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Introduction Bulbus Fritillaria unibracteata Hsiao et K.C. Hsia is an important traditional Chinese medicine, widely used for the treatment of coughs, phlegm and asthma for thousands of years. Due to an increasing demand in clinic practices, a variety of substitutes have appeared in the market, resulting in a big challenge in the differentiation of bulbus F. unibracteata and its substitutes. Aim To differentiate bulbus F. unibracteata and its substitutes (bulbus Fritillaria ussuriensis Maxim.) based on their main isosteroidal alkaloid contents, and to test the potentiality of chemometrics as a tool for discrimination. Materials and methods The nine isosteroidal alkaloids in 61 batches of Fritillariae bulbus were simultaneously quantitated by liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to classify the two kinds of Fritillariae bulbus. Results Quantitative analysis showed that there were differences in the content of the nine alkaloids between two kinds of Fritillariae bulbus. According to the content of nine isosteroidal alkaloids, bulbus of F. unibracteata and F. ussuriensis were successfully distinguished by PCA model. Among these isosteroidal alkaloids, verticine and verticinone may be used as potential chemical markers for the identification and differentiation between the two kinds of Fritillaria bulbus. Conclusion The LC-MS/MS method coupled with PCA would be a powerful strategy to differentiate bulbus F. unibracteata and substitute specimens for quality evaluation and control.

Introduction Glucosinolates (1-5) are important secondary metabolites found in Isatis indigotica roots. Due to their high hydrophilic and ionic nature, purified glucosinolates often contain salt impurities and moisture. Accurate assessment of their purities is important for glucosinolates being utilised as chemical markers. Objective To develop and validate quantitative proton (1 H) nuclear magnetic resonance (qHNMR) methods for purity assessments of aliphatic and indole glucosinolates (1-5). Method Several NMR parameters such as pulse program, relaxation time, and delay time were optimised. Three qHNMR methods were developed using gluconapin (3), neoglucobrassicin (4), and sinigrin (5) for method validation and with maleic acid as internal standard. Results The quantification was based on the integrated area ratios of an olefinic proton (H-4 for 1-3; H-6 for 4; and H-3 for 5) of the side chain from glucosinolates relative to the olefinic proton from the internal standard using deuterated water (D2 O) as the solvent. The qHNMR methods were successfully applied for purity assessments of four aliphatic glucosinolates (1-3 and 5: progoitrin, epiprogoitrin, gluconapin, and sinigrin), and an indole glucosinolate (4: neoglucobrassicin). Conclusion The purity of glucosinolates isolated from I. indigotica and commercial sinigrin was accurately assessed using the developed qHNMR method. The qHNMR provides a reliable and superior means to determine the purity of glucosinolates.

Introduction Angiogenesis is closely related to a variety of diseases, and therapies based on angiogenesis are intensely investigated. Studies have shown that the use of Gastrodiae Rhizoma (GR, Gastrodia elata) can benefit the treatment of ischemic cardiovascular diseases and atherosclerosis by stimulating angiogenesis. Objective This study tested the angiogenesis effects of a group of chemical markers isolated from GR. Material and methods Zebrafish model was used to evaluate angiogenesis by setting four groups: blank control group, model group, positive control group and treatment group (0.1, 1, and 100 μg/mL RGP). The Gray correlation analysis (GCA) was implemented to calculate the correlation coefficients of each compound between the peak area in liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) and the bioactivity, the top ten components with the correlation degree > 0.9 were listed. Results and discussion The optimum final concentration of GR on proangiogenesis effect was determined to be 100 μg/mL. Ten compounds, including gastrodin, parishin E, stigmasterol, p-hydroxybenzyl alcohol, citric acid, etc., were identified to have high correlation coefficients with proangiogenic activity. Furthermore, the network pharmacologic analysis of these compounds revealed that the compounds systematically regulate the formation of new blood vessels via networked vital targets and signalling pathways. Conclusion GR can promote the growth of blood vessels, ten chemical components discovered contribute to this proangiogenesis activity. These chemical markers of GR thus provide a foundation for further studies on medicinal substances and quality evaluation of GR, also providing a scientific basis for modern interpretation of the processing theory of traditional Chinese medicine.

INTRODUCTION Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate statistical analysis can provide tools to help detect differences in plant chemistry when grown under varying conditions. Hypericum perforatum, or Saint John's wort, plants are a suitable model to explore methods of discrimination between early stage plants grown in different conditions. OBJECTIVES The purpose of this work was to develop a method for identifying differences in chemical profiles between young Hypericum perforatum plants grown under different lighting conditions. MATERIAL AND METHODS Cuttings were grown for 3 weeks under different light conditions. Plant extracts were prepared in MeOD-d4 and analysed by 1 H-NMR. A multivariate analysis method of the NMR data was developed in an effort to determine variations in chemical profiles. RESULTS The method identified specific metabolites as drivers of difference between the plants grown under different light conditions. STOCSY (statistical total correlation spectroscopy) and quantification of highlighted metabolites supported the findings of the multivariate analysis. Glutamine, sucrose and fructose were found to be chemical markers of light quality in this study. CONCLUSION NMR metabolomics using a medium field instrument could find differences in plant chemistry when grown in different conditions. This method could easily be extended to benchtop instruments and be used for crop monitoring and growth condition optimisation.

