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Analytical and Preparative Hyphenation of Countercurrent Chromatography (CCC/CPC) to Structure Elucidation Methods in the Field of Natural Product Recovery and Identification

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Natural Products Chemistry".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 20448

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Dr. Gerold Jerz

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Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany
Interests: off-line/ on-line hyphenation of countercurrent (CCC) and Centrifugal Partition Chromatography (CPC) to structure elucidation method; two-dimensional metabolite analysis; food and medicinal plant analysis; structural elucidation of betacyanin pigments and saponins; evaluation of biological activities; natural products from agricultural residues
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Dear Colleagues,

Since Dr. Yoichiro Ito (NIH, Bethesda, USA) introduced “the coil planet centrifuge” in 1966 (published in Nature), countercurrent chromatography and related techniques have started to work at a higher efficiency using strong and fast-changing gravitation force fields. This technology was originally designed for the separation of human blood cells but then evolved rapidly into a powerful fractionation tool for the preparative recovery of bioactive products from all kinds of natural resources. The use of immiscible solvent systems, specifically designed for a current separation problem, is one of the unique features of this methodology where mobile and stationary chromatographic phases are in the liquid state.

Looking back over a development period of more than fifty years, many innovative approaches in CCC/CPC have evolved, including powerful system designs. The practicability of natural product chemists holds potential for the direct implementation of structure elucidation technology for the fast identification of target metabolites.

Therefore, this Special Issue will cover “Analytical and Preparative Hyphenation of Countercurrent Chromatography (CCC/CPC) to Structure Elucidation Methods in the Field of Natural Product Recovery and Identification” and will collect original research/review articles dealing with innovative developments in this field.

Some selected topics from the keyword compilation mentioned here are off-line and on-line hyphenation experiments to CCC/CPC using sensitive analytical detection methods, such as liquid mass-spectrometry (LC-ESI-, APCI-, and MALDI-MS) or gas chromatography (GC-MS) for molecular-weight based target recovery; structural elucidation by 1D/2D-NMR for direct identification or compound mapping; two dimensional CCC/CPC (heart-cutting) applications; and orthogonal couplings such as CCC/CPC to LC-MS or to GC-MS for deeper investigation of metabolite profiles of natural product extracts.

Another topic of relevance is the combination of CCC/CPC with bio-assay-guided fractionation methods for lead-structure search and drug development.

Dr. Gerold Jerz
Guest Editor

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Keywords

Off-line or on-line hyphenation of CCC/CPC to structure elucidation
methods (mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR))
Two-dimensional CCC or CPC (heart-cutting)
Two-dimensional metabolite analysis (CCC/CPC x GC or LC-MS)
Bio-assay guided CCC/CPC isolation procedures

Published Papers (9 papers)

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Research

12 pages, 1972 KiB

Open AccessArticle

Target-Guided Isolation of Progenitors of 1,1,6-Trimethyl-1,2-dihydronaphthalene (TDN) from Riesling Wine by High-Performance Countercurrent Chromatography

by Recep Gök, Pia Selhorst, Mats Kiene, Theresa Noske, Michael Ziegler, Ulrich Fischer and Peter Winterhalter

Molecules 2022, 27(17), 5378; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27175378 - 23 Aug 2022

Cited by 2 | Viewed by 1505

Abstract

High-performance countercurrent chromatography (HPCCC) was used for the target-guided isolation of precursors of 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) from Riesling wine. In separated HPCCC fractions of an Amberlite^® XAD^®-2 extract obtained from a German Riesling, TDN-generating fractions were identified by the acid-catalyzed hydrolysis of the progenitors at pH 3.0 and subsequent HS-GC-MS/MS analysis. The presence of multiple TDN-generating precursors in Riesling wine could be confirmed. From polar HPCCC fractions (11–13 and 14–16), 3,4-dihydroxy-7,8-dihydro-β-ionone 3-O-rutinoside and 3,4-dihydroxy-7,8-dihydro-β-ionone 3-O-β-d-glucopyranoside were isolated as major TDN-precursors at a sufficient amount for structure elucidation by NMR spectroscopic studies. In the medium polar HPCCC factions (27–35), enzymatic hydrolysis liberated the aglycones 3-hydroxy-β-ionone and 3-hydroxy-TDN in minor amounts. In further less polar TDN-generation fractions (36–44 and 45–50), glycosidic progenitors were absent; instead, a minor TDN formation most likely from non-conjugated constituents was observed. Full article

