelength fingerprints, an integrated fingerprint assessment strategy based on information entropy was set up involving a super-information characteristic digitized parameter of fingerprints, which reveals the total entropy value and absolute information amount about the fingerprints and, thus, offers an excellent method for fingerprint integration. The correlation results between quantified fingerprints and quantitative determination of 5 marker compounds, including glycyrrhizic acid (GLY), liquiritin (LQ), isoliquiritigenin (ILG), isoliquiritin (ILQ) and isoliquiritin apioside (ILA), indicated that multi-component quantification could be replaced by quantified fingerprints. The Fenton reaction was employed to determine the antioxidant activities of CBAT samples in vitro, and they were correlated with HPLC fingerprint components using the partial least squares regression (PLSR) method. In summary, the method of multi-wavelength fingerprints combined with antioxidant activities has been proved to be a feasible and scientific procedure for monitoring and evaluating the quality consistency of CBAT.     

金银花配方颗粒、标准汤剂含量测定及指纹图谱研究

本文主要为金银花配方颗粒的质量评价提供依据,并建立金银花配方颗粒、标准汤剂含量测定及HPLC指纹图谱的研究方法。主要采用HPLC法测定金银花饮片、标准汤剂、配方颗粒绿原酸、木犀草苷含量,建立三者指纹图谱共有模式。结果显示金银花饮片、标准汤剂、配方颗粒中绿原酸、木犀草苷含量差异较大;分别建立金银花饮片、标准汤剂、配方颗粒的指纹图谱;金银花配方颗粒HPLC指纹图谱中的12个共有峰均可在标准汤剂中追踪到,有11个可以在饮片中得到追踪,并指认出绿原酸、芦丁、木犀草苷3种成分。表明所建立的定性、定量分析方法简便,准确度高,专属性强,对金银花配方颗粒的鉴定及质量控制具有指导意义和参考价值。     

Development of capillary electrophoresis fingerprint for quality control of Rhizoma Smilacis Glabrae

Introduction – Rhizoma Smilacis Glabrae (RSG) is a Chinese herbal medicine used for detoxication and as a diuretic. However, in some regions of China, RSG is used confusedly with some other herbs. Objective – To develop a capillary electrophoresis (CE)‐DAD fingerprint method for quality evaluation, species differentiation and product identification of RSG. Methodology – The CE separation conditions and extraction procedure were optimised. Eighteen batches of RSG samples were analysed and the standard fingerprint used for authentication was simulated by the average of all tested samples. Results – The optimal CE separation conditions were developed with running buffer of 20 mm borax containing 3 mm β‐cyclodextrin at pH 9.4, voltage of 25 kV and temperature of 25°C. The separation could be completed within 8 min. Nine peaks were found in the electropherogram of RSG and five peaks were identified as astilbin, taxifolin, 5‐O‐caffeoylshikimic acid, shikimic acid and trans‐resveratrol, respectively. Methanol and sonication were recommended for the sample preparation. All RSG samples showed similar chromatographic profile and six ‘held in common’ peaks were found. By the standard fingerprint, RSG could be well distinguished from its two confusable species, Rhizoma Smilacis Chinae and Rhizoma Heterosmilacis. Conclusion – A CE‐DAD fingerprint analysis method was developed for the quality control of RSG. The standard fingerprint could represent the chemical profile of RSG and be used for its authentication. Copyright © 2010 John Wiley & Sons, Ltd.     

Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat

Daphnane-type diterpenoids (DDs) are the main types of plant diterpene orthoesters known and have remarkable biological activities. However, the in vivo toxicity and pharmacokinetic profile of DDs remains unkonwn. The aim of this study was to investigate the toxicity and pharmacokinetic profile of DDs from Genkwa Flos (Thymelaeaceae). The toxicity of diterpenoids was evaluated after oral administration of total diterpenoids extract from Genkwa Flos to rats, and the blood concentration of diterpenoids was analyzed by ultra performance liquid chromatography tandem triple-quadrupole mass spectrometry (UPLC–TQ-MS). The diterpenoids were confirmed to be the toxic components of Genkwa Flos. The pharmacokinetic profile of these diterpenoids was quite different due to their different structures. Although the contents of yuanhuafine and yuanhuapine were low in the extract, the blood concentrations were extremely high. In contrary, the contents of genkwanine F and Wikstroemia factor M₁ in the extract were much higher, but they could not be detected in the blood. This result implied that yuanhuafine and yuanhuapine but not genkwanine F and Wikstroemia factor M₁ were the potentail toxic components of Genkwa Flos in vivo. This paper shows for the first time the toxicity of diterpenoids from Genkwa Flos was correlated with their blood concentration and when DDs were used for medicinal purposes, their contents in herb as well as their blood concentrations should be considered.     

