Combined chromatographic strategy based on macroporous resin,high‐speed counter‐current chromatography and preparative HPLC for systematic separation of seven antioxidants from the fruit of Terminalia billerica

In the present study, combined chromatographic strategy based on macroporous resin, high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography for systematic separation of antioxidants from crude samples guided by high‐performance liquid chromatography with 1,1‐diphenyl‐2‐picrylhydrazyl has been successfully established. Based on this strategy, seven antioxidants including isorugosin A, _β‐1,2,3,6‐tetragalloyl‐D ‐glucose, chebulinic acid, 1,2,3,4,6‐penta‐O‐galloyl‐_β‐D‐glucose, chebulagic acid, ethyl gallate, and gallic acid were obtained from the fruit of Terminalia billerica. First, high‐performance liquid chromatography with 1,1‐diphenyl‐2‐picrylhydrazyl experiment showed the presence of seven main antioxidants in the crude extract of the fruit of Terminalia billerica. Then, a macroporous resin column chromatography method was developed for the enrichment of these seven antioxidants. Finally, an efficient method based on high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography was developed for the separation of these antioxidants. In the selection of solvent systems, it was found that acetic acid could be a good regulator for modifying the partition coefficient values of tannins. The present study provides a reference for systematic separation of antioxidants from crude samples. Considering the general existence of antioxidants in crude samples, this combined chromatographic strategy might lead to broader application prospects.     

Two‐dimensional hydrophilic interaction chromatography × reversed‐phase liquid chromatography for the preparative isolation of potential anti‐hepatitis phenylpropanoids from Salvia prattii

Traditional Tibetan medicine is important for discovery of drug precursors. However, knowledge of the chemical composition of traditional Tibetan medicines is very limited due to the lack of appropriate chromatographic purification methods. In the present work, Salvia prattii was taken as an example, and an off‐line hydrophilic interaction liquid chromatography/reversed‐phase liquid chromatography preparative method was developed for the purification of phenylpropanoids with high purity from a crude sample of Salvia prattii. Based on the separation results of four different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAmide preparative column with the crude sample concentration of 62.0 mg/mL, and five main fractions were obtained from the 12.4 g crude sample with a recovery of 54.8%. An XCharge C₁₈ preparative column was applied in the second‐dimensional preparation to further isolate the phenylpropanoids from the redissolved first‐dimensional fractions with concentration of approximately 50.0 mg/mL. The purities of the phenylpropanoids isolated from the crude sample of Salvia prattii were higher than 98%, indicating that the method was efficient for the purification of phenylpropanoids with high purity from Salvia prattii. Additionally, this method showed great potential in the preparation of phenylpropanoids and can serve as a good example for the purification of phenylpropanoids from other plant materials.     

Comparative in vivo pharmacokinetics study of affeic acid,isoferulic acid and ferulic acid in crude and three different prepared Cimicifuga foetida L.

Our study investigated the differences in pharmacokinetics of three major components of crude Cimicifuga foetida L. and its fried product and honey- and liquor-prepared products. A rapid and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry approach was established for determing caffeic acid, isoferulic acid and ferulic acid in rat plasma. The approach has good linearity, precision, accuracy, recovery and stability. Phenolic acid was rapidly absorbed. The times to peak concentration were shorter in the processed group than those for the crude product, with their values of <30 min. The peak concentration values of caffeic acid and isoferulic acid were higher in the crude group than in the processed groups (p < 0.05). Area under the curve values of the three phenolics in the crude group were significantly higher than those of the processed groups (p < 0.05).     

Preparative Isolation of Destruxins from Metarhizium Anisopliae by High Performance Liquid Chromatography

Abstract

A method has been developed for preparative isolation of destruxins, a group of major insecticidal cyclodepsipeptides from culture broth of Metarhizium anisopliae. Prepurification of the crude extract by flash chromatography on silica gel followed by HPLC on reverse phase C18 Column using linear gradients of acetonitrile-water permitted the isolation of ten destruxins.     

Application of High Temperature High Resolution Gas Chromatography to Crude Extracts of Propolis

The underivatized acetone and hexane fractions from propolis samples (predominant flora Citrus spp. and Vernonia polyanthes) were analyzed by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds, including flavonoid aglycones, phenolic acids, and high molecular weight compounds were characterized in crude extracts by HT-HRGC-MS. HT-HRGC and HT-HRGC-MS were shown to be quick and informative tools for rapid analysis of crude extracts without need for prior derivatization and purification.     

