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• An UHPLC-MS/MS dereplicative method for Farfarae Flos. • Identification of 74 sesquiterpenoids by diagnostic filtering. • Isolation of 11 compounds including three new ones for method validation. A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)-based dereplicative method was developed for identifying oplopane- and bisabolane-type sesquiterpenoids from buds of Tussilago farfara L. The analysis of these chemical analogues, sesquiterpene esters, is challenging by MS-based nontargeted metabolomic approaches because of their in-source fragmentation and structural diversity. To profile these sesquiterpenoids, four diagnostic ions (m / z 215.143, 217.158, 229.123, and 231.138) were suggested in the positive ion mode and the developed method utilized two sequential MS/MS scan modes to identify common skeletons and investigate the fragmentation patterns of their parent molecules. Precursor ion scan by triple quadrupole MS/MS provided the parent molecular ions from their diagnostic ions, and product ion scan by quadrupole time-of-flight MS/MS confirmed their fragmentation behaviors. Under the optimized UHPLC-MS/MS method, 74 sesquiterpenoids were characterized from the Farfarae Flos and 11 compounds were isolated for the method validation. Among those compounds, three sesquiterpenoids were newly separated from the Farfarae Flos. Furthermore, the diagnostic ions and the MS/MS fragment behaviors were applied to the accurate quantification of the 8 isolated sesquiterpenoids. Therefore, the developed LC–MS/MS-based method highlighted the chemical composition of the Farfarae Flos and could be extended to the screening and quantification of other sesquiterpene esters. [ABSTRACT FROM AUTHOR]

A novel application of counter-current chromatography (CCC) to enrich plant extracts using direct and continuous injection (CCC-DCI) was developed to fractionate sesquiterpenoids from the buds of Tussilago farfara L. In this study, an n -hexane-acetonitrile-water (HAcW) solvent system was separately pumped into the CCC column, and an extraction solution (45% acetonitrile) was directly and continuously injected into the CCC column. Since the extraction solution was used as a mobile phase in this method, solvent consumption could be greatly reduced. To enrich the extraction solution (315.9 g/5.4 L), only 4.2 L water, 4.6 L acetonitrile, and 1.2 L n -hexane were used, including the extraction step. Finally, 6.8 g of a sesquiterpenoid-enriched (STE) fraction was obtained from the crude extract (315.9 g) of Tussilago farfara (1 kg) in a single CCC run with a separation time of 8.5 h. The sample injection capacity of CCC-DCI was greater than 300 g; this amount of sample could not be handled in conventional CCC or other fractionation methods with the same column volume. Moreover, three major sesquiterpenoids ( 1 : tussilagone, 2 : 14-acetoxy-7β-(3′-ethyl cis -crotonoyloxy)-1α-(2′-methylburyryloxy)-notonipetranone, and 3 : 7β-(3′-ethyl cis -crotonoyloxy)-1α-(2′-methylburyryloxy)-3, 14-dehydro- Z -notonipetranone) were purified from the STE fraction by CCC, and their chemical structures were elucidated by 1 H NMR and 13 C NMR. A quantification study was conducted, and the contents of compounds 1 – 3 in the CCC-DCI fraction were higher than those of conventional multi-step fractionations performed in series: solvent partitioning and open column chromatography. Furthermore, the average CCC-DCI recoveries were 96.1% ( 1 ), 96.9% ( 2 ), and 94.6% ( 3 ), whereas the open column chromatography recoveries were 77.7% ( 1 ), 66.5% ( 2 ), and 58.4% ( 3 ). The developed method demonstrates that CCC is a useful technique for enriching target components from natural products. [ABSTRACT FROM AUTHOR]

Abstract: Platycosides extracted from Platycodi Radix were analyzed by HPLC coupled with electrospray ionization multistage tandem mass spectrometry (HPLC/ESI-MS n ). Predominant [M +Na]+ ions in positive mode and [M −H]− ions in negative mode in the direct ESI-MS spectra of extract provided information on molecular weights, but minor components and isomers could not be discriminated. However, combining HPLC and ESI-MS n , allowed eleven platycosides, including four acetylated platycodin isomers and two prosapogenines to be analyzed. During MS2 analysis conducted to elucidate the structures of platycosides, fragment ions provided information on sugar moieties attached at C-28 of triterpene structure of the platycosides. Glycosidic bond cleavages at C-3 were revealed by fragment ions in MS3 spectra. Some characteristic fragment ions not related to sugar bond cleavage revealed that an esterified triterpene is linked to sugars at C-28. The only sugar ring-cross cleavage corresponding to 90Da in the negative MS2 spectrum took place at an arabinosyl sugar moiety. By using HPLC/ESI-MS n , three acetylated platycosides in Platycodi Radix extract were newly identified. [Copyright &y& Elsevier]

Aralia continentalis (Araliaceae) is widely used as a medicinal plant in East Asia. Previous studies have indicated that diterpenoid isomers (kaurenoic acid, continentalic acid, and ent -continentalic acid) are the major bioactive compounds of this plant. A new strategy was developed to alleviate difficulties in the separation of these isomers from this plant. A three-phase solvent system was applied to separate the isomers, and furthermore, the distribution ratio ( K c ) was introduced as a substitute for the partition coefficient ( K D ). For compounds exhibiting a single equilibrium, their distributions in two immiscible phases were only affected by the partition coefficient of each solute. However, compounds that have a dissociating functional group (e.g., –COOH) are involved in two types of equilibrium in the two-phase system. In this case, the partitioning behaviors of the solutes are greatly affected by the pH of the solution. A mathematical prediction was applied for adjusting the solutions to the proper pH values. To prevent non-used phase (medium phase) waste, both the stationary phase (upper phase) and mobile phase (lower phase) were prepared on-demand without pre-saturation with the application of 1 H NMR. Each fraction obtained was collected and dried, yielding the following diterpenoid isomers from the 50 mg injected sample: kaurenoic acid (19.7 mg, yield: 39%) and ent -continentalic acid (21.3 mg, yield: 42%). [ABSTRACT FROM AUTHOR]