Prediction and interpretation of the antioxidant capacity of green tea from dissimilar chromatographic fingerprints

Previously, multivariate calibration techniques have been successfully applied to model and predict the antioxidant activity of green tea from its chromatographic fingerprint. Since the selectivity differences between dissimilar chromatographic systems have already been valuably used in several applications, in this paper it is studied whether combining the complementary information contained in two dissimilar fingerprints can improve the predictive capacity of the multivariate calibration model. The simplest way of combining the data is concatenating both fingerprints for each sample. The resulting matrix can then be subjected to Orthogonal Projections to Latent Structures (O-PLS). Unfortunately, this approach resulted in a more complex model with a prediction error of about the average of the errors obtained with the individual fingerprints. Secondly, only the peaks with high loading and low orthogonal loading from both chromatograms were included in the O-PLS model. This resulted in a reduced complexity, but not in better predictions, probably due to a lack of complementarity of the information concerning the antioxidant capacity. Finally, the concatenated fingerprints were subjected to stepwise multiple linear regression (MLR) in order to build a model based on the variables most correlated with the antioxidant capacity. The obtained prediction error was lower than those of both previous approaches, but still higher than the error of the model based on a single analysis. This is probably again caused by a lack of complementarity in the variables. Nevertheless, it was advantageous to develop fingerprints on dissimilar system, because it enables to choose the most suited chromatographic profile to build a multivariate calibration model for the considered purpose. In contrast to what was expected, the study showed that the most simple (so the worst separated) fingerprints resulted in the best predictions. On the other hand, a more complex fingerprint in which more compounds are separated is still important to improve the interpretability of the model.     

Secreted aspartate proteinases,a virulence factor of<Emphasis Type="Italic">Candida</Emphasis> spp.: Occurrence among clinical isolates

Production of secreted aspartate proteinases was determined in a set of 646 isolates of Candida and non-Candida yeast species collected from 465 patients of the University Hospital in Olomouc (Czechia) in the period 1995-2002, and Candida samples obtained from 64 healthy volunteers using solid media developed for this purpose. Using random amplified polymorphic DNA analysis (RAPD) 79 Candida isolates from blood were analyzed to show potential relationships between clustering of the fingerprints and extracellular proteolytic activity of these strains. C. albicans, C. tropicalis and C. parapsilosis possess always proteolytic activity while non-Candida species did not display any proteolysis. A tight relationship between fingerprints and extracellular proteolysis in the Candida isolates was not shown. A remarkable consistency between fingerprint clusters and proteolysis occurred in a subset of C. parapsilosis samples. Suboptimal pH of the growth medium was shown to facilitate the investigation of potential co-incidence of genotypic and phenotypic traits.     

DNA fingerprinting analysis of vegetative compatibility groups in Aspergillus caelatus

Forty-three isolates of Aspergillus caelatus, whose vegetative compatibility groups (VCGs) have been identified, were assessed by DNA fingerprinting using a repetitive sequence DNA probe (pAF28) cloned from A. flavus. Thirteen distinct DNA fingerprint groups or genotypes were identified among the 43 isolates. Twenty-four isolates belonging to VCG 1 produced identical DNA fingerprints and included isolates from the United States and Japan. Four other DNA fingerprint groups had multiple isolates sharing identical fingerprints corresponding to VCGs 2, 3, 12 and 13. Eight of the 13 fingerprint groups corresponding to VCGs 4-11 were represented by a single isolate with a unique fingerprint pattern. These results provide further confirmation that the pAF28 probe can distinguish VCGs of species within Aspergillus section Flavi based on DNA fingerprint patterns and that the probe can be used to estimate the number of VCGs in a sample population. Most of the A. caelatus isolates produced fewer restriction fragments and weakly hybridized with the repetitive DNA probe pAF28 compared to hybridization patterns obtained with A. flavus, suggesting less homology of the probe to A. caelatus genomic DNA.     

