Authentication of Cassia seeds on the basis of two-wavelength HPLC fingerprinting with the use of chemometrics

High performance liquid chromatographic(HPLC) fingerprints of Cassia seed,a traditional Chinese medicine(TCM),were developed by means of the chromatograms at two wavelengths of 238 and 282 nm.Then,the two data sets were combined into one matrix.The application of principal component analysis(PCA) for this data matrix showed that the samples were clustered into four groups in accordance with the plant sources and preparation procedures.Furthermore,partial least squares(PLS),back propagation artificial neural...

Chemical analysis of the Hedysarum multijugum root by HPLC fingerprinting

The root of Hedysarum multijugum（RHM） is recorded as a folk herbal medicine in China and is sometimes used as a substitute for Hedysari Radix, which is a famous traditional Chinese medicine derived from the roots of Hedysarum polybotrys. In the present study, a sensible, reliable, and reproducible HPLC-DAD fingerprint analysis method for RHM was developed and then subsequently applied to analyze RHM samples from different origins. The chemical constituents of the RHM samples were generally consistent, although it was slightly affected by the local environment of the plant. In addition, the chemical constituency of RHM was shown to be significantly different from that of Hedysari Radix, suggesting that RHM is not suitable as a substitute for Hedysari Radix, at least from the chemical point of view.

HPLC fingerprinting and quantification of gentiopicroside and loganic acid in Gentianae Macrophyllae Radix crude drugs

HPLC fingerprinting and quantification of gentiopicroside（GPS） and loganic acid（LA） in Gentianae Macrophyllae Radix（GMR） crude drugs were developed in this study.The samples were separated on Zorbax SB-C_（18） column（250 mm×4.6 mm, 5μm） with a linear gradient of acetonitrile and 0.04%phosphoric acid.The HPLC flow rate was 1.0 mL/min and a UV absorption was measured at 230 nm.An orthogonal L9（3^4） test was applied for the optimization of sample extraction conditions,and an aliquot of GMR sample（g） was extracted with 15-fold of 50%ethanol（mL） for 30 min by sonication.Quantitative analysis showed that the content of GPS（14.05 mg/g-74.61 mg/g） in all samples was obviously higher than that of LA（1.13 mg/g-40.46 mg/g）. Based on the content ratio of GPS over LA（1.8-11.4）,samples originated from Gentiana macrophylla（with content ratio of GPS over LA≤4.3） could be distinguished from those from G.dahurica and G.dahurica var.gracilipes（with content ratio of GPS over LA≥4.8）.The principle components analysis of the HPLC fingerprints showed that samples originated from G.macrophylla and G.dahurica（including G.dahurica var.gracilipes） could be divided into two groups.This established HPLC-DAD method could be efficiently used for the species identification and quality control of GMR crude drugs.

HPLC fingerprint of Nauclea officinalis stems in different harvesting period

Objective:To establish HPLC fingerprint of Nauclea officinalis stems in different harvesting periods,and analyze the effect of harvest time on the quality of the medicinal materials by combining with chemical pattern recognition.Methods:The analysis was performed on Sun Fire-C18(150 mm×4.6 mm,5μm)column.The mobile phase consisted of acetonitrile-0.1%phosphoric acid solution(gradient elution)at a flow rate of 1.0 mL/min.Results:The HPLC fingerprint of Nauclea officinalis stems was established and 12 common peaks were determined,and 3 chromatographic peaks were identified by comparison with the mixed references.There were some differences in the quality of Nauclea officinalis in different harvesting periods.The OPLS-DA analysis successfully predicted four main markers of quality difference.Conclusion:The established HPLC fingerprint could reflect the composition characteristics of Nauclea officinalis stems in different harvesting period,and the main markers that influence the composition difference of the stems could be used as key indicators for the quality control.

Correlation analysis between the HPLC fingerprints and in vitro antibacterial activity of ethyl acetate extracts from Radix isatidis

Aim To study the correlation between the HPLC fingerprints and in vitro antibacterial activities of EtOAc extracts of Radix isatidis from various sources. Methods Ten batches of Radix isatidis EtOAc extracts were analyzed with HPLC and the fingerprints were established. The influence of EtOAc extracts on the thermogenic curve of growth of Escherchia coli was obtained by microcalorimetry. The chemical differences of EtOAc extracts of Radix isatidis from various sources in the HPLC fingerprints were probed with hierarchical clustering analysis and similarity analysis. The correlation between the HPLC fingerprints and in vitro antibacterial activities was analyzed with multivafiant correlation analysis. Results Close correlation existed between the HPLC fingerprints and in vitro antibacterial activities of EtOAc extracts of Radix isatidis. Conlusion The combination of HPLC fingerprints and antibacterial activities can be used to discover principle components of Radix isatidis on bioactivity.

