Content of Two Major Steroidal Glycoalkaloids in Tomato (Solanum lycopersicum cv. Micro-Tom) Mutant Lines at Different Ripening Stages.

Esculeoside A and tomatine are two major steroidal alkaloids in tomato fruit (Solanum lycopersicum) that exhibit anti-inflammatory, anticancer, and anti-hyperlipidemia activities. Tomatine contained in immature tomato fruit is converted to esculeoside A as the fruit matures. To develop new tomato varieties based on the content analysis of functional secondary metabolites, 184 mutant lines were generated from the original cultivar (S. lycopersicum cv. Micro-Tom) by radiation breeding. Ultra-performance liquid chromatography coupled with evaporative light scattering detector was used to identify the mutant lines with good traits by analyzing tomatine and esculeoside A content. Compared with the original cultivar, candidates for highly functional cultivars with high esculeoside A content were identified in the mature fruit of the mutant lines. The mutant lines with low and high tomatine content at an immature stage were selected as edible cultivars due to toxicity reduction and as a source of tomatine with various pharmacological activities, respectively. During the process of ripening from green to red tomatoes, the rate of conversion of tomatine to esculeoside A was high in the green tomatoes with a low tomatine content, whereas green tomatoes with a high tomatine content exhibited a low conversion rate. Using methanol extracts prepared from unripe and ripe fruits of the original cultivar and its mutant lines and two major compounds, we examined their cytotoxicity against FaDu human hypopharynx squamous carcinoma cells. Only tomatine exhibited cytotoxicity with an IC50 value of 5.589 µM, whereas the other samples did not exhibit cytotoxicity. Therefore, radiation breeding represents a useful tool for developing new cultivars with high quality, and metabolite analysis is applicable for the rapid and objective selection of potential mutant lines.

Effect of Flower Removal on Content of Three Alkaloids in Different Parts of Fritillaria thunbergii from Different Regions and at Different Growth Stages / 中国实验方剂学杂志

ObjectiveTo study the effect of flower removal on the content of three alkaloids in different parts of Fritillaria thunbergii from different regions and at different growth stages. MethodThe content of peiminine， peimine， and peimisine in the bulb， root， stem， and leaf of F. thunbergii after flower removal and with flower un-removed at different growth stages and in different regions were determined simultaneously by ultra-performance liquid chromatography-evaporative light scattering detection （UPLC-ELSD） method. The UPLC was conducted on ACQUITY UPLC BEH C18 column （2.1 mm × 150 mm， 1.7 μm） with the mobile phase of 0.02% triethylamine aqueous solution （A） and methanol （B）elution gradient（0-2 min， 45%A； 2-5 min， 45%-25%A； 5-7 min， 25%A； 7-17 min， 25%-10%A； 17-20 min， 10%A）， flow velocity of 0.20 mL·min-1， column temperature 35 °C， sample room temperature of 20 °C， and injection volume of 3 µL. The ELSD was carried out at drift tube temperature 45 °C and with the sprayer parameter of 40%. ResultThe flower removal significantly increased the yield of F. thunbergii. At the budding stage， the alkaloid content in the bulb of F. thunbergii from Ningbo in Zhejiang， Pan'an in Zhejiang， and Nantong in Jiangsu after flower removal were significantly higher than that of flowering un-removal treatment， while it showed no significant difference between the flower removal and un-removal treatments for the samples from Fengjie in Chongqing. At the flowering stage， the alkaloid content in the bulb of F. thunbergii from Nantong in Jiangsu after flower removal was significantly higher than that of flower un-removal treatment， while it showed an opposite trend for the samples from Pan'an in Zhejiang and Fengjie in Chongqing and had no significant difference between the two treatments for the samples from Ningbo in Zhejiang. At the bulb expansion stage， the alkaloid content in the bulb of F. thunbergii from Ningbo in Zhejiang and Pan’an in Zhejiang after flower removal were significantly higher than that of flower un-removal treatment， which was opposite for the samples from Nantong in Jiangsu and had no significant difference between the treatments for the samples from Fengjie in Chongqing. At the harvest stage， except for the samples from Pan'an in Zhejiang， the samples from the rest 3 regions showed decreased alkaloid content in the bulb after flower removal compared with that of flower un-removal treatment. The alkaloid content in the leaf was higher than that in the bulb of F. thunbergii at all growth stages and from different origins. ConclusionFlower removal can increase the yield of F. thunbergii. The alkaloid content in the bulb of F. thunbergii with flower removed was higher than that with flower un-removed at the budding stage， while this trend was reversed at the harvest stage. Both the yield and the alkaloid content of F. thunbergii from Pan'an in Zhejiang were increased by flower removal. The above-ground part of F. thunbergii has a potential development value.