Holistic Evaluation of Quality Consistency of Ixeris sonchifolia (Bunge) Hance Injectables by Quantitative Fingerprinting in Combination with Antioxidant Activity and Chemometric Methods

A widely used herbal medicine, Ixeris sonchifolia (Bge.) Hance Injectable (ISHI) was investigated for quality consistency. Characteristic fingerprints of 23 batches of the ISHI samples were generated at five wavelengths and evaluated by the systematic quantitative fingerprint method (SQFM) as well as simultaneous analysis of the content of seven marker compounds. Chemometric methods, i.e., support vector machine (SVM) and principal component analysis (PCA) were performed to assist in fingerprint evaluation of the ISHI samples. Qualitative classification of the ISHI samples by SVM was consistent with PCA, and in agreement with the quantitative evaluation by SQFM. In addition, the antioxidant activities of the ISHI samples were determined by both the off-line and on-line DPPH (2, 2-diphenyl-1-picryldrazyl) radical scavenging assays. A fingerprint–efficacy relationship linking the chemical components and in vitro antioxidant activity was established and validated using the partial least squares (PLS) and orthogonal projection to latent structures (OPLS) models; and the online DPPH assay further revealed those components that had position contribution to the total antioxidant activity. Therefore, the combined use of the chemometric methods, quantitative fingerprint evaluation by SQFM, and multiple marker compound analysis in conjunction with the assay of antioxidant activity provides a powerful and holistic approach to evaluate quality consistency of herbal medicines and their preparations.     

Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI-MS/MS

Ginkgo biloba is one of the most popular herb nutrition supplements, with terpene lactones and flavonoids being the two major active components. A fingerprint profile method was developed using a capillary HPLC/MS method which can identify more than 70 components from the G. biloba product. The method allows the flavonoids and terpene lactones to be detected simultaneously and information of both the parent ion and its fragmentation can be obtained in just one HPLC/MS run. Targeted post-acquisition analysis allows mass spectrometric information regarding the identification of flavonoid components to be easily distinguished from other data, however the same approach for terpene lactones was less successful due to dimer formation and requires further development. The fingerprint profiles of five commercial G. biloba nutritional supplements were obtained and compared; variation of some components among the samples was observed and fortification could be detected. In the quality control analysis of the G. biloba product this method could be viewed as complementary to specific quantitative analysis of some bioactive components of the herb.     

中药XRD二阶导数指纹图谱的研究

以三种不同产地的丹参为研究对象,研究植物类中药XRD二阶导数指纹图谱方法学和植物类中药XRD二阶导数指纹图谱的特征。丹参XRD二阶导数指纹图谱具有峰位明确,峰型尖锐,特征性强的特点,不同产地丹参的差异性在二阶导数指纹图谱得以体现。通过相似度计算,三种丹参之间的相似度都小于0.8,可准确鉴定不同产地的丹参。结果表明:XRD二阶导数指纹图谱可用于植物类中药的XRD指纹图谱研究。     

Study on the Main Components Interaction from Flos Lonicerae and Fructus Forsythiae and Their Dissolution In Vitro and Intestinal Absorption in Rats