(This article belongs to the Special Issue Analytical and Preparative Hyphenation of Countercurrent Chromatography (CCC/CPC) to Structure Elucidation Methods in the Field of Natural Product Recovery and Identification)

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14 pages, 2247 KiB

Open AccessArticle

Separation of Dihydro-Isocoumarins and Dihydro-Stilbenoids from Hydrangea macrophylla ssp. serrata by Use of Counter-Current Chromatography

by Johannes Wellmann, Beate Hartmann, Esther-Corinna Schwarze, Silke Hillebrand, Stephan I. Brueckner, Jakob Ley, Gerold Jerz and Peter Winterhalter

Molecules 2022, 27(11), 3424; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27113424 - 26 May 2022

Cited by 3 | Viewed by 1614

Abstract

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar ‘Yae-no-amacha’ using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data. Full article

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14 pages, 5941 KiB

Open AccessArticle

Bioassay-Guided Fractionation of Siparuna glycycarpa n-Butanol Extract with Inhibitory Activity against Influenza A(H1N1)pdm09 Virus by Centrifugal Partition Chromatography (CPC)

by Carla Monteiro Leal, Suzana Guimarães Leitão, Leonardo Luiz Oliveira de Mello, Isabel de Castro Rangel, Carlos Vinicius Azevedo da Silva, Milene Dias Miranda, Amanda Resende Tucci, Camilla Blanco de Assis, Carolina de Queiroz Sacramento, Natalia Fintelman-Rodrigues, Hector Henrique Ferreira Koolen, Boniek Gontijo Vaz, Rosineide Costa Simas and Gilda Guimarães Leitão

Molecules 2022, 27(2), 399; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27020399 - 08 Jan 2022

Cited by 3 | Viewed by 2235

Abstract

Siparuna glycycarpa occurs in the Amazon region, and some species of this genus are used in Brazilian folk medicine. A recent study showed the inhibitory effect of this species against influenza A(H1N1)pdm09 virus, and in order to acquire active fractions, a polar solvent system n-butanol-methanol-water (9:1:10, v/v) was selected and used for bioassay-guided fractionation of n-butanol extract by centrifugal partition chromatography (CPC). The upper phase was used as stationary phase and the lower phase as mobile (descending mode). Among the collected fractions, the ones coded SGA, SGC, SGD, and SGO showed the highest antiviral inhibition levels (above 74%) at 100 µg·mL⁻¹ after 24 h of infection. The bioactive fractions chemical profiles were investigated by LC-HRMS/MS data in positive and negative ionization modes exploring the Global Natural Products Social Molecular Networking (GNPS) platform to build a molecular network. Benzylisoquinoline alkaloids were annotated in the fractions coded SGA, SGC, and SGD collected during elution step. Aporphine alkaloids, O-glycosylated flavonoids, and dihydrochalcones in SGO were acquired with the change of mobile phase from lower aqueous to upper organic. Benzylisoquinolinic and aporphine alkaloids as well as glycosylated flavonoids were annotated in the most bioactive fractions suggesting this group of compounds as responsible for antiviral activity. Full article

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14 pages, 1805 KiB

Open AccessArticle

Isolation of N-Ethyl-2-pyrrolidinone-Substituted Flavanols from White Tea Using Centrifugal Countercurrent Chromatography Off-Line ESI-MS Profiling and Semi-Preparative Liquid Chromatography

by Weidong Dai, Maria Ramos-Jerz, Dongchao Xie, Jiakun Peng, Peter Winterhalter, Gerold Jerz and Zhi Lin

Molecules 2021, 26(23), 7284; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26237284 - 30 Nov 2021

Cited by 4 | Viewed by 1765

Abstract

N-Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5′′′R- and 5′′′S-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (R-EGCG-cThea and S-EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. N-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) and 2. N-hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, v/v/v/v) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite partition ratio K_D values, selectivity factors α, and resolution factors R_S. After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of R-EGCG-cThea and 2.9 mg of S-EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that R- and S-EGCG-cThea possessed in vitro inhibition effects on α-glucosidase with IC₅₀ of 70.3 and 161.7 μM, respectively. Full article