Study on steroidal saponins in crude and stir‐fried Fructus Tribuli by ultra‐high‐performance liquid chromatography‐mass spectrometry coupled with multivariate statistical analysis

Fructus Tribuli is a traditional Chinese medicine used clinically for many years. Crude Fructus Tribuli and stir‐fried Fructus Tribuli are recorded in the Pharmacopoeia of the People′s Republic of China. However, the differences between steroidal saponins in crude Fructus Tribuli and stir‐fried Fructus Tribuli have not been compared. In this study, ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry along with multivariate statistical analysis was developed to discriminate the chemical profiles and identify the steroidal saponins of crude Fructus Tribuli and stir‐fried Fructus Tribuli. Additionally, an ultra‐high‐performance liquid chromatography triple‐quadrupole mass spectrometer was used for the simultaneous quantification of nine major steroidal saponins to analyze the variations between crude Fructus Tribuli and stir‐fried Fructus Tribuli. Finally, a total of 30 steroidal saponins whose structures or contents changed significantly after processing were found and identified. The mechanism of structural transformations deduced indicated that during the stir‐frying of Fructus Tribuli, C‐22 hydroxy furostanol saponins were converted to the corresponding furostanol saponins containing C‐20‐C‐22 double bonds by dehydroxylation and deglycosylation reactions that occurred in the spirostanol saponins causing the generation of steroidal sapogenins. This study was successfully applied to the global analysis of crude Fructus Tribuli and stir‐fried Fructus Tribuli. The results of this research will be beneficial to explore the processing mechanism of Fructus Tribuli.     

Preparative isolation of ganoderic acid S,ganoderic acid T and ganoderol B from Ganoderma lucidum mycelia by high‐speed counter‐current chromatography

Ganoderic acid S, ganoderic acid T and ganoderal B are the main bioactive triterpenes of Ganoderma lucidum. In this study, mycelia of G. lucidum were obtained by two‐stage fermentation and then extracted by ethanol and petroleum ether sequentially to obtain crude triterpenes. The crude sample was further purified by recycling high‐speed counter‐current chromatography with n‐hexane–ethyl acetate–methanol–water (7:12:11:5, v/v/v/v) as the optimized two‐phase solvent system. A 16.4 mg aliquot of ganoderol B with a purity of 90.4% was separated from 300 mg of the crude sample in a single run. After employing the recycling elution mode of HSCCC with n‐hexane–ethyl acetate–methanol–water (6:10:8:4.5, v/v/v/v) for five cycles, 25.7 mg ganoderic acid T and 3.7 mg ganoderic acid S with purities of 97.8 and 83.0%, respectively, were obtained. The purities of three compounds were determined by high‐performance liquid chromatography and their chemical structures were identified by NMR and MS data.     

Rapid separation of lactucin and lactucopicrin from Cichorium glandulosum by medium-pressure preparative liquid chromatography and quantitative analyses by high-performance thin-layer chromatography

Medium-pressure preparative liquid chromatography (MPPLC) was used to isolate and prepare lactucin and lactucopicrin from the whole herb of Cichorium glandulosum. After extracting the methanolic extract of the whole plant with petroleum ether and ethyl acetate several times to obtain ethyl acetate extract, the crude products, namely, lactucin and lactucopicrin were separated using MPPLC and thin-layer chromatography (TLC) tracking, and their purity rates reached more than 80%. The qualitative and quantitative analyses of lactucin and lactucopicrin were carried out by high-performance thin-layer chromatography (HPTLC) on the whole herb of C. glandulosum. The contents of lactucin and lactucopicrin were determined by scanning at 256 nm in the whole herb of C. glandulosum. The R_F values of lactucin and lactucopicrin were 0.42 ± 0.05 and 0.65 ± 0.05 with linear ranges of 0.498–2.988 and 0.499–2.994 μg/band, respectively. The correlation coefficients were 0.9938 and 0.9946, respectively, thereby showing a good linear relationship. The average recoveries were 99.96% and 99.52%, and the relative standard deviations (RSDs) were 2.49% and 2.45%, respectively. The crude products, namely, lactucin and lactucopicrin, can be isolated with high purity from the whole herbs of C. glandulosum by MPPLC. The lactucin and lactucopicrin contents of C. glandulosum can be determined rapidly and accurately by HPTLC.

     

Senecipyrrolidine,an unusual pyrrolidine alkaloid isolated from Jacobaea gigantea (Desf.) Pelser (Asteraceae)

Alkaloids and phenolic compounds are among the most biologically active natural products from the Jacobaea/Senecio genera (Asteraceae). To isolate original natural products directly from Jacobaea gigantea crude polar extracts, centrifugal partition chromatography (CPC) was used. Previously, we reported the phytochemical study of J. gigantea (syn. Senecio giganteus) n-butanol extract using various classical chromatographical techniques combined with CPC. Herein major constituents from the J. gigantea crude ethyl acetate extract and further compounds from the n-butanol extract were purified in only one step using this technique. A new pyrrolidine alkaloid, named senecipyrrolidine was isolated along with thirteen known compounds – chiro-inositol, three phenolic acids, six flavonoids, two quinones and emiline, another pyrrolidine alkaloid – from crude n-butanol or ethyl acetate extracts. Pyrrolidine alkaloids were isolated for the first time in the Jacobaea/Senecio genera and were probably biogenetically related to the two isolated quinones derivatives jacaranone and 3a-hydroxy-3,3a,7,7a-tetrahydrobenzofuran-2,6-dione, isolated in this species.     

