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Ultra high performance liquid chromatography tandem linear ion trap orbitrap mass spectrometry (UHPLC-LTQ-orbitrap-MS) was applied to analyze and identify flavonoids and phenylethanoid glycosides in the Tibetan herb Lagotis brevituba Maxim. A method of data-dependent scan coupling with dynamic exclusion was developed for analyzing flavonoids and phenylethanoid glycosides under positive and negative ion mode of electrospray ionization (ESI). The compounds of Lagotis brevituba Maxim. were systematically identified through exact molecular mass, fragmentation patterns, retention time and reported references. A total of 167 compounds were detected, of which 84 were flavonoids and 83 were phenylethanoid glycosides, which greatly enriched the number and types of flavonoids and phenylethanol glycosides in Lagotis genus medicinal plants. Baohuoside Ⅰ, 4 disaccharide O-glycoside flavonoids (composed of deoxyhexose and glucuronic acid), 9 C-glycoside flavonoids, 15 tetrasaccharide phenylethanoid glycosides and 5 phenylethanoid glycosides with substituents on the β-position of the phenylethyl group were identified in Lagotis genus medicinal plants for the first time. This study provides scientific support for elucidating the material basis and improving the quality control of Lagotis brevituba Maxim.
ABSTRACT
Objective: To establish HPLC fingerprints of Lagotis integra and Lagotis brevituba which were contained in National Drug Standard and compared to Lagotis ramalana, Lagotis alutacea and Lagotis brachystachya by the established method. Methods: The similarity of HPLC fingerprints of L. integra and L. brevituba was low, so the HPLC fingerprint methods were established for both. The HPLC analysis was performed on Waters X-Bridge C18 (250 mm × 4.6 mm, 5 μm), using acetonitrile and 0.2% formic acid solution as the mobile phase at a flow rate of 1.0 mL/min, the detection wave-length was 328 nm and column temperature was 35 ℃ (L. integra) and 25 ℃ (L. brevituba). Results: There were 14 common peaks in the fingerprint of L. integra, and plantamajoside, hemiphroside B, 10-O-trans-p-methoxycinnamoyl-catalpol and 10-O-[(E)-3,4-dimethoxycinna moyl]-catalpol were standardized. There were 13 common peaks in the fingerprint of L. brevituba, and echinacoside, plantamajoside and acteoside were standardized. The similarity of HPLC fingerprint was more than 0.9 between L. integra and L. alutacea, but the others had low similarity with them. The similarity of HPLC fingerprint was more than 0.9 between L. brevituba from different batches and L. ramalana, while the others had low similarity with them. Conclusion: The established method could effectively identify L. brevituba, L. integra and L. alutacea were advised to be recorded in Medical Standards of the Ministry of Health. Lagotis ramalana could be used as a new base for "Honglian" (origin: Lagotis brevituba).
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Objective:To study the components with urate anion transporter 1（URAT1） regulation effect and their combination mechanisms of Lagotis brevituba by integrating techniques of HK-2 cell capture，UPLC-Q-TOF-MS and molecular docking，so as to provide material and theory bases for the development of new hypouricemic medicines based on L. brevituba. Method:The HK-2 cells were applied to capture the components of L. brevituba. UPLC-Q-TOF-MS was used to identify those components. The molecular docking technique was adopted to study the interaction mechanism between the compounds and URAT1. Result:Eight components were successfully screened and identified as hyperoside，plantamajoside，kaempferol-3-O-glucoside，lugrandoside，nepitrin，isolugrandoside，homoplantaginin，luteolin，respectively. Those components could combine with URAT1 mainly through hydrogen bond，van der Waals force and hydrophobic action，which were closely related to structure and compound types. Furthermore，the LibDock score of phenylethanoids was higher than that of flavonoids. Conclusion:The integration of target cell capture，UPLC-Q-TOF-MS and molecular docking techniques could be successfully used to identify captured compounds of L. brevituba with URAT1 regulation effects and illustrate their potential combination mechanisms as well as the structure-activity relationships. The findings may provide material and theory bases for the development of new hypouricemic medicines based on L. brevituba.