First Determination of Glycidyl Ester Species in Edible Oils by Reverse-Phase Ultra-Performance Liquid Chromatography Coupled with an Evaporative Light-Scattering Detector.

In this work, a new ultra-performance liquid chromatograph-evaporative light-scattering detector (UPLC-ELSD) method for quantitation of glycidyl esters (GE) contents in edible oils is presented. The method features complete separation of five GE species within 20 min by a C18 column and gradient elution with a mobile phase consisting of 85% and 2.5% methanol aqueous solutions. The coefficients of regression (R2) were all ≥0.9999 for the linear-quadratic regression curves of GE species in a concentration range of 5~80 µg/mL. The intraday and interday recoveries (%) of GE species in solvent were in a range of 81.3~107.3%, and the intraday and interday coefficients of variation (CVs, %) were all ≤8.6%. The average recovery (%) of GE species spiked in extra-virgin olive oil samples ranged from 88.3~107.8% and the intermediate precision (CV, %) of ≤14% indicated acceptable accuracy and precision. The method exhibited limit of quantification (LOQ) for each GE species (0.6 µg glycidol equivalents/g oil). The method was applied to determine GE concentrations of six commercial oil samples, and total glycidol equivalents were consistent with data obtained by GC-MS method. This UPLC-ELSD method could be adopted for precursory screening and research purposes to improve food safety when MS detectors are unavailable.

[Simultaneous determination of six major isosteroidal alkaloids in Beimu by UPLC-ELSD].

An UPLC method was established for the direct determination of six major bioactive isosteroidal alkaloids, namely peimisine, imperialine, sipeimine-3-D-glucoside, verticinone, verticine and hupehenine from the bulbus of Fritillaria(Beimu), a commonly used antitussive traditional Chinese medicinal(TCM) herb. An Acquity UPLC~(TM) CSH C_(18) column(2.1 mm×100 mm, 1.7 µm) was used for all analysis. The investigated six compounds were all separated with gradient mobile phase consisting of 0.02% diethylamine-water-methanol at a flow rate of 0.3 mL·min~(-1). The temperature of sample manager was set at 20 ℃. Drift tube temperature was 45 ℃, and spray parameter was 40% with injection volume of 1 µL. Then, the further quality assessment of Beimu was carried out by cluster analysis(CA) and principal component analysis(PCA). The investigated all had good linearity(r≥0.998 9) over the tested ranges. The method is simple, accurate and reproducible, and can be used for determining the content of six major bioactive isosteroidal alkaloids.