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12 pages, 1837 KiB

Open AccessArticle

Combined Ultrahigh Pressure Extraction and High-Speed Counter-Current Chromatography for Separation and Purification of Three Glycoside Compounds from Dendrobium officinale Protocorm

by Wei Zhang, Yingjie Zhang, Jinying Wang, Wenjuan Duan and Feng Liu

Molecules 2021, 26(13), 3934; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26133934 - 28 Jun 2021

Cited by 2 | Viewed by 1784

Abstract

As an alternative to Dendrobium candidum, protocorm-like bodies (PLBs) of Dendrobium candidum are of great value due to their high yield and low cost. In this work, three glycoside compounds, β-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] (I), β-D-glucopyranose 1-[(E)-3-(3, 4-dihydroxyphenyl)-2-propenoat] (II), and 1-O-sinapoyl glucopyranoside (III), were extracted and isolated by ultrahigh pressure extraction (UPE) coupled with high-speed counter-current chromatography (HSCCC) from PLBs of D. officinale. First, the target compounds were optimized and prepared with 50% ethanol solution at a 1:30 (g/mL) solid/liquid ratio in 2 min under 300 MPa by UPE. Then, the crude extract was chromatographed with a silica gel column, and primary separation products were obtained. In addition, the products (150 mg) were separated by HSCCC under the solvent system of MTBE-n-butyl alcohol-acetonitrile-water (5:1:2:6, v/v/v/v), yielding 31.43 mg of compound I, 10.21 mg of compound II, and 24.75 mg of compound III. Their structures were further identified by ESI-MS, ¹H NMR, and ¹³C NMR. The antioxidant results showed that the three compounds expressed moderate effects on the DPPH· scavenging effect. Compound II had the best antioxidant capacity and its IC₅₀ value was 0.0497 mg/mL. Full article

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11 pages, 1469 KiB

Open AccessArticle

Scale-Up Preparation of Crocins I and II from Gardeniajasminoides by a Two-Step Chromatographic Approach and Their Inhibitory Activity Against ATP Citrate Lyase

by Shuguang Guan, Qiaoli Pu, Yinan Liu, Honghong Wu, Wenbo Yu, Zifeng Pi, Shu Liu, Fengrui Song, Jingya Li and De-An Guo

Molecules 2021, 26(11), 3137; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26113137 - 24 May 2021

Cited by 4 | Viewed by 1845

Abstract

Crocins are highly valuable natural compounds for treating human disorders, and they are also high-end spices and colorants in the food industry. Due to the limitation of obtaining this type of highly polar compound, the commercial prices of crocins I and II are expensive. In this study, macroporous resin column chromatography combined with high-speed counter-current chromatography (HSCCC) was used to purify crocins I and II from natural sources. With only two chromatographic steps, both compounds were simultaneously isolated from the dry fruit of Gardenia jasminoides, which is a cheap herbal medicine distributed in a number of countries. In an effort to shorten the isolation time and reduce solvent usage, forward and reverse rotations were successively utilized in the HSCCC isolation procedure. Crocins I and II were simultaneously obtained from a herbal resource with high recoveries of 0.5% and 0.1%, respectively, and high purities of 98.7% and 99.1%, respectively, by HPLC analysis. The optimized preparation method was proven to be highly efficient, convenient, and cost-effective. Crocins I and II exhibited inhibitory activity against ATP citrate lyase, and their IC₅₀ values were determined to be 36.3 ± 6.24 and 29.7 ± 7.41 μM, respectively. Full article

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17 pages, 3248 KiB

Open AccessArticle

Laguncularia racemosa Phenolics Profiling by Three-Phase Solvent System Step-Gradient Using High-Performance Countercurrent Chromatography with Off-Line Electrospray Mass-Spectrometry Detection

by Fernanda das Neves Costa, Gerold Jerz, Peter Hewitson, Fabiana de Souza Figueiredo and Svetlana Ignatova

Molecules 2021, 26(8), 2284; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26082284 - 15 Apr 2021

