Markers For Quality Control Research Articles

Integrated and global pseudotargeted metabolomics strategy applied to screening for quality control markers of Citrus TCMs.

Traditional Chinese medicine (TCM) exerts its therapeutic effect in a holistic fashion with the synergistic function of multiple characteristic constituents. The holism philosophy of TCM is coincident with global and systematic theories of metabolomics. The proposed pseudotargeted metabolomics methodologies were employed for the establishment of reliable quality control markers for use in the screening strategy of TCMs. Pseudotargeted metabolomics integrates the advantages of both targeted and untargeted methods. In the present study, targeted metabolomics equipped with the gold standard RRLC-QqQ-MS method was employed for in vivo quantitative plasma pharmacochemistry study of characteristic prototypic constituents. Meanwhile, untargeted metabolomics using UHPLC-QE Orbitrap HRMS with better specificity and selectivity was employed for identification of untargeted metabolites in the complex plasma matrix. In all, 32 prototypic metabolites were quantitatively determined, and 66 biotransformed metabolites were convincingly identified after being orally administered with standard extracts of four labeled Citrus TCMs. The global absorption and metabolism process of complex TCMs was depicted in a systematic manner.

Ultra-performance convergence chromatography for the quantitative determination of bioactive compounds in Aralia continentalis Kitagawa as quality control markers.

A rapid ultra-performance convergence chromatography method was developed for the quantitative determination of bioactive compounds in Aralia continentalis as quality control markers. Quantitative analysis indicated the presence of two major bioactive compounds: diterpenoid acids continentalic acid and kaurenoic acid. Using a Torus 1-aminoanthracene column, continentalic acid and kaurenoic acid were separated in less than 8min. The method was validated with respect to precision, accuracy, and linearity according to the International Conference on Harmonization guidelines. The optimized method exhibited a good linear correlation (r2 > 0.996), excellent precision (RSD<1.0%), and acceptable recoveries (99.97-100.26%). Limits of detection for continentalic acid and kaurenoic acid were 0.068and 0.097μg/mL, respectively, while their corresponding limits of quantitation were 0.207 and 0.295μg/mL. The system performance of ultra-performance convergence chromatography was compared with that of conventional high-performance liquid chromatography with respect to analysis time and efficiency. The proposed method was found to be reliable and convenient for the quantitative analysis of continentalic acid and kaurenoic acid in A. continentalis from South Korea and A. pubescens from China. This study is expected to serve as a guideline for the quality control of Aralia continentalis.

Characterization of antioxidants from Detarium microcarpum Guill. et Perr. leaves using HPLC-DAD coupled with pre-column DPPH assay

Detarium microcarpum is an African tree which is widely used as a folk medicine and food. A methanol extract from the leaves as well as its hexane, dichloromethane, ethyl acetate and n-butanol fractions were evaluated for their total phenolic content and radical scavenging activities. Additionally, a pre-column DPPH-HPLC assay was performed to screen and identify the compounds responsible for the antioxidant properties of the extract. Results indicated that the methanol extract contained a substantial amount of phenolic compounds with a value of 51.7 ± 0.2 mg gallic acid equivalent/g. It also exerted potent radical scavenging activities in both DPPH and ORAC assays, with respective values of 937 ± 16 and 2247 ± 63 μmol trolox equivalent/g. Its ethyl acetate and n-butanol fractions were shown to possess the highest TPC and radical scavenging activities. Pre-column DPPH-HPLC experiments led to the direct detection of five potential radical scavenging compounds which were identified as gallic acid, quercetin 3,7-O-dirhamnoside, myricetin 3-O-rhamnoside, quercetin 3-O-glucoside and quercetin 3-O-rhamnoside. All of them were confirmed to exert potent radical scavenging activities using common DPPH spectrophotometric assay. These findings revealed that D. microcarpum leaves possess potent antioxidant activity and could be recommended as a nutraceutical resource. Additionally, the identified bioactive compounds can be considered as appropriate markers for quality control.

