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

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.

SUBMITTER: Kim JH

PROVIDER: S-EPMC7412496 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Application of Linear Gradient Solvent System in Centrifugal Partition Chromatography Facilitating Bioassay-Guided Fractionation of Yongdamsagan-Tang, Traditional Oriental Decoction.

Project description:As important pharmaceutical resources, traditional herbal medicines retain continuous attention. To do that, isolation and identification of bioactive molecules from traditional herbal decoction are important. However, conventional fractionation through octadecyl silica column faces irreversible sample adsorption that causes a bias in bioactivity assessment. However, liquid-liquid chromatographic system suffers tedious K value calculation as well as insufficient capacity in separation power when crude extract composed of widely ranging polarities. Here, we developed a comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) to aid bioassay-guided isolation. The lower aqueous phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary, whereas its upper organic phase followed by the upper phase of ethyl acetate-acetonitrile-water and water-saturated n-butanol-acetonitrile-water in the same ratio were eluted in a linear gradient mode, thereby increasing polarity in the mobile phase. The HPLC profiling of CPC fraction showed that proposed gradient CPC was suitable to separate metabolites from Yongdamsagan-Tang, a traditional medicinal decoction made of ten herbal plants. Exhibiting a high recovery yield of 98.3%, antioxidant response element (ARE) luciferase-inducing assay in HepG2 cells indicated that the fractions composed of baicalein and wogonin, the marker natural products of Scutellaria baicalensis, were to be the most effective molecules from Yongdamsagan-Tang. The presented results demonstrated that bioassay-guided separation that assisted with a linear gradient CPC is an incomparable alternative to HPLC and biphasic CPC in terms of higher yield rate and redundant K value calculation, respectively, which led to an unbiased/time-saving separation and identification of bioactive molecules from the complex crude extract of natural products.

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Project description:The increasing drug resistance of malaria parasites challenges the treatment of this life-threatening disease. Consequently, the development of innovative and effective antimalarial drugs is inevitable. O-tigloylcyclovirobuxeine-B, a nor-cycloartane alkaloid from Buxussempervirens L., has shown promising and selective in vitro activity in previous studies against Plasmodiumfalciparum (Pf), causative agent of Malaria tropica. For further investigations, it is indispensable to develop an advanced and efficient isolation procedure of this valuable natural product. Accordingly, we used liquid-liquid chromatography including centrifugal partition chromatography (CPC) to obtain the pure alkaloid on a semi-preparative scale. Identification and characterization of the target compound was accomplished by UHPLC/+ESI-QqTOF-MS/MS, 1H NMR and 13C NMR. In conclusion, this work provides a new and efficient method to obtain O-tigloylcyclovirobuxeine-B, a valuable natural product, as a promising antiplasmodial lead structure for the development of innovative and safe medicinal agents.

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Centipeda minima

Project description:Centipeda minima Raw sequence reads

| PRJNA745255 | ENA

Online hyphenation of centrifugal partition chromatography with countercurrent chromatography (CPC-CCC) and its application to the separation of saturated alkylresorcinols.

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| S-EPMC9234026 | biostudies-literature

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

Project description: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.

| S-EPMC8781433 | biostudies-literature

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