Cited by 8 | Viewed by 2070

Abstract

The detailed metabolite profiling of Laguncularia racemosa was accomplished by high-performance countercurrent chromatography (HPCCC) using the three-phase system n-hexane–tert-butyl methyl ether–acetonitrile–water 2:3:3:2 (v/v/v/v) in step-gradient elution mode. The gradient elution was adjusted to the chemical complexity of the L. racemosa ethyl acetate partition and strongly improved the polarity range of chromatography. The three-phase solvent system was chosen for the gradient to avoid equilibrium problems when changing mobile phase compositions encountered between the gradient steps. The tentative recognition of metabolites including the identification of novel ones was possible due to the off-line injection of fractions to electrospray ionization mass spectrometry (ESI-MS/MS) in the sequence of recovery. The off-line hyphenation profiling experiment of HPCCC and ESI-MS projected the preparative elution by selected single ion traces in the negative ionization mode. Co-elution effects were monitored and MS/MS fragmentation data of more than 100 substances were used for structural characterization and identification. The metabolite profile in the L. racemosa extract comprised flavonoids, hydrolysable tannins, condensed tannins and low molecular weight polyphenols. Full article

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21 pages, 2915 KiB

Open AccessArticle

Polyphenolic Compounds Extracted and Purified from Buddleja Globosa Hope (Buddlejaceae) Leaves Using Natural Deep Eutectic Solvents and Centrifugal Partition Chromatography

by Jeniffer Torres-Vega, Sergio Gómez-Alonso, José Pérez-Navarro, Julio Alarcón-Enos and Edgar Pastene-Navarrete

Molecules 2021, 26(8), 2192; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26082192 - 10 Apr 2021

Cited by 14 | Viewed by 3586

Abstract

Chemical profiling of Buddleja globosa was performed by high-performance liquid chromatography coupled to electrospray ionization (HPLC-DAD-ESI-IT/MS) and quadrupole time-of-flight high-resolution mass spectrometry (HPLC-ESI-QTOF/MS). The identification of 17 main phenolic compounds in B. globosa leaf extracts was achieved. Along with caffeoyl glucoside isomers, caffeoylshikimic acid and several verbascoside derivatives (β-hydroxyverbascoside and β-hydroxyisoverbascoside) were identified. Among flavonoid compounds, the presence of 6-hydroxyluteolin-7-O-glucoside, quercetin-3-O-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside was confirmed. Campneoside I, forsythoside B, lipedoside A and forsythoside A were identified along with verbascoside, isoverbascoside, eukovoside and martynoside. The isolation of two bioactive phenolic compounds verbascoside and forsythoside B from Buddleja globosa (Buddlejaceae) was successfully achieved by centrifugal partition chromatography (CPC). Both compounds were obtained in one-step using optimized CPC methodology with the two-phase solvent system comprising ethyl acetate-n-butanol-ethanol-water (0.25:0.75:0.1:1, v/v). Additionally, eight Natural Deep Eutectic Solvents (NADESs) were tested for the extraction of polyphenols and compared with 80% methanol. The contents of verbascoside and luteolin 7-O-glucoside after extraction with 80% methanol were 26.165 and 3.206 mg/g, respectively. Among the NADESs tested in this study, proline- citric acid (1:1) and choline chloride-1, 2- propanediol (1:2) were the most promising solvents. With these NADES, extraction yields for verbascoside and luteolin 7-O-glucoside were 51.045 and 4.387 mg/g, respectively. Taken together, the results of this study confirm that CPC enabled the fast isolation of bioactive polyphenols from B. globosa. NADESs displayed higher extraction efficiency of phenolic and therefore could be used as an ecofriendly alternative to classic organic solvents. Full article

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10 pages, 1610 KiB

Open AccessArticle

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography

by Ji Hoon Kim, Eun Ju Jung, Yun Jung Lee, En Mei Gao, Ahmed Shah Syed and Chul Young Kim

Molecules 2020, 25(13), 3077; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25133077 - 06 Jul 2020

Cited by 5 | Viewed by 2701

Abstract

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane–acetonitrile–water (10:2:8, v/v), ethyl acetate–acetonitrile–water (10:2:8, v/v), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v). The lower phase of the n-hexane–acetonitrile–water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate–acetonitrile–water (10:2:8), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products. Full article

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Analytical and Preparative Hyphenation of Countercurrent Chromatography (CCC/CPC) to Structure Elucidation Methods in the Field of Natural Product Recovery and Identification

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