A simple and effective method based on enzyme-ligand complex for affinity analysis of lipase inhibitor from Schisandra chinensis (Turcz.) Baill

https://doi.org/10.1016/j.jchromb.2022.123393Get rights and content

Highlights

  • A simple method was developed for affinity screening of active component.

  • Schisandra chinensis possessed the potential lipase inhibitory activity.

  • 5-Hydroxymethyl-2-furaldehyde is a non-competitive inhibitor.

  • 5-Hydroxymethyl-2-furaldehyde interacted with lipase by van der Waals force, H-bonds.

Abstract

Schisandra chinensis (Turcz.) Baill has various biological activities including anti-obesity. Rapid analysis and screening of active compounds from natural extracts is one of the challenges faced by natural drug research. In order to analyze and screen lipase inhibitor from Schisandra chinensis extract, a method inspired by the specific binding of enzyme to ligand was developed and established. Through optimization of incubation conditions, such as time, temperature, and pH, the potential active compound was locked by comparing the change of the chemical components of the S. chinensis extract before and after incubation with lipase. Subsequently, the target compound was isolated by high-speed counter-current chromatography and was identified as 5-hydroxymethyl-2-furaldehyde. Moreover, in vitro activity determination confirmed that 5-hydroxymethyl-2-furaldehyde with an IC50 value of 284.78 ± 16.45 μg/mL interacted with the lipase through non-competitive inhibition. Furthermore, molecular docking further revealed that 5-hydroxymethyl-2-furaldehyde can be embedded into the active pocket of lipase via multiple hydrogen bonds and other interactions. This study not only screened a potential lipase inhibitor from S. chinensis through the newly developed method, but also can be used as a typical reference for the discovery of active components from functional foods and natural products.

Introduction

Lipase inhibitor is one of the effective drugs to treat obesity and has important applications in industrial production. During the extraction and storage of industrial oil, triglycerides are easily converted to free fatty acids and glycerol by lipases in the environment, which affected the quality of industrial oil [1]. In the human body, the lipases catalyse the hydrolysis of complex oligosaccharides and triglycerides into digestible molecules, and the excessive absorption of these molecules may lead to obesity [2]. Furthermore, insulin resistance caused by fatty acids accumulation can also lead to various complications [3]. Lipase inhibitors can be used to prevent various undesirable changes by inhibiting the lipase activity [4]. The research of lipase inhibitors can not only affect specific reactions at the level of industrial application, but also can explore the molecules with pharmacological or biotechnological value [5].

Functional foods and natural products are the important sources of bioactive compounds [6]. Many lipase inhibitors were found in natural products, such as the alkaloid stephalagine, a lipase inhibitor extracted from the fruit peel of Annona crassiflora, and pentagalloylglucose extracted from Toona sinensis and so on [7], [8].

Schisandra chinensis, a deciduous broad-leaved vine, is mainly distributed in China, Japan and Korea. The fruits of S. chinensis have a variety of active ingredients, including lignans, triterpenes, and flavonoids [9], [10]. The existence of different components correspond to a variety of biological effects, such as antilipolysis, anticancer, antibacterial, antioxidant, anti-obesity etc [10], [11], [12], [13], [14], [15], [16], [17]. Due to the pharmacological activities [18], its fruits are served as a representative traditional Chinese medicine. In this study, the extract of S. chinensis fruits showed potential lipase inhibitory activity, which inspired us to further discover the active compounds.

Rapid screening of active substances in complex mixtures was not only related to the development of lipase inhibitors, but also posed a great challenge to the research of natural active substances. To solve this problem, many methods have been developed, such as ligand fishing technology based on immobilized enzyme [19], on-line activity detection technology [20], [21], [22], chromatography mass spectrometry combined with virtual screening [23], [24], and so on. Although these methods had obvious advantages to improve the efficiency of screening active components from complex extracts, they also had some disadvantages. For example, the immobilized enzyme relied on advanced nano-carriers and the immobilization process was relatively complex [17], [19]. On-line activity detection and chromatography mass spectrometry combined with virtual screening also needed advanced instruments, such LC-MS (high-resolution), GC–MS, NMR, etc [21]. In addition, it was also a challenging task to successfully isolate the target compounds from complex systems for the verification of their activities [2].

In this study, a simple and rapid screening method and an effective separation strategy was established successfully to discover the lipase inhibitors from S. chinensis fruits. Based on the interactions between enzyme and ligand, the content of the active compounds in the mixture before and after incubation were different. By comparing the contents changes, a potential lipase inhibitor from S. chinensis fruits was found. And then, the active compound was purposely separated by HSCCC. Besides, the activity of the separated compound was verified by in vitro experiment and the interactions were explained by molecular docking.

Section snippets

Materials and chemicals

Aspergillus niger lipase was purchased in Solarbio (Beijing, China). Arabic gum and 4-Nitrophenyl palmitate (p-NPP) were obtained from Yuanye Biotechnology and Energy Technology (Shanghai, China), respectively. Schisandra chinensis (Turcz.) Baill was obtained from Dandong, China, and authenticated by Yi Yang, Dalian Polytechnic University (Voucher No. FBML-20210514).

Preparation of the ethanol extract

The dried and crushed S. chinensis (Turcz.) Baill (660 g) was refluxed twice for 3 h with petroleum ether, ethyl acetate, and

Inhibitory effects of ethanol extract on lipase

The extracts and chemical components of S. chinensis had a wide range of biological activities, including the potential to prevent and treat obesity [29]. Therefore, the lipase activity of S. chinensis ethanol extract was investigated. The substrate p-NPP corresponding to lipase was colorless, but the colored p-NP can be released after lipase hydrolysis. Thus, ethanol extracts (five concentrations) were selected to determine the inhibition rate of lipase, as shown in Fig. 1A. The inhibition

Conclusions

In this study, a lipase inhibitor in the ethanol extract of S. chinensis fruit was successfully found by ligand fishing and HSCCC separation, and was identified as 5-HMF. In vitro activity experiments confirmed that 5-HMF was a non-competitive inhibitor of lipase and molecular docking showed that 5-HMF bound to the key residues of lipase in the catalytic site through van der Waals force, hydrogen bonds, etc. This study not only screened a potential lipase inhibitor from S. chinensis through the

CRediT authorship contribution statement

Shanshan Tang: Data curation, Investigation, Validation, Writing – original draft. Shuo Liu: Software, Investigation. Yunxiao Wang: Data curation, Methodology. Dongyu Gu: Supervision, Writing – review & editing. Jing Tian: Resources, Project administration. Yi Yang: Formal analysis, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from the Natural Science Foundation of Liaoning Province (No. 2019-MS-033).

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