Efficient extraction of polysaccharides from Lycium barbarum L. by aqueous two-phase system combined with tissue-smashing extraction

Highlights

• 17.86% (NH₄)₂SO₄ (w/w) and 28.86% ethanol (w/w) is a satisfactory ATPS system.

• By comparing HWE and UAE, TSE-ATPS showed a higher extraction yield.

• LBPs extracted by different methods have different physicochemical properties.

• TSE assisted ATPS method provides a novel insight for extracting polysaccharides.

Abstract

An effective method based on tissue-smashing extraction assisted aqueous two-phase system (TSE-ATPS) was developed for extracting Lycium barbarum L. fruits polysaccharides (LBPs). The ethanol/salt aqueous two-phase system (ATPS) composed of three different salts was evaluated, from which the system of 17.86% (NH₄)₂SO₄ (w/w) and 28.86% ethanol (w/w) was chosen. The conditions were as follows: solid-to-solvent ratio 1:30 (m/v), tissue-smashing power 8000 rpm (8 krpm), extraction time 4 min and the extraction yield of LBPs was 24.79 mg/g. Compared with hot water extraction (HWE) and ultrasound-assisted extraction (UAE), TSE-ATPS ((NH₄)₂SO₄/ethanol) improved the extraction yield of LBPs. LBPs extracted using various methods have different chemical structures and monosaccharide compositions. Superior antioxidant activity was demonstrated when the ATPS was composed of K₂HPO₄/ethanol and NaH₂PO₄/ethanol. Therefore, as an innovative, promising, and rapid crushing extraction technology, TSE-ATPS provides a better choice for extraction and separation of polysaccharides from natural products.

Introduction

Lycium barbarum L. (LB) fruits, also known as goji berries or wolfberries, mainly grow in arid and semiarid regions of Northwest China, such as Ningxia Province (Wang et al., 2019). Ni Zhumo, a famous Chinese herbalist, recorded in His “Convergent Speech on the Materia Medica” (Ben Cao Hui Yan) that “goji berry has the functions of supplementing energy and blood, regulating Yin and Yang, reducing internal heat, resisting wind and humidity”. LB fruits commonly serve as the raw material for dietary nutritional supplements that are widely used to regulate homoeostasis and prevent and treat diseases, including strengthening the kidneys, repairing the liver, and improving eyesight (Pires et al., 2018). The bioactive components of LB fruits are complex and include carotenoids, carbohydrates, vitamins, phenolic acids, flavonoids, amino acids, and other ingredients. Reportedly, Lycium barbarum L. fruits polysaccharides (LBPs) are the primary active components of LB fruits, which are estimated to comprise 5–8% of the dried fruits, and have many pharmacological activities and provide nutritional value, including improved immunity, anti-aging, protection against cancer, antioxidation, and protection of the liver and cardiovascular system (Hu et al., 2021, Qian et al., 2017, Zheng et al., 2021;). The antioxidant properties of LBPs. have been widely studied. In our earlier report, the antioxidant capacity of LBPs was achieved through the removal of superoxide anions from the intracellular mitochondria (Liu et al., 2021). According to the latest achievement in our laboratory, LBPs protect human adult retinal pigmented epithelium-19 cells from H₂O₂-induced oxidative damage by regulating the expression of antioxidant enzymes and downregulating matrix metalloprotein kinase expression (Liu et al., 2022). LBPs significantly increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and decrease malondialdehyde (MDA) levels and creatine kinase activity in the skeletal muscle of exhaustive exercise rats (Niu et al., 2008, Shan et al., 2011). In recent years, LBPs have been widely used in nutraceutical and pharmaceutical research. Therefore, the development of high-efficiency extraction methods for LBPs is crucial.

LBPs are usually obtained by conventional methods, such as hot water extraction (HWE), enzymatic extraction, and alkali extraction (Li et al., 2019, Wang et al., 2012). Although HWE is simple, it is time-consuming, inefficient, and consumes considerable energy. Enzymatic extraction requires demanding operating conditions, such as proper temperature and pH, which also increase production costs. Although alkali extraction can improve the dissolution rate of polysaccharides, the entire production process requires strong alkali-resistant materials and non-green production technology. A single solvent phase also extracts coexisting impurities, leading to a series of complex purification procedures, such as repeated precipitation with a high concentration of ethanol, which not only requires a long precipitation process, but also causes a large amount of ethanol consumption and increases production costs (Zhang et al., 2018).