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Potential xanthine oxidase (XOD) inhibitors in Lagotis brevituba were captured by using affinity and ultrafiltration. The structures of the captured components were identified by ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC-Q-TOF-MS). The binding intensity and binding mechanism between the captured components and XOD were analyzed by using molecular docking software Autodock 4.2. A total of 17 compounds were identified, including 9 flavonoids, 5 phenolic acids and 3 triterpenes. Molecular docking results showed that all the captured components could be spontaneously bound with XOD mainly via hydrogen bond, Van der Waals' force and hydrophobic interaction. From the perspective of binding energy and scoring function, the collected fractions all had potential prospects for XOD inhibitors, and the flavonoid luteolin-3',7 glucuronide had the best effect. The results also showed that affinity and ultrafiltration, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and molecular docking technology can provide a powerful tool for the analysis of XOD inhibitor components in natural products.
ABSTRACT
The chemical constituents of Lagotis brevituba were rapidly determined and analyzed by using ultra performance liquid chromatography tandem quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS) method, providing material basis for the clinical application of L. brevituba. The separation was performed on UPLC YMC-Triart C₁₈ (2.1 mm×100 mm, 1.9 μm) column, with acetonitrile-water containing 0.2% formic acid as mobile phase for gradient elution. The flow rate was 0.4 mL•min-1 gradient elution and column temperature was 40 ℃, the injection volume was 2 μL. ESI ion source was used to ensure the data collected in a negative ion mode. The chemical components of L. brevituba were identified through retention time, exact relative molecular mass, cleavage fragments of MS/MS and reported data. The results showed that a total of 22 compounds were identified, including 11 flavones, 6 phenylethanoid glycosides, 1 iridoid glucosides, and 4 organic acid. The UPLC-Q-TOF-MS/MS method could fast identify the chemical components of L. brevituba, providing valuable information about L. brevituba for its clinical application.
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Objective: To study the chemical constituents from Lagotis brevituba. Methods: The chemical constituents were isolated by solvent extraction, repeated silica gel chromatography, and preparative-HPLC. Their structures were elucidated by physicochemical properties and NMR data. Results: Eleven compounds were isolated from the ethyl acetate and n-butanol fraction of L. brevituba and their structures were identified as arvenin I (1), 3,4-dihydroxyphenylethanol (2), bis (2-ethylhexyl) phthalate (3), plantamajoside (4), mannitol (5), ethyl gallate (6), ethyl 3,4-dihydroxybenzoate (7), dibutyl phthalate (8), β-sitosterol (9), acteoside (10), and mussaenosidic acid (11). Conclusion: Compounds 1-3, 6, and 7 are obtained from the plants in Lagotis Gaertn. for the first time. Compounds 4 and 5 are isolated from L. brevituba for the first time.
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AIM: To study inhibitive effect of Lagotis Brevituba Maxim extracted on human stomach cancer SGC-7901 cells proliferation and/or induce apoptosis. METHODS: Human stomach cancer SGC-7901 cells were treated with Lagotis Brevituba Maxim extracted from n-butanol of 0.34～21.60 mg/mL for 24 h～72 h.Cell proliferation was evaluated by MTT assay.Morphological changes of apoptosis were examined by fluorescence microscope and electron microscope.DNA fragmentation was visualized by agarose gel electrophoresis.The amount of apoptosis cells was measured by flow cytometry. RESULTS: Growth of SGC-7901 cells was obviously inhibited by Lagotis Brevituba Maxim extract with value from 21.60 mg/mL to 0.34 mg/mL.After incubation of SGC-7901 cells with 2.7 mg/mL Lagotis Brevituba Maxim extract for 48 h, morphological changes of apoptosis were observed.DNA ladder was identified by agarose gel electrophoresis of DNA ladder. CONCLUSION: The n-butanol extract of Lagotis Brevituba Maxim can inhibit SGC-7901 cell growth and induce apoptosis.