Efficient Discovery of Quality Control Markers for Gastrodia elata Tuber by Fingerprint‐Efficacy Relationship Modelling

Gastrodia elata tuber (GET) has been widely used in China as a famous herbal medicine. However, the quality control markers (QCMs) for GET still need further investigation. To develop a rational strategy based on fingerprint-efficacy relationship modelling to discover the efficacy-related QCMs, using GET as a case study. The high-performance liquid chromatography (HPLC) fingerprints of 13 batches of GET extracts were established and 10 common peaks were structurally characterised by HPLC coupled with quadrupole time-of-flight mass spectrometry (QTOF-MS). The neuroprotective effects of GET samples were evaluated using the in vitro model of β-amyloid peptide (Aβ25-35 )-induced PC12 cell death. The fingerprint-efficacy relationship of chemical fingerprints and neuroprotective effects was linked by orthogonal projection to latent structure-discriminant analysis (OPLS-DA) model. The chemical combination of 5-hydroxymethyl-2-furaldehyde (5-HMF), parishin B (PB) and parishin C (PC) was discovered and confirmed as QCMs of GET by the OPLS-DA method. The selected QCMs allowed to evaluate the relative quality of GET samples. This study has demonstrated that the rational strategy based on fingerprint-efficacy relationship modelling might be generally applicable for the quality control of herbal medicines. Copyright © 2017 John Wiley & Sons, Ltd.

Artepillin C, is it a good marker for quality control of Brazilian green propolis?

As commercialisation of Brazilian green propolis is going on, quality evaluation and authenticity are important. The result demonstrated that artepillin C found by far in Brazilian green propolis by HPLC-ESI-MS/MS analysis, while a small interferent may be mistaken as artepillin C in some propolis from China. A new HPLC quality control method as artepillin C for marker was developed, which is the primary assessment criteria for quality control of Brazilian green propolis.

Discovery of discriminatory quality control markers for Chinese herbal medicines and related processed products by combination of chromatographic analysis and chemometrics methods: Radix Scutellariae as a case study

The processing procedure of traditional Chinese herbal medicines (CHMs) plays an essential role in clinical applications. However, little progress has been made on the quality control of crude and processed products. The present work, taking Radix Scutellariae (RS), wine-processed RS and carbonized RS as a typical case, developed a comprehensive strategy integrating chromatographic analysis and chemometric methods for quality evaluation and discrimination of crude RS and its processed products. Chemical fingerprints were established by high-performance liquid chromatography coupled with photodiode array detector and quadrupole time-of-flight mass spectrometry, and similarity analyses were calculated based on eleven common characteristic peaks. Subsequently, four chemical markers were discovered by back propagation-artificial neural network (BP-ANN) modeling. The selected markers were quantified by the ‘single standard to determine multi-components’ (SSDMC) method, and then the quantitative data were subjected to principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Furthermore, support vector machine (SVM) was employed to predict the different processed products of RS. Finally, a hotmap visualization was conducted for clarifying the distribution of major flavonoids among different drugs. Collectively, the proposed strategy might be well-acceptable for quality control of CHMs and their related processed products from the processing mechanism-based perspective.

Efficient Preparation of Ephedrine Alkaloids-free Ephedra Herb Extract and Its Antitumor Effect and Putative Marker Compound.

Ephedra Herb is a crude drug for the treatment of headache, bronchial asthma, nasal inflammation, and the common cold. Although it has been considered that ephedrine alkaloids (EAs) are the principal active ingredients of Ephedra Herb, EAs are known to induce palpitations, hypertension, insomnia, and dysuria as major side effects. Therefore, the administration of EAs-containing drugs to patients with cardiovascular-related diseases is strongly contraindicated. Previously, we isolated herbacetin 7-O-neohesperidoside from Ephedra Herb. In addition, we found that herbacetin, a flavonoid aglycone in Ephedra Herb, had antiproliferative and analgesic effects. Therefore, the prospect of preparing safer natural medicines without the adverse effects associated with EAs was appealing. In this symposium review, to achieve the aim of producting a clinically useful Ephedra Herb extract with none of the adverse effects associated with EAs, I present an efficient preparation method of EAs-free Ephedra Herb extract, together with its chemical composition, antiproliferative effects, and a putative marker for quality control.