Simultaneous determination of six major isosteroidal alkaloids in Beimu by UPLC-ELSD / 中国中药杂志

An UPLC method was established for the direct determination of six major bioactive isosteroidal alkaloids, namely peimisine, imperialine, sipeimine-3-D-glucoside, verticinone, verticine and hupehenine from the bulbus of Fritillaria(Beimu), a commonly used antitussive traditional Chinese medicinal(TCM) herb. An Acquity UPLC~(TM) CSH C_(18) column(2.1 mm×100 mm, 1.7 μm) was used for all analysis. The investigated six compounds were all separated with gradient mobile phase consisting of 0.02% diethylamine-water-methanol at a flow rate of 0.3 mL·min~(-1). The temperature of sample manager was set at 20 ℃. Drift tube temperature was 45 ℃, and spray parameter was 40% with injection volume of 1 μL. Then, the further quality assessment of Beimu was carried out by cluster analysis(CA) and principal component analysis(PCA). The investigated all had good linearity(r≥0.998 9) over the tested ranges. The method is simple, accurate and reproducible, and can be used for determining the content of six major bioactive isosteroidal alkaloids.

[Quality control of Guci tablets using UPLC-ELSD fingerprint analysis coupled with chemometrics].

Ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) fingerprint analysis method was established for quality control of Guci tablets. Chromatographic separation was performed on Waters Acquity UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) at 30 °C of column temperature. Acetonitrile-0.1% formic acid solution was adopted as mobile phase for gradient elution. The flow rate was set at 0.3 mL·min⁻¹, and the injection volume was 3 µL. Detection was carried out on an ELSD with a nitrogen pressure of 0.28 MPa, drift tube temperature of 60 °C, and gain of 400. A total of 39 batches of samples produced by six manufacturers were measured by using the above method and the data were analyzed by ChemPattern software. The peak present in more than 75% of the samples was defined as a common peak, and 30 common peaks were determined. Among them, 19 peaks were identified by rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) method, 16 of which were confirmed by reference substances. The similarity of the tested samples was 0.47-0.98, suggesting that the quality of the samples from different manufacturers varied greatly. Furthermore, principal component analysis (PCA) and hierarchical analysis (HCA) were performed to clarify the main different components in samples. The results indicated that there might be some feeding problems about Paeoniae Radix Alba, Notoginseng Radix et Rhizoma, and Clematidis Radix et Rhizoma in a few manufacturers. This study provided some evidences for the overall quality control of Guci tablets, as well as its quality standard improvements.

Quality control of Guci tablets using UPLC-ELSD fingerprint analysis coupled with chemometrics / 中国中药杂志

Ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) fingerprint analysis method was established for quality control of Guci tablets. Chromatographic separation was performed on Waters Acquity UPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) at 30 °C of column temperature. Acetonitrile-0.1% formic acid solution was adopted as mobile phase for gradient elution. The flow rate was set at 0.3 mL·min⁻¹, and the injection volume was 3 μL. Detection was carried out on an ELSD with a nitrogen pressure of 0.28 MPa, drift tube temperature of 60 °C, and gain of 400. A total of 39 batches of samples produced by six manufacturers were measured by using the above method and the data were analyzed by ChemPattern software. The peak present in more than 75% of the samples was defined as a common peak, and 30 common peaks were determined. Among them, 19 peaks were identified by rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) method, 16 of which were confirmed by reference substances. The similarity of the tested samples was 0.47-0.98, suggesting that the quality of the samples from different manufacturers varied greatly. Furthermore, principal component analysis (PCA) and hierarchical analysis (HCA) were performed to clarify the main different components in samples. The results indicated that there might be some feeding problems about Paeoniae Radix Alba, Notoginseng Radix et Rhizoma, and Clematidis Radix et Rhizoma in a few manufacturers. This study provided some evidences for the overall quality control of Guci tablets, as well as its quality standard improvements.

Chemical Profiling and Bioactivity of Body Wall Lipids from Strongylocentrotus droebachiensis.