Separation of three phenolic high‐molecular‐weight compounds from the crude extract of Terminalia Chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography

This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography. The ultrasound‐assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high‐speed counter‐current chromatography without any pretreatment using n‐hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by ¹H NMR spectroscopy.     

Droplet counter-current chromatography for the preparative isolation of various glycosides

Droplet counter-current chromatography has proven to be an efficient method for separating polar compounds in the range of a few mg to gram quantities, and in this respect is complementary to the popular high-performance liquid chromatography. This is exemplified in the following by the direct obtention of four bioactive saponins in pure form from the crude extract of Hedera helix berries, isolation of an extremely unstable catechin glycoside from Dalbergia nitidula and the separation of flavone glycosides. A rapid thin-layer chromatography method for selecting the solvent system is also described.     

An efficient high‐speed countercurrent chromatography method for preparative separation of javanicin from Fusarium solani,a fungus isolated from the fruiting body of the mushroom Trametes trogii

To develop an efficient method for large preparation of javanicin from Fusarium solani, a rapid and simple method by high‐speed countercurrent chromatography was established based on average polarity (P′ values) and partition coefficients (K values) of crude samples. A suitable solvent system for high‐speed countercurrent chromatography was selected from many possible biphasic solvent systems. HSCCC was successfully applied to separate and purify javanicin, the main bioactive component of solid cultures of the fungus F. solani isolated from the fruiting body of Trametes trogii, with petroleum ether–ethyl acetate–methanol–water (4:3:2:1, v/v) as solvent system. A total amount of 40.6 mg of javanicin was obtained from 100 mg crude sample. The purity of javanicin was 92.2% with a recovery of 95.1%, as determined by high‐performance liquid chromatrography. The molecular structure was identified primarily by NMR and MS methods. The results indicated that high‐speed countercurrent chromatography could be a powerful technology for separating naphthoquinones from the solid cultures of the fungus F. solani. It is also of significance that the separation of javanicin from natural source was carried out for the first time utilizing high‐speed countercurrent chromatography.     

Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter‐current chromatography

Resveratrol dimers belong to a group of compounds called stilbenes, which along with proanthocyanidins, anthocyanins, catechins, and flavonols are natural phenolic compounds found in grapes and red wine. Stilbenes have a variety of structural isomers, all of which exhibit various biological properties. Counter‐current chromatography with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (2:5:4:5, v/v/v/v) was applied to isolate and purify stilbene from the stems of wine grape. Two isomers of resveratrol dimers trans‐ε‐viniferin and trans‐δ‐viniferin were obtained from the crude sample in a one‐step separation, with purities of 93.2 and 97.5%, respectively, as determined by high‐performance liquid chromatography. The structures of these two compounds were identified by ¹H and ¹³C NMR spectroscopy. In addition, their antioxidant activities were assessed by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay. The antioxidant activities of trans‐δ‐viniferin were higher than that of trans‐ε‐viniferin in this model. This work demonstrated that counter‐current chromatography is a powerful and effective method for the isolation and purification of polyphenols from wine grape. Additionally, the DPPH radical assay showed that the isolated component trans‐δ‐viniferin exhibited stronger antioxidant activities than trans‐ε‐viniferin and a little bit weaker than vitamin E at the same concentration.     

p-Hydroxymercuribenzoate as thiol-protein modifier for simultaneous determination of glycolytic enzymes by hydrophobic-interaction chromatography

Summary The use of hydrophobic-interaction chromatography (HIC) is proposed for the simultaneous determination of more than one thiol-protein after formation of the corresponding mercury mercaptides withp-hydroxymercuribenzoate (PHMB). The new chromatographic procedure, based on the HIC separation of the modified proteins from each other and from excess organomercury reagent has been successfully applied to the quantitative determination of phosphoglucose isomerase (PGI) and phosphoglucose mutase (PGM) in crude PGI powder, and of L-lactate dehydrogenase, PGM and aldolase in crude pyruvate kinase from rabbit muscle. The suitability of²⁰³Hg-labelled PHMB has been tested in the analysis of mixtures, which give barely distinguishable UV-peaks owing to the presence of other non-thiol components in the sample. For this purpose glyceraldehyde 3-phosphate dehydrogenase (GAPDHy) and PGIy from bakers yeast have been considered. Results obtained in experiments performed by both procedures are reported.     