Aralia continentalis (Araliaceae) is widely used as a medicinal plant in East Asia. Previous studies have indicated that diterpenoid isomers (kaurenoic acid, continentalic acid, and ent -continentalic acid) are the major bioactive compounds of this plant. A new strategy was developed to alleviate difficulties in the separation of these isomers from this plant. A three-phase solvent system was applied to separate the isomers, and furthermore, the distribution ratio ( K c ) was introduced as a substitute for the partition coefficient ( K D ). For compounds exhibiting a single equilibrium, their distributions in two immiscible phases were only affected by the partition coefficient of each solute. However, compounds that have a dissociating functional group (e.g., –COOH) are involved in two types of equilibrium in the two-phase system. In this case, the partitioning behaviors of the solutes are greatly affected by the pH of the solution. A mathematical prediction was applied for adjusting the solutions to the proper pH values. To prevent non-used phase (medium phase) waste, both the stationary phase (upper phase) and mobile phase (lower phase) were prepared on-demand without pre-saturation with the application of 1 H NMR. Each fraction obtained was collected and dried, yielding the following diterpenoid isomers from the 50 mg injected sample: kaurenoic acid (19.7 mg, yield: 39%) and ent -continentalic acid (21.3 mg, yield: 42%). [ABSTRACT FROM AUTHOR]

Highlights: [•] An integrated model of flow-rate gradient and solvent gradient was developed. [•] The model efficacy was improved by optimizing the operational parameters. [•] The model was applied to CCC for one-step separation of eight coumarins from Seseli resinosum. [•] The roles of stepwise gradient, system temperature, and GC-FID were discussed. [•] The separation mechanism was discussed on the basis of the final data. [Copyright &y& Elsevier]

Abstract: Comprehensive two-dimensional chromatography (LC×LC) using combinations of two columns (C18 ×CN and C18 ×NH2) was employed with electrospray (ESI) mass spectrometry to analyze platycosides from root extract. Based on the capability of the C18, CN and NH2 columns to separate the platycosides, the orthogonality in two-dimensional space according to each combination of columns was predicted from the correlation coefficients between the retention times of the 17 compounds separated by the independent CN and C18 columns, and NH2 and C18 columns. The expected distribution of the peaks was also compared with the two-dimensional plots obtained by practical separation in an LC×LC system. The increased peak capacities using C18 ×NH2 allowed three minor components and five isomers of the platycosides to be newly separated, which were not identified with 1D-LC using the individual C18 column, whereas the combination of C18 ×CN did not result in any improvement of the separation performance. [Copyright &y& Elsevier]

Abstract: We determined the minute amount of non-polar ginsenosides in red ginseng with a reversed-phase high-performance liquid chromatography-pulsed amperometric detection (RP-HPLC-PAD) method. Non-polar ginsenosides efficiently extracted by ethyl acetate were well separated in 40min using a water–acetonitrile gradient eluent and detected by PAD under NaOH alkaline conditions. The ginsenoside detection limits (S/N=3) were 0.03–0.10ng. The coefficients of linear regression were 0.9972–0.9990. Intra- and inter-day precision (RSDs) was less than 8.34% and average recovery was 98.06–102.73%. The total amount of non-polar ginsenosides in hairy root of red ginseng was slightly higher than in the main root. [Copyright &y& Elsevier]

Abstract: Ginseng (Panax ginseng C. A. Meyer) has been well known to have a variety of ginsenosides that show diverse biological activities. Especially, the components of ginsenosides are quite different depending on the processing method. Recently, there have been several reports showing that less polar ginsenosides from Korean red ginseng (steam-treated Panax ginseng) have potent biological activities such as radical scavenging, vasodilating and anti-tumor activities. In this study, we have isolated four known ginsenosides Rg3, Rk1, Rg5 and F4 from Korean red ginseng by high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD). The enriched saponin fraction (350mg) was separated by using methylene chloride–methanol–water–isopropanol (6:6:4:1, v/v) as the two-phase solvent system and yielded 28.6mg of Rg5, 26.6mg of Rk1, 32.2mg of Rg3 and 8.1mg of F4. The purity of these ginsenosides was assessed by HPLC–ELSD to be over 95%, and their structures were characterized by electrospray ionization mass spectrometry (ESI-MS), 1H NMR and 13C NMR. [Copyright &y& Elsevier]

Abstract: Saponins in Platycodi Radix (platycosides) exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammatory, immunomodulatory and anti-obesity activities. In this study, we developed a new HPLC separation coupled with evaporative light scattering detector (ELSD) for the simultaneous quantitative determination of ten major saponins in Platycodi Radix. Simultaneous separation of these saponins was achieved on a C18 analytical column. The mobile phase consisted of a gradient of aqueous acetonitrile. The method was validated for linearity, precision, accuracy, limit of detection and quantification. Electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography coupled with on-line mass spectrometry (LC-ESI MS/MS) were applied to identify platycosides in the purified fractions and in the crude extract. Under ESI-MS/MS conditions, the fragmentation patterns of [M−H]− ions exclusively show signals corresponding to cleavage of the glycosidic bonds, thus allowing a rapid identification of saponins in the crude extract of Platycodi Radix. The validated HPLC method provides a new basis of overall assessment on quality of Platycodi Radix, and ESI-MS/MS and LC-ESI MS/MS approaches offers analytical tools for a rapid screening of platycosides in the crude extract. [Copyright &y& Elsevier]