The Flos Lonicerae-Fructus Forsythiae herb couple is the basic components of Chinese herbal preparations (Shuang-Huang-Lian tablet, Yin-Qiao-Jie-Du tablet and Fufang Qin-Lan oral liquid), and its pharmacological effects were significantly higher than that in Flos Lonicerae or Fructus Forsythiae, but the reasons remained unknown. In the present study, pattern recognition analysis (hierarchical cluster analysis (HCA) and principal component analysis (PCA)) combined with UHPLC-ESI/LTQ-Orbitrap MS system were performed to study the chemical constitution difference between co-decoction and mixed decoction in the term of chemistry. Besides, the pharmacokinetics in vivo and intestinal absorption in vitro combined with pattern recognition analysis were used to reveal the discrepancy between herb couple and single herbs in the view of biology. The observation from the chemical view in vitro showed that there was significant difference in quantity between co-decoction and mixed decoction by HCA, and the exposure level of isoforsythoside and 3, 5-dicaffeoylquinic acid in co-decoction, higher than that in mixed decoction, directly resulted in the discrepancy between co-decoction and mixed decoction using both PCA and HCA. The observation from the pharmacokinetics displayed that the exposure level in vivo of neochlorogenic acid, 3, 4-dicaffeoylquinic acid, isoforsythoside and forsythoside A, higher than that in single herbs, was the main factor contributing to the difference by both PCA and HCA, interestingly consistent with the results obtained from Caco-2 cells in vitro, which indicated that it was because of intestinal absorption improvement of neochlorogenic acid, 3, 4-dicaffeoylquinic acid, isoforsythoside and forsythoside A that resulted in a better efficacy of herb couple than that of single herbs from the perspective of biology. The results above illustrated that caffeic acid derivatives in Flos Lonicerae-Fructus Forsythiae herb couple could be considered as chemical markers for quality control of its preparations.     

A tree-based method for the rapid screening of chemical fingerprints

Background  

The fingerprint of a molecule is a bitstring based on its structure, constructed such that structurally similar molecules will have similar fingerprints. Molecular fingerprints can be used in an initial phase of drug development for identifying novel drug candidates by screening large databases for molecules with fingerprints similar to a query fingerprint.     

Clonal composition of the peach-potato aphid Myzus persicae (Homoptera: Aphididae) in France and Scotland: Comparative analysis with IGS fingerprinting and microsatellite markers

Fourteen colonies of the peach‐potato aphid, Myzus persicae, were taken either from French peach trees or weeds in 2001. Thirty five apomictic parthenogenetic lineages (APLs) were established. Ribosomal DNA intergenic spacer (IGS) fingerprinting was used to characterise these and 28 fingerprints were duly obtained. Those lineages with different fingerprints were considered different genotypes and those with the same fingerprint as the same. The genetic identity of APLs was further tested using four microsatellite loci. APLs that differed by IGS fingerprint had distinct microsatellite allele combinations and those that had the same IGS fingerprint had the same microsatellite allele combinations. The results confirmed that IGS types corresponded to different aphid genotypes. Independent APLs with identical IGS and microsatellite genotype were therefore considered different representatives of the same clone. APLs from M. persicae found on Scottish crops in 1995, 1996 and 2001, as well as a long‐term laboratory line were also examined by the same methods. Their IGS fingerprints were similar or identical suggesting that they all belonged to the same clone. Microsatellite markers also suggested that these lineages were derived from a single clone. Some field lineages exhibited slight modifications to their IGS fingerprints confirming that the IGS evolves more rapidly than these microsatellite alleles. Thus, IGS will continue to provide a useful marker for aphid fieldwork.     

SVM-Based Synthetic Fingerprint Discrimination Algorithm and Quantitative Optimization Strategy

Synthetic fingerprints are a potential threat to automatic fingerprint identification systems (AFISs). In this paper, we propose an algorithm to discriminate synthetic fingerprints from real ones. First, four typical characteristic factors—the ridge distance features, global gray features, frequency feature and Harris Corner feature—are extracted. Then, a support vector machine (SVM) is used to distinguish synthetic fingerprints from real fingerprints. The experiments demonstrate that this method can achieve a recognition accuracy rate of over 98% for two discrete synthetic fingerprint databases as well as a mixed database. Furthermore, a performance factor that can evaluate the SVM''s accuracy and efficiency is presented, and a quantitative optimization strategy is established for the first time. After the optimization of our synthetic fingerprint discrimination task, the polynomial kernel with a training sample proportion of 5% is the optimized value when the minimum accuracy requirement is 95%. The radial basis function (RBF) kernel with a training sample proportion of 15% is a more suitable choice when the minimum accuracy requirement is 98%.     