Retine Part I. Purification and Partial Characterization of Retine from Pig Liver

A modified method has been developed to purify retine from pig liver. The crude extract of retine can be separated into five fractions by paper chromatography, each of them having been tested for the effect on the proliferation of protozoal Tetrahymena Pyriformis. One of the fractions, G₃, has the strongest effect. The present results show that G₃ fraction showed positive reactions toward 2,4-dinitro-phenyl hydrazine, ethylene diamine, and ninhydrin. Other properties of the fraction has also been studied.     

LC–DAD–ESI-MS-MS for Characterization and Quantitative Analysis of Diterpenoids from Coleus forskohlii

Qualitative characterization and quantitative analysis of labdane diterpenoids from Coleus forskohlii have been achieved by liquid chromatography hyphenated with photodiode-array detection and tandem electrospray ionization mass spectrometry (LC–DAD–ESI-MSⁿ). By use of this method, thirteen forskolin-type diterpenoids were identified in the crude extract on the basis of their fragmentation mechanisms. Fragmentation rules were deduced from nine forskolin-type standards by ESI-MS in positive-ion mode. It was found that fragmentation behavior varied with the position and number of the substituents on the skeleton; this could be used for convenient identification of this type of compound. Six marker diterpenoids were also quantified, and the quality of both the cultivated and wild plants was evaluated.

     

Direct Obtention of Pure Compounds from Crude Plant Extracts by Preparative Liquid Chromatography

It has been possible to submit the crude hexane extract of the barks of a medicinal plant directly to preparative liquid chromatography. A two step solvent elution gave 6 peaks, four of which readily afforded crystalline material. The entire process required 2–3 hours whereas conventional chromatography required ca. 2 weeks.     

Rapid screening and identification of caffeic acid and its esters in Erigeron breviscapus by ultra‐performance liquid chromatography/tandem mass spectrometry

Caffeic acid and its esters (CAEs) are widely distributed in the plant kingdom and have been reported to elicit a wide range of exceptional biological activities. Present methods for screening and characterization of CAEs normally need the use of liquid chromatography diode‐array detection/multistage mass spectrometry (LC‐DAD/MSⁿ). In this report, a rapid and efficient method coupling ultra‐performance liquid chromatography (UPLC) with fragment‐targeted multi‐reaction monitoring (MRM) has been developed for screening CAEs in a crude extract of Erigeron breviscapus, while a UPLC/quasi‐MSⁿ method has been applied in the structural identification of these compounds. Furthermore, a simple quasi‐UPLC/MS/MS method based on in‐source collision‐induced dissociation (CID) has been proposed for rapid identification of the CAEs. As a result, a total of more than 34 CAEs were detected and their structures characterized. Nine of them were reported from E. breviscapus for the first time. Applications of these strategies in the chemical investigation of an injection of E. breviscapus resulted in the identifications of 16 CAEs. These strategies, if appropriate modifications are made, will be very useful in screening and characterization not only of CAEs, but of other structural types of compounds in various complex matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Gradient elution method in centrifugal partition chromatography for the separation of a complex sophorolipid mixture obtained from Candida bombicola yeasts

Sophorolipids represent an important class of natural surfactants with a variety of environmental, cosmetic, and pharmaceutical applications. Despite their promising physicochemical and biological properties, the use of sophorolipids is hampered by the lack of information regarding their individual structure‐activity relationships. The major difficulty in isolating pure sophorolipids arises from the high complexity of crude fermentation media composition and from their strong structural similarities. In this work, a centrifugal partition chromatography method was developed in an original gradient elution mode for the separation of sophorolipids produced by the yeast Candida bombicola. Experiments were realized by using three sets of solvent systems composed of n‐heptane, ethyl acetate, n‐butanol, methanol, and water in different proportions. The separation was performed at 5 mL/min in the ascending mode by increasing progressively the polarity of the organic mobile phase. In these conditions, more than 80% of the sophorolipids present in the initial crude fermentation extract were eluted successively from the most hydrophobic lactone forms to the most hydrophilic acid forms. The structures of the isolated sophorolipids were further elucidated by HPLC and NMR analyses.     

Preparative isolation of neomangiferin and mangiferin from Rhizoma anemarrhenae by high‐speed countercurrent chromatography using ionic liquids as a two‐phase solvent system modifier

A preparative high‐speed countercurrent chromatography method for isolation and purification of neomangiferin and mangiferin from Rhizoma anemarrhenae was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Neomangiferin and mangiferin were purified from the crude extract of R. anemarrhenae by using ethyl acetate‐water‐[C₄mim][PF₆] (5:5:0.2 v/v) as two‐phase solvent system. In total, 22.5 mg of neomangiferin and 70.6 mg of mangiferin were obtained from 150 mg of the crude extract. The purities of neomangiferin and mangiferin were 97.2 and 98.1%, respectively, as determined by HPLC. The chemical structures of the isolated compounds were identified by ¹H‐NMR and ¹³C‐NMR.