Nutritional Compositions of Maca Grown in Ebian and HPLC Fingerprints of Macaene and Macamide

In the present study, the nutritional compositions of maca which was grown in a mountain area at an elevation of 2 200-2 800 m of Ebian County,Sichuan Province were measured, and then HPLC analysis on two representative active compounds（macaene and macamide） in the maca sample was performed.The results revealed that there were 24.20% total protein, 18.40% total amino acids（including 3.84% arginine）, 42.80% total sugars, 1.36% fat and kinds of minerals（including 1.14% potassium） in Ebian maca. HPLC fingerprints of macaene and macamide of Ebian maca were similar to those of Peru maca. The results suggested that maca could be cultivated with good quality in some mountain areas with an altitude1 000 m lower than the origin place in Peru.

Fingerprint analysis of different Panax herbal species by HPLC-UV method

Aim To establish a method for differentiating commercial samples of Panax species including notoginseng, cultivated ginseng （Chinese ginseng and Korean ginseng）, wild ginseng, red ginseng, three types of American ginsengs, and one American ginseng preparation with their HPLC fingerprints for assnrning the quality of different commercial samples of Panax species. Methods HPLC-UV method was used to establish their fingerprints, Zorbax Extend C18 （250 mm×4.6 mm, 5 μm） was used as the analytical column, and acetonitrile/KH2PO4 aqueous solution was used as the mobile phase with gradient elution. Results The fingerprints of different commercial samples of Panax species varied in their holistic chromatograms and some specific constituents. Conclusion This method is reliable, reproducible and simple, It could be applied in the routine authentication of different commercial samples of Panax species

HPLC Fingerprint with Multi-components Analysis for Quality Consistency Evaluation of Traditional Chinese Medicine Si-Mo-Tang Oral Liquid Preparation

Si-Mo-Tang（SMT） oral liquid preparation, a traditional Chinese medicine, was prepared from four crude herbal drugs, Fructus Aurantii Submaturus, Radix Aucklandiae, Semen Arecae and Radix Linderae Aggregatae. A combinative method using HPLC fingerprint and quantitative analysis was developed and validated for quality consistency evaluation of SMT. Individual HPLC chromatograms were evaluated against the mean chromatogram generated via a similarity evaluation computer program. Data from chromatographic fingerprints were also processed with principal component analysis（PCA） and hierarchical cluster analysis（HCA）. Additionally, six components （naringin, isonaringin, hesperidin, neohesperidin, norisoboldine and potassium sorbate） in SMT were simultaneously determined to interpret the quality consistency. For fingerprint analysis, 20 peaks were selected as the characteristic peaks to evaluate the similarities of 26 SMT collected from different manufacturers. Among the 20 characteristic peaks, 10 peaks were assigned to be naringin, hesperidin, neohesperidin, isonaringin, neoeriocitrin, tangeretin, nobiletin, norisoboldine, 5-（ethoxymethyl）furan-2-carbaldehyde and potassium sorbate, respectively. The results of similarity analysis, PCA and HCA, indicate that the samples from different manufacturers were consistent with each other in composition. The results from the quantitative data show that the contents of six compounds were significantly different in SMT oral liquid preparations from different manufacturers. The combinative method of chromatographic fingerprint with quantitative analysis developed here offered an efficient way for the quality consistency evaluation of the traditional Chinese medicine SMT.

HPLC/MS Fingerprint Analysis of Tangshenosides

Radix codonopsis(党参)，which is derived fromCodonopsis pilosula, C.pilosula subsp. Modesta andC. Tangshen(ChP,2000),has been used as a remedy fora decrease of appetite, psychoneurosis, fatigue, dyspepsiaetc.or as a substitute for Panax ginseng in traditionalChinese medicine. Relating to the constituents of theseherbs, triterpenes, phytosterols, furaldehyde, sesquitesand some phenolic glycosides have been reported, butnone of these compounds seems to be responsible for thebiological activities of this drug. As regards the quality evaluation of Radix Codonopsis, a HPLC analysis of atractylenolide Ⅲ has been described, but this sesquiterpene lactone exists in a very low content and is difficultfor conventional analysis. The determination of polysaccharide also met limitation because of its non-specialization. In this paper, we report the HPLC/MS fingerprintsanalysis of phelolic glycosides from Radix Codonopsis including three species.