Introduction Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium, two morphologically similar herbal medicines derived from Artemisia genus. Although the two Artemisia herbs have been used as medicines for a long time in China, the study of their phytochemical and bioactive composition is limited. Objective To comprehensively compare and evaluate the composition of Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium, and find the chemical makers for quality evaluation of the two Artemisia herbal medicines. Methodology Eight compounds including six phenolic acids and two flavonoids were assayed by a single reference standard for simultaneous determination of multi-components method using 3-caffeoylquinic acid as the reference standard. The quantitative data were further analysed by chemometric approaches to compare and distinguish the two herbal medicines. Results The credibility and feasibility of the single reference standard for simultaneous determination of the multi-components method were carefully validated. The validated method was applied to analyse 16 batches of Artemisiae argyi Folium and 10 batches of Artemisiae lavandulaefoliae Folium samples. The quantitative results showed that 3,5-di-O-caffeoylquinic acid was the most abundant constituent, and the contents of flavonoids were notably different between the two herbal medicines. The chemometric analysis results indicated the two flavonoids, jaceosidin and eupatilin could be used as chemical markers for quality evaluation of the two herbal medicines. Conclusion The single reference standard for simultaneous determination of the multi-components method coupled with chemometrics analysis could be a well-acceptable strategy to compare and evaluate the quality of Artemisiae argyi Folium and Artemisiae lavandulaefoliae Folium.

Introduction Bulbus fritillariae cirrhosae (known as Chuān bei mǔ in China, BFC) contain fritillaria steroidal alkaloids as the bioactive ingredients and are widely used as traditional Chinese medicine for the treatment of cough and phlegm. Due to limited wild resources, the cultivated species are becoming predominantly used in Chinese traditional medicine markets. Objective To assess the impact of different growth stages on the alkaloids of cultivated BFC and establish a reference for quality control and guidance for appropriate harvesting practices. Methods The ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) metabolomic strategy was applied to determine potential chemical markers for the discrimination and quality control of cultivated BFC in different growth stages. The molecular feature extraction and multivariate statistical analysis were applied to alkaloid extraction and full metabolomic profiling of cultivated BFC for classification and marker compound characterisation. Result This approach allowed the establishment of a fast and efficient comparative multivariate analysis of the metabolite composition of 42 samples covering growth of cultivated BFC ranging in age from one to seven years old. Four alkaloid compounds were identified in cultivated BFC based on accurate mass, retention time, and MS/MS fragments. These compounds may be used as potential chemical markers for the classification and discrimination of cultivated BFC samples indifferent growth stages. Conclusions The proposed analytical method in combination with multivariate statistical analysis comprised a useful and powerful strategy to explore the chemical ingredients and transforming mechanisms of cultivated BFC and for quality evaluation and control. Copyright © 2018 John Wiley & Sons, Ltd.

Introduction Tinospora cordifolia is a widely distributed medicinal plant used in various traditional and commercial Ayurvedic formulations. Due to the wide use of this plant it is important to know the extent of variability in the metabolite profile resulting from geographical location, season and gender. Objective To develop a statistical approach based on phytochemical markers for confident prediction of variations in metabolic profile and cytotoxicity due to geographical, seasonal and gender difference in T. cordifolia stem. Methods A HPLC-ESI-QTOF-MS method was used for the metabolite profiling of T. cordifolia stem. The data were analysed using chemometric methods including Student's t-test, ANOVA, FA/PCA and ROC curve analysis and validated for the identification of chemical variations. The bioactivity of selected samples was also tested using a cell cytotoxicity assay to assess the functional aspect of the phytochemical variability. Results The chemometric approach applied here identified marker ions for geographical locations (m/z 294.1139 and 445.2136), seasons (m/z 344.1482, 359.1501, and 373.1305) and gender (m/z 257.1380) with 100% statistical sensitivity and specificity. An in vitro cytotoxicity evaluation revealed that male T. cordifolia stem was the most effective in inhibiting the growth of cancerous cell lines. Conclusions The developed and validated chemometric approach identified the analytical markers for phytochemical variations in unknown T. cordifolia stem samples from male or female plants and samples collected from different geographical locations and seasons. The results are supported by comparative cytotoxic activity data. Copyright © 2017 John Wiley & Sons, Ltd.