Multiple Activities of Punica granatum Linne against Acne Vulgaris.

Acne is a common skin condition with sebum overproduction, hyperkeratosis, Propionibacterium acnes (P. acnes) and Staphylococcus aureus, and inflammation. Punica granatum (pomegranate) is well-known for its anti-inflammatory effects; however, few studies have discussed the anti-acne effects of pomegranate. In this study, we found that pomegranate extract (PG-E) significantly reduced P. acnes-induced edema in Wistar rat ears. Therefore, an evaluation platform using multiple pathogenic mechanisms of acne was established to explore the anti-acne effects of pomegranate. Results showed that PG-E inhibited bacterial growth and lipase activity. Through a bioguided-fractionation-isolation system, four hydrolysable tannins, punicalagin (1), punicalin (2), strictinin A (3), and granatin B (4), were isolated. Compounds 1 and 2 had greater anti-bacterial activities and anti-testosterone-induced HaCaT proliferative effects than the others. Compounds 1, 3, and 4 displayed lipase inhibitory effects. Compound 4 decreased cyclooxygenase-2 expression and downregulated prostaglandin E2 production in heat-killed P. acnes-treated RAW 246.7 cells. In conclusion, PG-E is abundant in hydrolysable tannins that display multiple anti-acne capacities, including anti-bacterial, anti-lipase, anti-keratinocyte proliferation, and anti-inflammatory actions. Hence, PG-E has great potential in the application of anti-acne and skin-care products, and punicalagin (1), the most effective component in PG-E, can be employed as a quality control marker.

Rapid determination of 30 bioactive constituents in XueBiJing injection using ultra high performance liquid chromatography-high resolution hybrid quadrupole-orbitrap mass spectrometry coupled with principal component analysis

Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. Extensive chemical studies revealed that XBJ injection contains amino acids, phenolic acids, flavonoid glycosides, terpeneglycosides and phthalides. In this study, the applicability of ultra high performance liquid chromatography coupled with high resolution hybrid quadruple-orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) for the simultaneous quantitative analysis of 30 bioactive constituents in XueBiJing injection (XBJ) was investigated. The mass spectrometer was operated in full MS scan mode. The use of 70,000FWHM mass resolution and narrow mass windows (5ppm) could effectively improve the selectivity of the method. Separation was achieved on a Waters ACQUITY UPLC® HSS C18 column (2.1mm×100mm, 1.8μm) with a gradient mobile phase consisting of acetonitrile-water (containing 10mM ammonium acetate) at a flow rate of 0.2mL/min. Satisfactory linearity was achieved within wide linear range and all correlation coefficients (r) of analytes were more than 0.9996. The limits of detection (LODs) were in the range of 0.1180–27.82ng/mL for different analytes. The relative standard deviations (RSDs) of inter- and intra-day precisions were less than 3.0% and the recoveries of the assay were in the range of 98.5%–101.5%. The validated method was successfully applied for simultaneous determination of 30 bioactive compounds in XueBiJing injection from 10 batches samples by UHPLC-Q-Orbitrap MS within 10min. Moreover, the results were evaluated principal component analysis and two compounds might be the most important chemical markers for chemical quality control of XBJ injection. The novel Q-Orbitrap mass spectrometry has been proved to be a very promising and powerful tool for routine screening of bioactive compounds in traditional Chinese medicine injection, ensuring drug safety and public health.

Quality Evaluation of Lepidium meyenii (Maca) Based on HPLC and LC-MS Analysis of its Glucosinolates from Roots

In order to evaluate the quality of maca samples, 15 batches of maca hypocotyls from different geographical origins were analyzed by using HPLC and liquid chromatography tandem mass spectrometry (LC-MS). Their glucosinolates (GLs) were identified, profiled, and quantified. Three aromatic GLs, glucosinalbin (GSB), glucotropaeolin (GTL), and glucolimnanthin (GLH), were identified from maca roots. It was found that the HPLC profiles of maca samples showed a similar qualitative pattern except for some differences in their contents. The amount ranges (μmol g−1, DW) of GSB, GTL, and GLH were 2.62–5.55, 12.99–57.12, and 3.15–12.12, respectively. Furthermore, a HCA (hierarchical clustering analysis) was used to classify the quality of maca samples according to their cultivated sources and GLs contents. The results showed that the combination of GSB, GTL, and GLH could be a marker for accurate determination and quality control of maca samples. It was concluded that the multi-component analysis in combination with HCA could be a supplement to assess the quality of maca materials.