Because they support environmental protection, continuous operation, and easy amplification, aqueous two-phase systems (ATPS) have been used to separate alkaloids, carbohydrates, proteins, enzymes, and isoflavones (Saddique et al., 2020, Zhang et al., 2021b). The extraction capability of an ATPS depends on its composition, which usually consists of a polymer and a salt, or short-chain ethanol and a salt. ATPS made of short-chain alcohols and salts have attracted increasing attention because of their low viscosity and high mass transfer efficiency, particularly for ATPS compositions that can form stable two-term systems and have a wide phase formation window (Lin et al., 2019). The ATPS composed of (NH₄)₂SO₄ was used to extract Lentinus edodes polysaccharides (Lin et al., 2019), the ATPS composed of K₂HPO₄ was used to extract polysaccharides from lilium davidiivar unicolor Salisb. In addition, Cheng et al. used an ATPS composed of NaH₂PO₄ and ethanol to extract polysaccharides from Gentiana scabra Bunge (Cheng et al., 2017). In the process of ATPS extraction, coexisting impurities and target substances can be selectively transferred to the top or bottom phase by adjusting the content of each component to achieve synchronous extraction and separation of target substances (Cheng et al., 2016).

In addition to the extraction solvents, the extraction technique is another important factor in natural product extraction. Ultrasonic-assisted extraction (Meng et al., 2021), microwave-assisted extraction (Lin et al., 2019), and pressure-assisted extraction (Zhang et al., 2021a) have been widely used to extract polysaccharides. However, these methods require significant time, solvent consumption, experimental instrumentation, and labour intensiveness.

Tissue-smashing extraction (TSE) is an innovative extraction technology that mainly consists of a stator with blades and a rotor with hole slots. This technique has been applied in previous studies by our research group (Gong et al., 2020). A schematic diagram and its working principle are shown in Supplementary Material Figs. S1 and S2, respectively. Compared with the above traditional extraction methods, TSE can accelerate the contact between the solvent and target by rapidly pulverising the target particles while strongly stirring the solution, and can achieve rapid extraction at normal temperature conditions (Jin et al., 2017). Owing to its high extraction yield, low solvent and energy consumption, simple operation, and extremely short extraction time (Yin et al., 2020), TSE is the preferred extraction method for natural products. TSE technology has made great progress in the extraction of anthocyanins, lignans, triterpenoid saponins, flavonoids (Yin et al., 2020), and other substances from parts of plants.

RSM is an integration of statistical and mathematical methods, and is an effective tool for process optimisation when many factors interact in the extraction process (Luo, 2012). In order to further understand a range of factors affecting the process and reduce the tests number, the RSM experiment was adopted to determine the optimal process parameters (Zhang et al., 2011). At present, it is widely used for the extraction of polysaccharides (Chen et al., 2012, Feng and Zhang, 2020, Zhu et al., 2015).

Herein, the objective of this study was to establish a green, economical and simple tissue-smashing extraction-assisted aqueous two-phase system (TSE-ATPS) method for extracting LBPs from LB fruits. To the best of our knowledge, there are no reports on the combination of TSE and ATPS used for extracting LBPs. A single-factor experiment and response surface methodology (RSM) were used to optimise the composition of the ATPS, solid-to-solvent ratio, tissue-smashing power, and time. In addition, HWE and ultrasound-assisted extraction (UAE) were compared with TSE-ATPS. The structures of polysaccharides extracted by different methods were characterised by Fourier transform infrared (FT-IR) spectroscopy, and the compositions of monosaccharides were determined by ion chromatography (IC). To evaluate the potential value of LBPs in the biomedical and health food industries, the antioxidant activities of LBPs were determined by DPPH and hydroxyl radical scavenging assays.