Strategy for the separation of strongly polar antioxidant compounds from Lycium barbarum L. via high-speed counter-current chromatography
Introduction
Strongly polar compounds from natural products are increasingly important as herbal drugs for the treatment of chronic diseases [1], [2] and as the most important resources for drug discovery [3], [4]. Studies of strongly polar compounds have created an increasing demand for efficient bioassay-guided separation methods. In a previous study, a method that uses high-speed counter-current chromatography (HSCCC) that is target-guided by 2,2-diphenyl-1-picrylhydrazyl (DPPH) high-performance liquid chromatography (HPLC) experimentation was developed for the separation of antioxidant compounds [5], [6], [7], [8]. After reacting with DPPH·, the HPLC peak area of compounds with potential antioxidant activity would be reduced, possibly to zero, while for compounds without antioxidant activity, the peak area would not change substantially [6], [7]. The best solvent system for separating antioxidant target compounds was selected via the calculation of the partition coefficient (KU/L) value based on DPPH-HPLC experimentation. The KU/L value was the ratio of the peak areas of a target compound in the upper phase and the lower phase that were obtained using the method [6]. However, the low adsorption ability in the chromatographic separation process was inevitable due to the strong polarity of the target compounds. No strongly polar antioxidant target compounds were discovered via DPPH-HPLC experimentation. Therefore, it was difficult to separate strongly polar antioxidant compounds using HSCCC that was target-guided by DPPH-HPLC experimentation. In this study, a novel calculation of the KU/L value was proposed for the identification of strongly polar antioxidant components. Then, these components were separated via HSCCC.
Recently, studies proposed the use of a hydrophilic organic/salt-containing aqueous two-phase system to isolate strongly polar compounds [9], [10], [11], [12]. It was experimentally proved that a hydrophilic organic/salt-containing aqueous two-phase system was an alternative solvent system for CCC separation of natural products with high polarity because two hydrophilic organic/salt-containing aqueous two-phase systems were successfully used to separate caffeoylquinic acid isomers and dihydroxybenzoic acid isomers [9]. In a study by Zeng et al. [10], an ultra-polar solvent series for separating polar compounds via HSCCC was introduced, and the utility of the method was demonstrated in the separation of 3 catecholamines. As another example of the isolation of polar compounds using a hydrophilic organic/salt-containing aqueous two-phase solvent system for counter-current chromatography, anethanol–ammonium sulfate–water aqueous two-phase system was used to separate nucleosides on multilayer coil HSCCC [11]. However, although hydrophilic organic/salt-containing aqueous two-phase solvent systems were used to separate strongly polar compounds, these solvent systems were not used in bioassay-guided separation via HSCCC. In this study, a hydrophilic organic/salt-containing aqueous two-phase solvent system was combined with HSCCC and used to facilitate bioassay-guided separation for the identification of strongly polar antioxidant compounds.
In the present study, a strategy of novel KU/L calculation was proposed for overcoming the separation of strongly polar antioxidant compounds. The KU/L value was expressed as the ratio of the antioxidant activities of the upper phase and the lower phase. This strategy was used in HSCCC with a hydrophilic organic/salt-containing aqueous two-phase system for the bioassay-guided separation of strongly polar antioxidant compounds from Lycium barbarum L.
Section snippets
Chemicals and reagents
The DPPH radical was purchased from Sigma Aldrich Co., St. Louis, USA. The HPLC-grade methanol was obtained from Merck KGaA, Darmstadt, Germany. All analytical-grade reagents were purchased from Sinopharm Chemical Reagent, Beijing, China. Deionized water was obtained from a Spring-R10 water purification system from Xiamen Research Water Purification Technology Co., Ltd, Xiamen, China.
Instruments
A TBE-300C HSCCC system that was equipped with a TBP-5002 constant-flow pump, a model TBD-2000 detector that
Antioxidant activity of the Lycium barbarum L. extract
In this study, the antioxidant activity of the Lycium barbarum L. extract was evaluated by measuring the DPPH radical scavenging rate. As shown in Fig. 1B, the DPPH radical scavenging rates of Lycium barbarum L. extract were 3.96%, 10.04%, 21.04%, 36.78% and 59.75% at the concentrations of 15.63, 31.25, 62.50, 125.00 and 250.00 μg/mL, respectively. The DPPH radical scavenging rate increased with the increment of the concentration of the Lycium barbarum L. extract. The standard curve equation
Conclusions
In this study, a strategy for defining the KU/L value was proposed for the selection of two-phase solvent system, namely, the KU/L value was defined as the ratio of the antioxidant capacities of the upper and lower phases. A solvent system of 95% EtOH-sat.(NH4)2SO4 (1:1.8, v/v) was successfully selected and used in HSCCC. The strategy was used in HSCCC with a hydrophilic organic/salt-containing aqueous two-phase system for the bioassay-guided separation of strongly polar antioxidant compounds
CRediT authorship contribution statement
Ningli Wang: Conceptualization, Methodology, Validation, Investigation, Software, Writing - original draft, Writing - review & editing. Dong Pei: Supervision, Project administration, Funding acquisition, Writing - original draft, Writing - review & editing. Peiliang Yu: Resources, Data curation, Investigation. Xinyi Huang: Formal analysis, Writing - review & editing. Lichun Zhao: Supervision, Writing - review & editing. : . Jianteng Wei: Resources, Writing - review & editing. Jianfei Liu:
Declaration of Competing Interest
The authors have declared no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China [grant number 201605149]; Chinese Academy of Sciences, “Western Young Scholars (Class A)” project; the Key Research and Development Program of Gansu Province [grant number 18YF1FA126]; and Key Research and Development Program of Ningxia Hui Autonomous Region [grant number 2019BEF02006].
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The first two authors contributed equally to this paper.