Traditional Uses, Chemical Constituents and Biological Activities of Plants from the Genus Sanguisorba L.

Plants from the genus Sanguisorba have been treated as medicinal ingredients for over 2000 years. This paper reviews advances in the botanical, phytochemical and pharmacological studies of the genus. To date, more than 120 chemical constituents have been isolated and identified from these plants, especially from S. officinalis and S. minor. Among these compounds, triterpenoids, phenols and flavonoids are the primary biologically active constituents. Triterpenoids can be used as quality control markers to determine the quality of medicinal materials and their preparations. In vivo and in vitro studies have shown that plants from the genus Sanguisorba exhibit a wide range of pharmacological properties, including hemostatic, antibacterial, antitumor, neuroprotective and hypoglycemic activities. In Chinese medical practice, many drugs (e.g., tablets and powders) that contain S. officinalis roots have been used to treat leukopenia, hemorrhaging and burns. However, there is still a multitude of Sanguisorba species that have garnered little or no attention. Indeed, there are few reports concerning the clinical use and toxic effects of these plants. Further attention should be focused on the study of these species in order to gather information on their respective toxicology data, any relevant quality-control measures, and the clinical value of the crude extracts, active compounds, and bioactive metabolites from Genus Sanguisorba.

Prediction of Radix Astragali Immunomodulatory Effect of CD80 Expression from Chromatograms by Quantitative Pattern-Activity Relationship.

The current use of a single chemical component as the representative quality control marker of herbal food supplement is inadequate. In this CD80-Quantitative-Pattern-Activity-Relationship (QPAR) study, we built a bioactivity predictive model that can be applicable for complex mixtures. Through integrating the chemical fingerprinting profiles of the immunomodulating herb Radix Astragali (RA) extracts, and their related biological data of immunological marker CD80 expression on dendritic cells, a chemometric model using the Elastic Net Partial Least Square (EN-PLS) algorithm was established. The EN-PLS algorithm increased the biological predictive capability with lower value of RMSEP (11.66) and higher values of Rp2 (0.55) when compared to the standard PLS model. This CD80-QPAR platform provides a useful predictive model for unknown RA extract's bioactivities using the chemical fingerprint inputs. Furthermore, this bioactivity prediction platform facilitates identification of key bioactivity-related chemical components within complex mixtures for future drug discovery and understanding of the batch-to-batch consistency for quality clinical trials.

Development of specific and quantitative methods for the quality control of the polysaccharides from sea-tangle and sargassum

Polysaccharides from numerous traditional Chinese medicines have been proven as the bioactive ingredients and are hence used as the quality control markers. However, the assessment criteria always show a poor specificity, due to the lack of systematic comparison among the analogous herbs. In the present study, two similar materials, namely sea-tangle and sargassum, were selected as the model herbs to develop more specific methods for quality control. Two well-established methods, determination of the total polysaccharides content and monosaccharides composition analysis, were both employed. Based upon the quantitative results, the evaluation criteria of the polysaccharides contents of not less than 2.0% and 1.7% were proposed for sea-tangle and sargassum, respectively. Nine identical monosaccharide derivatives appeared on the HPLC chromatograms of the hydrolysis and derivatized solutions of the two drugs. Principal component analysis and orthogonal partial least squares discriminant analysis using the peak areas of monosaccharides derivatives as the variables were performed, and the results indicated that mannuronic acid and xylose with the opposite concentrations in the two drugs were the differential components. A discriminative criterion using the peak area ratio of these two monosaccharides derivatives was proposed for the qualitative identification. In conclusion, a more specific and quantitative quality control method was developed for sea-tangle and sargassum.

Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting.

