Extraction, Structural Characterization, and Biological Functions of Lycium Barbarum Polysaccharides: A Review

Lycium barbarum polysaccharides (LBPs), as bioactive compounds extracted from L. barbarum L. fruit, have been widely explored for their potential health properties. The extraction and structural characterization methods of LBPs were reviewed to accurately understand the extraction method and structural and biological functions of LBPs. An overview of the biological functions of LBPs, such as antioxidant function, antitumor activity, neuroprotective effects, immune regulating function, and other functions, were summarized. This review provides an overview of LBPs and a theoretical basis for further studying and extending the applications of LBPs in the fields of medicine and food.

glycoconjugates with a molecular weight of 10-2300 kDa and comprise 5-8% of the dried fruits [11]. The beneficial health effects of LBPs, including antioxidant and antiaging effects, increased metabolism, antiglaucoma effects, immune regulation, anticancer effects, neuroprotective properties, and antidiabetic effects, have been reported [12][13][14][15][16]. According to the Chinese understanding of Lycium extracts and products, the content of LBPs is important for the efficacy of L. barbarum [17][18][19]. Therefore, as bioactive constituents of L. barbarum, LBPs have many biological functions to improve people's health. The biological functions of LBPs are complex and multifaceted because of the relationship between the physiological structure and functions of LBPs. The relationship and mechanism between LBPs and human health should be fully understood. In order to give a comprehensive understanding of LBPS, the extraction methods, structure, composition, and biological functions of LBPs were summarized and discussed in this review. We collected and summarized the relative contents from previous reports to provide a theory basis for comprehensively understanding and utilizing LBPs in medical and food fields.

Extraction Methods of LBPs
Previous reports have shown that the chemical ingredients of L. barbarum fruit include polysaccharides, proteins, and phenylpropanoids. Among these ingredients, LBPs account for 5-8% of the dried fruit and elicit biological effects [2]. LBPs are subjected to extraction, purification, and analysis. The flowchart of the extraction, purification, and analysis of LBPs is shown in Figure 2.
LBPs are extracted by destroying and degrading the cell wall under mild conditions without changing the properties of the polysaccharides in accordance with basic extraction principles [20]. Many LBP extraction methods, such as the water extraction method, enzyme-assisted extraction method, microwaveassisted extraction method, ultrasonic-assisted extraction method, and supercritical fluid extraction method, have been developed on the basis of this principle [21,22]. Traditional LBP extraction methods have some advantages and disadvantages. Novel LBP extraction technologies, such as ultrasound-assisted extraction method (UAE) [23,24], enzyme-assisted extraction method (EAM) [25,26], microwave-assisted extraction method (MAM) [27], and supercritical fluid extraction method (SFM) [28], have been developed to address the disadvantages of traditional extraction methods. The high extraction yield and high biological activity of LBPs are considered in choosing an extraction method [29].
These extraction methods have unique strengths and weaknesses [21]. Hot water extraction (HWE) is the traditional method for polysaccharide extraction. The yield of HWE is largely affected by extraction time, temperature, and the ratio of water to raw material. Also, the long duration and high temperature may lead to the degradation of the polysaccharides and decrease their biological activity. EAM possesses the advantages of environmental friendliness, high efficiency, ease of operation, and low investment cost and energy. However, the enzyme is characterized by specificity and selectivity, while several factors, such as enzyme concentration, temperature, time, and pH also affect the biological function of polysaccharides. MAM is a physical technique that is used for the extraction of polysaccharides. MAE has noticeable