Species-specific AFLP markers for identification of Zingiber officinale, Z. montanum and Z. zerumbet (Zingiberaceae)

The Zingiber genus, which includes the herbs known as gingers, commonly used in cooking, is well known for its medicinal properties, as described in the Indian pharmacopoeia. Different members of this genus, although somewhat similar in morphology, differ widely in their pharmacological and therapeutic properties. The most important species of this genus, with maximal therapeutic properties, is Zingiber officinale (garden ginger), which is often adulterated with other less-potent Zingiber sp. There is an existing demand in the herbal drug industry for an authentication system for the Zingiber sp in order to facilitate their commercial use as genuine phytoceuticals. To this end, we used amplified fragment length polymorphism (AFLP) to produce DNA fingerprints for three Zingiber species. Sixteen collections (six of Z. officinale, five of Z. montanum, and five of Z. zerumbet) were used in the study. Seven selective primer pairs were found to be useful for all the accessions. A total of 837 fragments were produced by these primer pairs. Species-specific markers were identified for all three Zingiber species (91 for Z. officinale, 82 for Z. montanum, and 55 for Z. zerumbet). The dendogram analysis generated from AFLP patterns showed that Z. montanum and Z. zerumbet are phylogenetically closer to each other than to Z. officinale. The AFLP fingerprints of the Zingiber species could be used to authenticate Zingiber sp-derived drugs and to resolve adulteration-related problems faced by the commercial users of these herbs.     

指纹与年龄相关性的量化分析

本文利用Leica M125体视显微镜对1510份样本中右手拇指的皱纹、乳突纹线密度、乳突纹线和小犁沟的宽度、细点线、屈肌褶纹等特征进行测量,通过SPSS软件分析年龄信息特征的相关性,选取其中相关性大的变量进行多元线性回归,并得出推断公式以分析并量化随年龄的增长指纹与年龄相关信息的变化特点及规律。研究结果显示,手印中的皱纹、乳突纹线密度、乳突纹线和小犁沟的宽度、细点线、屈肌褶纹等特征与年龄具有相关性,但利用这些变量构建的多元回归模型拟合优度并不高;皱纹、乳突纹线密度、乳突纹线和小犁沟的宽度、细点线、屈肌褶纹等可作为手印分析年龄的参考和辅助特征。乳突纹线的边缘形态、手印印痕的模糊程度、汗孔等以当前科技手段难以用测量描述特征的年龄信息,在手印分析年龄中或可发挥核心价值,具体还需进一步探究。     

Near Homogeneity of PR2-Bias Fingerprints in the Human Genome and Their Implications in Phylogenetic Analyses

Genes of a multicellular organism are heterogeneous in the G+C content, which is particularly true in the third codon position. The extent of deviation from intra-strand equality rule of A = T and G = C (Parity Rule 2, or PR2) is specific for individual amino acids and has been expressed as the PR2-bias fingerprint. Previous results suggested that the PR2-bias fingerprints tend to be similar among the genes of an organism, and the fingerprint of the organism is specific for different taxa, reflecting phylogenetic relationships of organisms. In this study, using coding sequences of a large number of human genes, we examined the intragenomic heterogeneity of their PR2-bias fingerprints in relation to the G+C content of the third codon position (P ₃ ). Result shows that the PR2-bias fingerprint is similar in the wide range of the G+C content at the third codon position (0.30–0.80). This range covers approximately 89% of the genes, and further analysis of the high G+C range (0.80–1.00), where genes with normal PR2-bias fingerprints and those with anomalous fingerprints are mixed, shows that the total of 95% of genes have the similar finger prints. The result indicates that the PR2-bias fingerprint is a unique property of an organism and represents the overall characteristics of the genome. Combined with the previous results that the evolutionary change of the PR2-bias fingerprint is a slow process, PR2-bias fingerprints may be used for the phylogenetic analyses to supplement and augment the conventional methods that use the differences of the sequences of orthologous proteins and nucleic acids. Potential advantages and disadvantages of the PR2-bias fingerprint analysis are discussed. Received: 21 December 2000 / Accepted: 16 February 2001