Phytoecdysteroids like 20-hydroxyecdysone (“ecdysterone”) can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly ‘guarantee’ product efficacy.

Spondias tuberosa (Anacardiaceae) leaves: profiling phenolic compounds by HPLC-DAD and LC-MS/MS and in vivo anti-inflammatory activity.

Spondias tuberosa is a medicinal plant used by several local communities in northeast Brazil to treat infections, digestive disorders and inflammatory conditions. The study aimed to identify and quantify the major phenolic in hydroethanolic extract of leaves from S. tuberosa and to evaluate its anti-inflammatory potential. The chemical profile of extract was analyzed by HPLC-DAD and HPLC-MS. The in vivo anti-inflammatory activity was investigated in carrageenan-induced hind paw edema and peritonitis models in mice. Identified and quantified through HPLC-DAD or HPLC-MS analyses of S. tuberosa extract were the following compounds: chlorogenic acid, caffeic acid, rutin and isoquercitrin. The inflammatory response to carrageenan was significantly reduced in both models by S. tuberosa extract. In hind paw edema, the edematogenic response was reduced by up to 63.6% and the myeloperoxidase activity was completely inhibited. In the peritonitis model, the total cell migration into the peritoneal cavity was reduced by up to 65%. The results obtained give evidence of the anti-inflammatory action of S. tuberosa and suggest the potential therapeutic benefit of this plant on inflammatory conditions. The chlorogenic acid, caffeic acid, rutin and isoquercitrin identified and quantified in S. tuberosa leaves enable us to suggest that these compounds could be used as chemical markers for quality control of derivative products from this species. Copyright © 2016 John Wiley & Sons, Ltd.

Developing an Absorption-Based Quality Control Method for Hu-Gan-Kang-Yuan Capsules by UFLC-QTOF-MS/MS Screening and HPLC-DAD Quantitative Determination.

Traditional Chinese Medicine Preparations (TCMPs) contain massive numbers of ingredients responsible for their multiple efficacies. An absorption–based quality control method for complicated TCMPs using Hu–gan–kang–yuan Capsule (HGKYC) as an example was developed. To select proper chemical markers for quality control of HGKYC, an ultra–fast liquid chromatography (UFLC) coupled with electrospray ionization quadrupole time–off light mass spectrometry (UFLC–QTOF–MS/MS) method was used for the rapid separation and structural identification of the constituents in the HGKYC extract and the rat serum after oral administration of HGKYC. As a result, one hundred and seven prototype constituents including flavonoids, organic acid, phenylpropanoids, anthraquinones, saponins, alkaloids, terpenes, phenols and amino acids in HGKYC extract, and 43 compounds found in rat serum after oral administration of HGKYC were unambiguously identified or tentatively characterized by comparing retention times and MS information with those of authentic standards or available literature references. Finally, a simple, low–cost and effective method of simultaneous determination for baicalein, wogonin, paeonol and emodin in HGKYC was developed using high performance liquid chromatography coupled with a diode array detector. In conclusion, an absorption–based quality control pattern was developed and successfully used for evaluating HGKYC.

Identification and quantification of the anti-inflammatory constituents in Pian-Tze-Huang by liquid chromatography combined with quadrupole time-of-flight and triple quadrupole mass spectrometry.

Both qualitative and quantitative analyses combined with activity evaluation were used for the integrated quality control study of Pien-Tze-Huang (PZH), a precious lozenge used for 460 years in China. In qualitative analysis, a high performance LC-quadrupole time-of-flight MS method was developed for the identification of chemical profiling in PZH. 27 compounds were unambiguously identified by comparing with reference substances, and 12 compounds were tentatively deduced based on their MS data and comparing with literatures. Then the in vitro anti-inflammatory activity of 27 compounds were evaluated, and 9 saponins, 11 bile acids, taurine and muscone exhibited significant inhibitory activities on TNF-α production with IC50 values ranging from 12.34 to 147.24μM. In quantitative analysis, 21 active compounds, selected as quality control markers, were simultaneously detected in 15 batches of PZH samples by ultra-performance LC-triple quadrupole MS. All the analytes were detected within 15min. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r(2)>0.9990). The respective relative standard deviations of intra- and inter-day precisions, repeatability, stability, and recovery were less than 4.32%, 4.94%, 4.78%, 4.87%, and 4.91%. This study established a high sensitive and efficient method for the integrated quality control of PZH, including qualitative and quantitative analyses combined with pharmacological activity evaluation.