Optimization of high pressure machine decocting process for Dachengqi Tang using HPLC fingerprints combined with the Box–Behnken experimental design

Using Dachengqi Tang（DCQT） as a model, high performance liquid chromatography（HPLC）fingerprints were applied to optimize machine extracting process with the Box–Behnken experimental design. HPLC fingerprints were carried out to investigate the chemical ingredients of DCQT; synthetic weighing method based on analytic hierarchy process（AHP） and criteria importance through intercriteria correlation（CRITIC） was performed to calculate synthetic scores of fingerprints; using the mark ingredients contents and synthetic scores as indicators, the Box–Behnken design was carried out to optimize the process parameters of machine decocting process under high pressure for DCQT. Results of optimal process showed that the herb materials were soaked for 45 min and extracted with 9 folds volume of water in the decocting machine under the temperature of 140 1C till the pressure arrived at 0.25 MPa;then hot decoction was excreted to soak Dahuang and Mangxiao for 5 min. Finally, obtained solutions were mixed, filtrated and packed. It concluded that HPLC fingerprints combined with the Box–Behnken experimental design could be used to optimize extracting process of traditional Chinese medicine（TCM）.

An HPLC Fingerprint Identification of Dried Barks of Ilex rotunda and Ilex godajam

[Objectives] A simple and reliable HPLC fingerprint method was developed for the identification of dried barks of Ilex rotunda and I. godajam. [Methods] Nine batches of dried barks of I. rotunda,and seven batches of dried barks of I. godajam collected from different pharmacies and arboretums in different regions of China were used to establish fingerprints. The software Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine( 2004 A Edition) was used to evaluate the fingerprints. [Results]The fingerprints of dried barks of I. rotunda and I. godajam were established. Methodological study met the technical requirements of fingerprints. The similarities of the fingerprints of dried barks of I. rotunda and I. godajam were all more than 0. 8 and 0. 9 respectively. There were 31 and 28 common peaks in I. rotunda and I. godajam,which could be classified into two clusters by principal component analysis( PCA) and hierarchical cluster analysis. [Conclusions] The feasibility and advantages of used HPLC fingerprints were verified,and the results indicated that the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation,principal component analysis and hierarchical cluster analysis can be successfully used to identify the authenticity of dried barks of I. rotunda and I. godajam.

The fingerprint and flavonoids contents by HPLC and the UPLC-DAD-Q-TOF-MS analysis of Sedum aizoon L.

Fingerprint analysis of Sedum aizoon L.was carried by High-performance liquid chromatography.There were 27 common peaks in the fingerprint of 10 samples.Eight of these common peaks,peaks 1(arbutin),4(gallic acid),10(myricetin-3-O-β-D-glucopyranoside),14(myricetin-3-O-α-L-rhamnopyranoside),19(quercetin-3-O-α-L-rhamnopyranoside),22(kaempferol-3-O-α-L-rhamnopyranoside),24(quercetin)and 26(kaempferol)were identified by reference substances.Moreover,six flavonoid compounds were simultaneously determined by the above method.By comparing with the constituents in the aerial parts,roots and flowers,it’s easy found that the aerial parts of S.aizoon had abundant flavonoids and the flavonoid aglycones mostly exist in the roots,while the flavonoid glycosides largely existing in the flowers.In addition,an UPLC-DAD-Q-TOF-MS method was developed for qualitative analysis of the aerial parts of S.aizoon.This study may provide a preliminary reference for the quality control of S.aizoon.

Anti--inflammatory effect of ethyl acetate extract of Sedum aizoon L. in LPS-stimulated RAW 264.7 macrophages and its HPLC fingerprint

Sedum aizoon L.（SA） has been widely used for treatment of various hemorrhages, insomnia, pain and trauma？ however, its anti-inflammatory activity is unknown. In this study, we firstly investigated the anti--inflammatory activity of SA extracts by petroleum ether（PE）, ethyl acetate（EtOAc）, and H2O in LPS-stimulated RAW 264.7 cells. Results showed that the EtOAc extract rich in phenolics and flavonoids significantly inhibited LPS-induced NO, TNF--α, and IL-6 production in RAW 264.7 cells（P0.01）, suggesting that this extract possessed potent anti--inflammatory activity. The phytochemical profile of the effective extract was subsequently analyzed by HPLC fingerprint with 11 standards. The results indicated that gallic acid, protocatechuic acid, p--hydroxybenzoic acid, ethyl gallate, iriflophene, 5,7-dihydroxy chromone, quercitrin, quercetin, luteolin, kaempferol, and isorhamnetin were present in this extract, which might contribute to its anti-inflammatory activity. Thus, the Et -OAc extract of SA showed anti-inflammatory activity and could be used as a potential natural anti--inflammatory agent.