The lipids from gonads and polyhydroxynaphthoquinone pigments from body walls of sea urchins are intensively studied. However, little is known about the body wall (BW) lipids. Ethanol extract (55 °C) contained about equal amounts of saturated (SaFA) and monounsaturated fatty acids (MUFA) representing 60% of total fatty acids, with myristic, palmitic and eicosenoic acids as major SaFAs and MUFAs, respectively. Non-methylene-interrupted dienes (13%) were composed of eicosadienoic and docosadienoic acids. Long-chain polyunsaturated fatty acids (LC-PUFA) included two main components, n6 arachidonic and n3 eicosapentaenoic acids, even with equal concentrations (15 µg/mg) and a balanced n6/n3 PUFA ratio (0.86). The UPLC-ELSD analysis showed that a great majority of the lipids (80%) in the ethanolic extract were phosphatidylcholine (60 µg/mg) and phosphatidylethanolamine (40 µg/mg), while the proportion of neutral lipids remained lower than 20%. In addition, alkoxyglycerol derivatives-chimyl, selachyl, and batyl alcohols-were quantified. We have assumed that the mechanism of action of body wall lipids in the present study is via the inhibition of MAPK p38, COX-1, and COX-2. Our findings open the prospective to utilize this lipid fraction as a source for the development of drugs with anti-inflammatory activity.

Simultaneous Determination of Saponins in Dripping Pills Made from Astragali Radix and Panax notoginseng by UPLC-ELSD / 中草药·英文版

Objective To develop a simple and fast method for removing polyethylene glycol (PEG) and simultaneous determination of fives saponins, i.e. astragaloside IV, notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Rd in dripping pills made from Astragali Radix and Panax notoginseng. Methods The extraction method was based on a liquid-liquid extraction using water-saturated n-butanol and the quantitative determination was based on ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC-ELSD). The chromatographic analysis was performed on an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) with a gradient elution of acetonitrile-0.1% formic acid aqueous solution within a runtime of 15 min. Results Compared to different methods, the proposed method could remove the interference of PEG in formulation. And the calibration curves showed good linearity during the test ranges. The method was validated for limits of detection and quantification, precision, and reproducibility. The recoveries were within the range of 96.87% – 99.97%. In addition, the verified method was firstly applied to determination of the five active ingredients in Qishen Yiqi Dripping Pills (QYDP) simultaneously. Conclusion The contents of five active ingredients are stable and homogeneous in QYDP, which indicates that the method could be readily utilized as a quality evaluation method for this traditional Chinese medicine dripping pills made from Astragali Radix and Panax notoginseng.

Diversity-oriented natural product platform identifies plant constituents targeting Plasmodium falciparum.

BACKGROUND: A diverse library of pre-fractionated plant extracts, generated by an automated high-throughput system, was tested using an in vitro anti-malarial screening platform to identify known or new natural products for lead development. The platform identifies hits on the basis of in vitro growth inhibition of Plasmodium falciparum and counter-screens for cytotoxicity to human foreskin fibroblast or embryonic kidney cell lines. The physical library was supplemented by early-stage collection of analytical data for each fraction to aid rapid identification of the active components within each screening hit. RESULTS: A total of 16,177 fractions from 1300 plants were screened, identifying several P. falciparum inhibitory fractions from 35 plants. Although individual fractions were screened for bioactivity to ensure adequate signal in the analytical characterizations, fractions containing less than 2.0 mg of dry weight were combined to produce combined fractions (COMBIs). Fractions of active COMBIs had EC50 values of 0.21-50.28 and 0.08-20.04 µg/mL against chloroquine-sensitive and -resistant strains, respectively. In Berberis thunbergii, eight known alkaloids were dereplicated quickly from its COMBIs, but berberine was the most-active constituent against P. falciparum. The triterpenoids α-betulinic acid and ß-betulinic acid of Eugenia rigida were also isolated as hits. Validation of the anti-malarial discovery platform was confirmed by these scaled isolations from B. thunbergii and E. rigida. CONCLUSIONS: These results demonstrate the value of curating and exploring a library of natural products for small molecule drug discovery. Attention given to the diversity of plant species represented in the library, focus on practical analytical data collection, and the use of counter-screens all facilitate the identification of anti-malarial compounds for lead development or new tools for chemical biology.