Gerold Jerz

Isolation of dammarane saponins from Panax notoginseng by high-speed counter-current chromatography

The Chinese phytomedicinal formulation Sanqi Zongdai Pian, traditionally prepared from crude extracts from roots of Panax notoginseng (Araliaceae), contains highly polar dammarane saponins which were separated at a preparative scale using high-speed counter-current chromatography (HSCCC). In each operation, 283 mg methanolic extract of five tablets was separated and yielded pure 157, 17, 13 and 56 mg of ginsenoside-Rb1, notoginsenoside-R1, ginsenoside-Re and ginsenoside-Rg1, respectively, n-hexane-n-butanol-water (3:4:7, v/v/v) was used for the two-phase solvent system of the HSCCC separation. The chemical structures of three ginsenosides and one notoginsenoside were elaborated by means of electrospray ionization MS-MS and NMR analysis.

Separation of andrographolide and neoandrographolide from the leaves of Andrographis paniculata using high-speed counter-current chromatography

The bioactive diterpenes andrographolide and neoandrographolide from the leaves of Andrographis paniculata NEES (Acanthaceae) were successfully separated by counter-current chromatography. A single 280-min separation yielded 189 mg of 99.9% andrographolide and 9.5 mg of 98.5% neoandrographolide applying water-methanol-ethyl acetate-n-hexane (2.5:2.5:4:1) solvent system. Structure confirmation was done by electrospray MS, one-dimensional NMR experiments, circular dichroism, optical rotation dispersion, and specific optical rotation [alpha]D.

Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography.

The first preparative fractionation of betalain pigments by means of ion-pair high-speed counter-current chromatography (IP-HSCCC) from berry extracts of Phytolacca americana (Phytolaccaceae) is presented. A novel HSCCC solvent system consisting of 1-butanol-acetonitrile-water (5:1:6, v/v/v) was applied using ion-pair forming trifluoroacetic acid at low concentration (0.7%, v/v). Affinity of polar betacyanins and betaxanthins to the organic stationary phase of the biphasic HSCCC solvent mixture was considerably improved. Partitioning coefficient values and influence of increasing trifluoroacetic acid additions to the biphasic solvent mixture were measured for all identified betacyanins and betaxanthins. Gentle separation by IP-HSCCC of the injected pigment extract (900 mg) yielded sufficient amounts of the principal pigments 15S-betanin/15R-isobetanin. The pure epimers separated by C18-HPLC were immediately studied by one- and two-dimensional NMR. In the recovered fractions, minor concentrated betacyanins and betaxanthins were significantly enriched by IP-HSCCC and were detected for the first time in the extracts of P. americana. IP-HSCCC and C18-HPLC were shown to be complementary techniques in the isolation procedure of recovering minor concentrated, highly polar and chemically instable betacyanins and betaxanthin from complex plant matrices. Altogether, identification of 17 betalains was achieved by HPLC-diode array detection-electrospray ionization MS/MS in the HSCCC fractions with their respective isomers, also resulting in the tentative elucidation of betacyanins with novel salicylic acid substitution pattern in the berry extracts of P. americana.

Preparation of Ursane Triterpenoids from Centella asiatica Using High Speed Countercurrent Chromatography with Step‐Gradient Elution

Abstract Pentacyclic triterpene aglycones and glycosides of the ursane type were successfully separated from 600 mg of a polar extract from Centella asiatica (Apiaceae) by high speed countercurrent chromatography (HSCCC), applying a mobile phase gradient with a step‐wise increase of elution strength. The lower phase of the biphasic liquid system composed of n‐hexane–n‐butanol–0.05 M NaOH (5:1:6, v/v/v) was used as the stationary phase, the upper phase was used as the initial mobile phase. The mobile phase was changed systematically into 1:1, 1:2 and 1:4 consisting of n‐hexane–n‐butanol saturated by 0.05 M NaOH. The separation mainly yielded five fractions with asiatic acid (18 mg), madecassic acid (13 mg), asiaticoside (140 mg), and madecassoside (75 mg). The chemical structures of the four compounds were confirmed by means of electrospray ionization ion trap multiple mass spectrometry (ESI–MS–MS) and NMR analysis.

Isolation of isomangiferin from honeybush (Cyclopia subternata) using high-speed counter-current chromatography and high-performance liquid chromatography.

Isomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid-liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid-liquid partitioning with ethyl acetate-methanol-water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether-n-butanol-acetonitrile-water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC-high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.

Target-guided separation of Bougainvillea glabra betacyanins by direct coupling of preparative ion-pair high-speed countercurrent chromatography and electrospray ionization mass-spectrometry