HPLC fingerprint-oriented preparative separation of major flavonoids from safflower extract by preparative pressurized liquid chromatography

The aim of this study was to rapidly isolate the major effective flavanoids from the extract of safflower（Carthamus tinctorius） using ODS medium pressure liquid chromatography（MPLC） and semi-preparative HPLC, guided by a developed fingerprint. Twelve compounds were isolated and their structures were elucidated as kaempferol 3-O-β-D-rutinoside（1）, kaempferol 3-O-β-D-glucoside（2）, rutin（3）, quercetin 3-O-β-D-glucoside（4）, 6-hydroxykaempferol 3,6,7-tri-O-β-D-glucoside（5）, 6-hydroxykaempferol 3-O-β-D-glucoside（6）, 6-hydroxykaempferol 6,7-di-O-β-D-glucoside（7）, 6-hydroxykaempferol 3-O-β-Drutinoside（8）, 6-hydroxykaempferol 3,6-di-O-β-D-glucosyl 7-O-β-D-glucuronide（9）, isosafflomin C（10）, safflomin C（11） and hydroxysafflor yellow A（12） by spectroscopic analysis and comparing with the literature. Our results demonstrated that preparative pressurized liquid chromatography combined with HPLC fingerprint guide is an efficient tool to isolate the target compounds quickly.

Identification and discrimination of Ziziphi Spinosae Semen and Ziziphi Mauritianae Semen based on HPLC fingerprint analysis

A simple, reproducible and reliable HPLC fingerprint analysis method of Ziziphi Spinosae Semen(ZSS) and Ziziphi Mauritianae Semen(ZMS) was established, and then it was applied to analyze samples collected from different locations. A total of 12 common fingerprint peaks were designated in ZSS and ZMS, respectively, and five of which were definitely identified. Of these common peaks, 10 peaks were presented in both ZSS and ZMS, whereas two were characteristic for ZSS and ZMS, respectively. Furthermore, the two characteristic peaks of ZMS were definitely assigned as 6′′′-sinapoylspinosin and frangufoline by HPLC guided purification and structural elucidation. The HPLC fingerprint analysis was proved to be useful in identifying and discriminating ZSS and ZMS, which was beneficial for quality control of ZSS and its products.

HPLC fingerprints and quality assessment of Rhizoma Coptidis from different areas

HPLC fingerprinting and determination methods were applied to evaluate 88 batches of Rhizoma Coptidis from 27 different areas. The results showed that HPLC fingerprint of Rhizoma Coptidis was established with good separation and repeatability, which could be used for quality assessment of Rhizoma Coptidis. Twelve common peaks were defined in characteristic fingerprints, and similarity evaluation system was applied to evaluate the fingerprints of 88 batches of Rhizoma Coptidis, the contents of six alkaloids have been determined. The method of assessing Rhizoma Coptidis and evaluation of its quality has been established.

Chemical Characteristics Combined with Bioactivity for Comprehensive Evaluation of Tumuxiang Based on HPLC-DAD and Multivariate Statistical Methods

Background: The dried roots of Inula helenium L.(IH) and Inula racemosa Hook f.(IR) are used commonly as folk medicine under the name of "tumuxiang(TMX)". Phenolic acid compounds and their derivatives, as main active constituents in IH and IR, exhibit prominent anti-inflammation effect.Objective: To develop a holistic method based on chemical characteristic and anti-inflammation effect for systematically evaluating the quality of twenty-seven TMX samples(including 18 IH samples and 9 IR samples) from different origins.Methods: HPLC fingerprints data of AL(Aucklandia lappa Decne.) whose dried root was similar with HR was added for classification analysis. The HPLC fingerprints of twenty-seven TMX samples and four AL samples were evaluated using hierarchical clustering analysis(HCA) and principle component analysis(PCA). The spectrum-efficacy model between HPLC fingerprints and anti-inflammatory activities was investigated by principal component regression(PCR) and partial least squares(PLS).Results: All samples were successfully divided into three main clusters and peaks 7, 9, 11, 22, 24 and 26 had a primary contribution to classify these medicinal herbs. The results were in accord with the appraisal results of herbs. The spectrum-efficacy relationship results indicated that citric acid, quinic acid, caffeic acid-β-D-glucopyranoside, chlorogenic acid, caffeic acid, 1,3-O-dicaffeoyl quinic acid, tianshic acid and 3β-Hydroxypterondontic acid had main contribution to anti-inflammatory activities.Conclusion: This comprehensive strategy was successfully used for identification of IH, IR and AL, which provided a reliable and adequate theoretical basis for the bioactivity relevant quality standards and studying the material basis of anti-inflammatory effect of TMX.