Introduction Polygonum capitatum is a well-known Chinese medicinal plant widely used by the Miao people for the treatment of various urologic disorders. Previous investigations have shown the presence of various types of phenolics. Our ultrahigh-performance liquid chromatography with photodiode array detection and mass spectrometry (UPLC–PDA–MS) analysis indicated that flavonoid glycosides and polyphenolic glycosides were its major constituents and quite a number of phenolic compounds have not yet been identified. Identification or characterisation of the major compounds of this plant will contribute to the scientific understanding of the medicinal plant and the authentication of the plant material and its pharmaceutical preparations. Objective To develop an efficient method for the identification and structural characterisation of the major compounds present in P. capitatum. Methods Elution of the 70% ethanol extract of P. capitatum by 80% ethanol on a D101 macroporous resin column afforded a phenolics-enriched fraction, separation of which by Sephadex LH-20 column chromatography eluted with absolute ethanol gave a tannin-free phenolic fraction (TFPF). Compounds present in TFPF were identified and structurally characterised by UPLC–PDA–ESI–MS/MS. Results An amino acid and 40 phenolics including a number of flavonoid glycosides and polyphenolic glycosides were identified or structurally characterised in TFPF. Among these compounds, four were new and 19 were described in the plant for the first time. Conclusion The study showed that TFPF of P. capitatum contained flavonoid glycosides and polyphenolic glycosides as its major principles. Polyphenolic glycosides were perhaps the most typical chemical markers of P. capitatum. Copyright © 2013 John Wiley & Sons, Ltd.

Introduction Angelicae dahuricae radix (Baizhi) is a widely used traditional Chinese medicine, for which the traditional processing method has been sun-drying after harvesting. In recent years this method has been replaced by sulphur-fumigation for the sake of improving its appearance and pest control. Correct identification of sulphur-fumigated Baizhi and rapid finding of appropriate chemical markers for the quality control of Baizhi are of very great importance. Objective To develop a rapid and efficient method of rapid resolution liquid chromatography (RRLC) combined with chemometrics for discriminating sulphur-fumigated Baizhi and controlling its quality. Methodology The samples were separated on a Zorbax SB-C18-column with a gradient elution programme using methanol and water. The optimal RRLC method was validated in terms of linearity, precision, stability and recovery test. In addition, hierarchical clustering analysis (HCA) and principle component analysis (PCA) were applied to differentiate the different samples. Results All the constituents were well separated within 18 min. The results of chemical fingerprinting and quantitative analysis showed that the sulphur-fumigated method obviously destroyed the phytochemical profiles of Baizhi. Moreover, the sun-dried and sulphur-fumigated Baizhi could be clearly separated by HCA and PCA, and the critical markers such as oxypeucedanin and imperatorin accountable for such differences were ultimately screened out. Conclusion This study revealed that sulphur-fumigation is an inappropriate approach for processing crude drugs and should be prohibited, and that the proposed RRLC method combined with chemometrics demonstrated an efficient strategy for quality control of Baizhi. Copyright © 2011 John Wiley & Sons, Ltd.

Brazilian propolis contains several phenolic compounds among which 5 diprenyl-4-hydroxycinnamic acid (artepillin-C) is commonly found in areas where flora is rich in Baccharis species. The quantification of artepillin-C has become an important factor as an indicator of Brazilian propolis quality and the compound may be used as a chemical marker for quality control in exportating green propolis. This work was to validate the method and evaluate the content of artepillin-C from 33 samples collected in different Brazilian regions. The method used was HPLC with UV-vis detection and a reversed-phase C(18) column. The validation parameters studied were: linearity, accuracy, precision, quantification and detection limits. The results obtained were: detection limit = 0.0036 microg/mL, quantification limit = 0.012 microg/mL, accuracy = 0.0064 and 0.078, recovery 98-102%. Artepillin-C content varied from 0 to 11% depending on the geographical origin. Propolis from the southeast region presented the highest level of artepillin-C (5.0-11.0%). Whist that from the northeast region did not show any artepillin-C.

For over a century, Catuaba has been used in Brazilian folk medicine as an aphrodisiac even though the identity of the plant material employed is often uncertain. The species recommended by the Brazilian Pharmacopeia is Anemopaegma arvense (Bignoniaceae), but many other plants, regionally known as Catuaba, are commercialised. Frequently, the quality control of such a complex system is based on chemical markers that do not supply a general idea of the system. With the advent of the metabolomics approach, a global analysis of samples becomes possible. It appears that (1)H-NMR is the most useful method for such application, since it can be used as a wide-spectrum chemical analysis technique. Unfortunately, the generated spectra is complex so a possible approach is to look at the metabolite profile as a whole using multivariate methods, for example, by application of principal component analysis (PCA). In the present paper, we describe for the first time a proton high-resolution magic angle spinning nuclear magnetic resonance ((1)H-HR-MAS NMR) method coupled with PCA for the metabolomic analysis of some commercial Catuaba samples, which provided a reduction in the time required for such analysis. A comparative study of HPLC, HR-MAS and liquid-NMR techniques is also reported.