In this study, preparative ion-pair high-speed countercurrent chromatography was directly coupled to an electrospray ionization mass-spectrometry device (IP-HSCCC/ESI-MS-MS) for target-guided fractionation of high molecular weight acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra (Nyctaginaceae). The direct identification of six principal acyl-oligosaccharide linked betacyanins in the mass range between m/z 859 and m/z 1359 was achieved by positive ESI-MS ionization and gave access to the genuine pigment profile already during the proceeding of the preparative separation. Inclusively, all MS/MS-fragmentation data were provided during the chromatographic run for a complete analysis of substitution pattern. On-line purity evaluation of the recovered fractions is of high value in target-guided screening procedures and for immediate decisions about suitable fractions used for further structural analysis. The applied preparative hyphenation was shown to be a versatile screening method for on-line monitoring of countercurrent chromatographic separations of polar crude pigment extracts and also traced some minor concentrated compounds. For the separation of 760mg crude pigment extract the biphasic solvent system tert.-butylmethylether/n-butanol/acetonitrile/water 2:2:1:5 (v/v/v/v) was used with addition of ion-pair forming reagent trifluoroacetic acid. The preparative HSCCC-eluate had to be modified by post-column addition of a make-up solvent stream containing formic acid to reduce ion-suppression caused by trifluoroacetic acid and later significantly maximized response of ESI-MS/MS detection of target substances. A variable low-pressure split-unit guided a micro-eluate to the ESI-MS-interface for sensitive and direct on-line detection, and the major volume of the effluent stream was directed to the fraction collector for preparative sample recovery. The applied make-up solvent mixture significantly improved smoothness of the continuously measured IP-HSCCC-ESI-MS base peak ion trace in the experimental range of m/z 50-2200 by masking stationary phase bleeding and generating a stable single solvent phase for ESI-MS/MS detection. Immediate structural data were retrieved throughout the countercurrent chromatography run containing complete MS/MS-fragmentation pattern of the separated acyl-substituted betanidin oligoglycosides. Single ion monitoring indicated clearly the base-line separation of higher concentrated acylated betacyanin components.

Metabolite profiling of polyphenols in peels of Citrus limetta Risso by combination of preparative high-speed countercurrent chromatography and LC–ESI–MS/MS

The polar constituents of peels from Citrus limetta variety Risso (Rutaceae) were investigated by a combination of two complementary chromatographic techniques consisting of preparative high-speed countercurrent chromatography (HSCCC), and off-line LC-ESI-MS/MS analysis to design a two-dimensional metabolite profile. Countercurrent chromatography (CCC) using solely immiscible solvent systems allowed the fractionation of principal components and an enrichment of minor concentrated metabolites from a crude polar solvent partition of C. limetta peels for subsequent structural identification by LC-ESI-MS/MS analysis. The combination of two very different chromatographic techniques resulted in lower detection limits for electrospray mass-spectrometry and revealed eighty-five compounds, including three abscisic acid derivatives, five limonoid glycosides, twenty-six dihydro-cinnamic and cinnamic acid glycosides, eleven flavanone glycosides, seven flavone glycosides, seventeen flavonol glycosides, including limocitrol and limocitrin derivatives. As a chemocharacteristic for C. limetta metabolites, many of the detected structures were linked to single and multiple 3-hydroxy-3-methyl-glutaryl (HMG) substitutions. C. limetta peels are a by-product of juice production, and not only the antioxidant fractions but also some of the fortified compounds could be used for food and pharmaceutical purposes.

Activity-guided isolation of resveratrol oligomers from a grapevine-shoot extract using countercurrent chromatography.

An activity-guided isolation of bioactive stilbenes has been carried out with the grapevine-shoot extract Vineatrol 30. After hexane precipitation of the polymeric constituents, the stilbene mixture was separated on a preparative scale using low-speed rotary countercurrent chromatography (LSRCCC). The antiproliferative activity of the separated LSRCCC fractions was then screened in the human cancer cell line A-431, and trans-resveratrol, trans-ε-viniferin, r-2-viniferin, hopeaphenol, and miyabenol C were identified as active principles. In addition, a new class of stilbene derivatives, which exhibit a γ-lactam ring structure and exert a weak growth-inhibiting activity in A-431 cells, has been identified.

Di-2-ethylhexyl phthalate in the fruits of Benincasa hispida

Di-2-ethylhexyl phthalate (DEHP) is a commonly used plasticizer that is harmful to human health. magnetic resonance spectroscopy from the edible fruit flesh of Benincasa hispida (wax gourd) of the plant family Curcurbitaceae. The DEHP content of seven wax gourd samples collected from southern and northern provinces in China was determined as (mean ± SD): 18.3 ± 0.43, 2.64 ± 0.44, 44.0 ± 0.34, 62.5 ± 0.48, 52.0 ± 0.42, 58.3 ± 0.55 and 75.5 ± 0.63 mg kg−1 fresh weight, respectively, indicating that most wax gourds were severely contaminated with DEHP.

Pyrrolizidine alkaloids in herbal teas for infants, pregnant or lactating women.

A general contamination of tea with pyrrolizidine alkaloids (PA) has just become known. Here, we report the application and modification of a new HPLC-ESI-MS/MS sum parameter method to quantitate PA content of herbal teas intended for infants, pregnant and lactating women. Using p-toluenesulfonyl isocyanate for derivatization and a stable isotope labeled internal standard, the total retronecine-/heliotridine-type PA content of the samples is expressed in form of a single sum parameter (retronecine equivalents: RE). The new methods were applied to analyze 44 tea samples for such consumer groups. Thirty eight products (86%) were tested PA positive showing PA concentrations ranging from 0 to 391 μg RE/kg (average: 50 μg RE/kg). The dataset is discussed in the view of the current discussion on PA in the food chain with special focus on those particular vulnerable consumer groups.