Quality evaluation of Farfarae Flos by ITS/ITS2 and HPLC fingerprint analysis

The flower bud of Tussilago farfara L.,also known as Farfarae Flos(FF),is commonly used in the traditional Chinese medicine(TCM)for the treatment of cough,bronchitis and asthmatic disorders.In this study,26 samples from five provinces across northern China were collected,and internal transcribed spacer-polymerase chain reaction(ITS-PCR)coupled with high performance liquid chromatography(HPLC)fingerprint profiling was used for the quality evaluation of FF.The results of the ITS sequence analysis showed the high similarity values among these samples.ITS2 sequence exhibited less molecular diversity compared with ITS1,and no obvious correlation was found between the variation of ITS with the production areas or cultivation methods.The results of HPLC fingerprints in combination with the similarity analysis suggested that relative contents of phenylpropanoids and flavonoids varied among the 26 FF samples,and there was also no obvious difference among the different habitats.The phenylpropanoids and the flavonoids showed similar fluctuating patterns,and positive correlation among them was also observed.The results presented in this study suggested that FF from different habits showed high similarities,and ITS-PCR coupled with HPLC fingerprint profiling could be used as a valuable approach for the quality evaluation of FF.

Comparative chemical characters of Pseudostellaria heterophylla from geographical origins of China

Objective:Pseudostellaria heterophylla has been paid more attention in recent years,mainly as a medicine food homology plant.The content determination of P.heterophylla is not specified in the Chinese Pharmacopoeia(version 2020).The environmental conditions in different production areas could exert an influence on the quality of P.heterophylla.The purpose of this study is to discriminate P.heterophylla collected from different geographical origins of China.Methods:In this study,the content of polysaccharide in 28 batches of P.heterophylla was determined using phenol–sulfuric acid.HPLC fingerprints were established under optimised HPLC-PDA methods.Subsequently,the similarity analysis(SA)and the quantification of heterophyllin B were analyzed.The metabolites of P.heterophylla were identified and evaluated using UHPLC-Q Exactive HF orbitrap MS system.Principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA),hierarchical cluster analysis(HCA)and orthogonal PLS-DA(OPLS-DA)were performed based on all peak areas.Results:The polysaccharide content in Guizhou and Jiangsu was higher than that of other production areas,which varied significant from different origins.While the content of heterophyllin B in Anhui and Jiangsu was high.The correlation coefficients of HPLC fingerprints for 28 batches samples ranged from 0.877 to 0.990,and the characteristic map can be used to identify and evaluate the quality of P.heterophylla.The samples from Fujian,Guizhou,Jiangsu provinces can be relatively separated using multivariate statistical analysis including PCA,PLS-DA,HCA,OPLS-DA,indicating that their metabolic compositions were significantly different.Ultimately,a total of 15 metabolites which were filtrated by a VIPvalue>1 and a P-value<0.05 associated with the separation of different origins were identified.Conclusion:HPLC fingerprint was established to evaluate the quality and authenticity of P.heterophylla.The present work showed that the difference of geographic distributions had an influence on the internal chemical compositions.A sensitive and rapid untargeted metabolomics approach by UHPLC-Q Exactive HF orbitrap MS was utilized to evaluate P.heterophylla from different origins in China for the first time.Overall,this study provides insights to metabolomics of P.heterophylla and supplies important reference values for the development of functional foods.

Development of the fingerprints of crude Pu-erh tea and ripened Pu-erh tea by high-performance liquid chromatography

A simple and accurate high-performance liquid chromatography（HPLC）method to generate the fingerprints of crude Pu-erh tea（CPT）and ripened Pu-erh tea（RPT）is described.A suitable chromatographic system was established using a linear gradient elution with acetonitrile and water containing 0.6% formic acid as the mobile phase and a detection wavelength of 300 nm.HPLC analysis identified 24 common peaks among RPT and 21 common peaks among CPT.This study revealed that crude Pu-erh tea and ripened Pu-erh tea contained some similar major constituents,but with distinctive peaks,which may be caused by the difference in the fermentation process.This HPLC fingerprint method could be used to evaluate and authenticate crude Pu-erh tea and ripened Pu-erh tea.