Antioxidant power as a quality control marker for completeness of amotosalen and ultraviolet A photochemical treatments in platelet concentrates and plasma units.

Pathogen inactivation treatments such as INTERCEPT aim to make sure blood and blood-derived products are free of pathogens before using them for transfusion purposes. At present, there is no established quality control assay that assesses the completeness of the treatment. As INTERCEPT is a photochemical treatment known to generate reactive oxygen species we sought to use the antioxidant power (AOP) of the blood product as a marker of treatment execution. In this perspective, we evaluated an electrochemically based miniaturized system, the EDEL technology, for measuring the AOP in both platelet concentrates (PCs) and plasma. Aliquots were withdrawn from PCs or plasma units before and after INTERCEPT treatment and a few microliters were directly deposited into the EDEL sensor for the AOP measurement. The result is expressed in EDEL, an arbitrary unit (micromolar equivalent of ascorbic acid). The INTERCEPT treatment resulted in a significant decrease of the AOP. An AOP threshold of 66.5, 89.0, 59.8, and 131.5 EDEL was determined for apheresis PCs collected from female and male donors, buffy coat PCs, and plasma units, respectively. Below the threshold value, INTERCEPT treatment is considered to be executed. Additionally, we showed that the presence of the photosensitizer in combination with the ultraviolet A illumination is required to observe the AOP decrease. The measurement of the AOP of PCs and plasma units can be used to document the completeness of the INTERCEPT treatment.

LacaScore: a novel plasma sample quality control tool based on ascorbic acid and lactic acid levels.

IntroductionMetabolome analysis is complicated by the continuous dynamic changes of metabolites in vivo and ex vivo. One of the main challenges in metabolomics is the robustness and reproducibility of results, partially driven by pre-analytical variations.ObjectivesThe objective of this study was to analyse the impact of pre-centrifugation time and temperature, and to determine a quality control marker in plasma samples.MethodsPlasma metabolites were measured by gas chromatography-mass spectrometry (GC–MS) and analysed with the MetaboliteDetector software. The metabolites, which were the most labile to pre-analytical variations, were further measured by enzymatic assays. A score was calculated for their use as quality control markers.ResultsThe pre-centrifugation temperature was shown to be critical in the stability of plasma samples and had a significant impact on metabolite concentration profiles. In contrast, pre-centrifugation delay had only a minor impact. Based on the results of this study, whole blood should be kept on wet ice and centrifuged within maximum 3 h as a prerequisite for preparing EDTA plasma samples fit for the purpose of metabolome analysis.ConclusionsWe have established a novel blood sample quality control marker, the LacaScore, based on the ascorbic acid to lactic acid ratio in plasma, which can be used as an indicator of the blood pre-centrifugation conditions, and hence the suitability of the sample for metabolome analyses. This method can be applied in research institutes and biobanks, enabling assessment of the quality of their plasma sample collections.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-016-1038-1) contains supplementary material, which is available to authorized users.

Qualitative and quantitative analysis of chemical constituents of Centipeda minima by HPLC-QTOF-MS & HPLC-DAD

A high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) method in both positive and negative ion modes was established to investigate the major constituents in the ethanolic extract of Centipeda minima (EBSC). Twelve common components including flavones and their glycosides, phenolic and polyphenolic acids, and sesquiterpene lactone were identified in ten batches of samples based on comparison with the retention time and accurate mass of external standards (mass accuracy within 3ppm) or the fragmentation patterns of tandem MS. Meanwhile, a simple, accurate and reliable HPLC-DAD method was also developed to determine the content of 10 chemical markers simultaneously. Results obtained from method validations including linearity, accuracy and precision showed that this new method is reliable and robust. Isochlorogenic acid A and brevilin A were found to be the most abundant in the ethanol extract of EBSC and could be served as markers for quality control of EBSC.