Extraction Methods of LBPs
Previous reports have shown that the chemical ingredients of L. barbarum fruit include polysaccharides, proteins, and phenylpropanoids. Among these ingredients, LBPs account for 5-8% of the dried fruit and elicit biological effects [2]. LBPs are subjected to extraction, purification, and analysis. The flowchart of the extraction, purification, and analysis of LBPs is shown in Figure 2.
LBPs are extracted by destroying and degrading the cell wall under mild conditions without changing the properties of the polysaccharides in accordance with basic extraction principles [20]. Many LBP extraction methods, such as the water extraction method, enzyme-assisted extraction method, microwave-assisted extraction method, ultrasonic-assisted extraction method, and supercritical fluid extraction method, have been developed on the basis of this principle [21,22]. Traditional LBP extraction methods have some advantages and disadvantages. Novel LBP extraction technologies, such as ultrasound-assisted extraction method (UAE) [23,24], enzyme-assisted extraction method (EAM) [25,26], microwave-assisted extraction method (MAM) [27], and supercritical fluid extraction method (SFM) [28], have been developed to address the disadvantages of traditional extraction methods. The high extraction yield and high biological activity of LBPs are considered in choosing an extraction method [29].
These extraction methods have unique strengths and weaknesses [21]. Hot water extraction (HWE) is the traditional method for polysaccharide extraction. The yield of HWE is largely affected by extraction time, temperature, and the ratio of water to raw material. Also, the long duration and high temperature may lead to the degradation of the polysaccharides and decrease their biological activity. EAM possesses the advantages of environmental friendliness, high efficiency, ease of operation, and low investment cost and energy. However, the enzyme is characterized by specificity and selectivity, while several factors, such as enzyme concentration, temperature, time, and pH also affect the biological function of polysaccharides. MAM is a physical technique that is used for the extraction of polysaccharides. MAE has noticeable advantages, such as shorter extraction time, higher extraction yield, lower cost, and less solvent consumption. UAM has the advantage of improving penetration and capillary effects, leading to an increase of polysaccharides' extractability. However, ultrasonic treatment could affect the structure and molecular weight (MW) of polysaccharides, which would cause a change in the biological activity. The extraction conditions and yield of LBP extract by different extraction methods are shown in Table 1. advantages, such as shorter extraction time, higher extraction yield, lower cost, and less solvent consumption. UAM has the advantage of improving penetration and capillary effects, leading to an increase of polysaccharides' extractability. However, ultrasonic treatment could affect the structure and molecular weight (MW) of polysaccharides, which would cause a change in the biological activity. The extraction conditions and yield of LBP extract by different extraction methods are shown in Table 1.
As the traditional extraction method is widely used in the extraction of LBPs, the yield of the water extraction method (WEM) is 5.87% under the experimental conditions [30]. The yields of LBPs via UAM, EAM, and MAM are 2.286-5.701%, 6.81% ± 0.10%, and 8.25% ± 0.07%, respectively [31][32][33][34][35]. The yield of LBPs of novel extraction methods is higher than that of the water extraction method. Moreover, the combination of different extraction methods can obtain enhanced yields of LBPs; when the ultrasoundenhanced subcritical water extraction method (USWE) is used to extract the LBPs, high recovery yields of LBPs are produced [24].  As the traditional extraction method is widely used in the extraction of LBPs, the yield of the water extraction method (WEM) is 5.87% under the experimental conditions [30]. The yields of LBPs via UAM, EAM, and MAM are 2.286-5.701%, 6.81% ± 0.10%, and 8.25% ± 0.07%, respectively [31][32][33][34][35]. The yield of LBPs of novel extraction methods is higher than that of the water extraction method. Moreover, the combination of different extraction methods can obtain enhanced yields of LBPs; when the ultrasound-enhanced subcritical water extraction method (USWE) is used to extract the LBPs, high recovery yields of LBPs are produced [24]. Biomolecules 2019, 9, 389 5 of 17

Structure and Composition of LBPs
The separation and structural characterization methods of purified LBPs have been developed. As active ingredients, LBPs possess various biological functions. More than 33 polysaccharides have been analyzed and identified from L. barbarum L.
The main applied and performance techniques for the structural characterization of LBP fraction include the following techniques: (1) high-performance gel permeation chromatography (HPGPC), which is used to determine the homogeneity and molecular weight of macromolecules; (2) partial acid or enzymatic hydrolysis, oxidation with periodic acid, Smith degradation, high-performance liquid chromatography (HPLC), gas chromatography (GC), polysaccharide analysis by gel electrophoresis (PACE), and high-performance thin-layer chromatography (HPTLC). These techniques are used to determine monosaccharide composition and map the glycidic component of glycoconjugates; (3) infrared (IR) spectral analysis permits the identification of pyranosyl or furanosyl ring form and α or β anomeric configuration in monosaccharide residues; (4) 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy used to assign the ratios of monosaccharides present and ratios of their anomeric bonds; (5) gas chromatography-mass spectrometry (GC-MS), employed to determine the linkage positions.
Studies have widely explored the structure and composition of LBPs and demonstrated that LBPs are polysaccharides, including some ingredients of acidic heteropolysaccharides, polypeptides, or proteins [37]. The molecular weight of LBPs at the range of 10-2300 kDa. The methods used for the isolation and purification of LBPs from L. barbarum include DEAE ion-exchange cellulose, gel-permeation chromatography, and high-performance liquid chromatography (HPLC) [31,38]. About 20 types of polysaccharides, including Rha, Fuc, Ara, Gal, and GalA, have been investigated. Studies have shown that monosaccharide and amino acid residues constitute glycoconjugates, and, relative to glycosidic linkage analysis of glycan backbone, branching sites and side chains were considered as the structure of LBPs. The possible structure of repeat units, molecular weights, and analysis technique of previously investigated polysaccharides in L. barbarum are shown in Table 2. Table 2. The possible structure of repeat units, molecular weights, and analysis technique of polysaccharides in L. barbarum [39] . Definitions: SEC = size exclusive chromatography, GC-MS = gas chromatography-mass spectrometry, NMR = nuclear magnetic resonance, IR = infrared, GC = gas chromatography, ESI-MS = electrospray ionization mass spectrometry, HPGPC = high-performance gel permeation chromatography.
The biological function of LBPs have many potential functions relative to the antioxidant activity in many tissues [62][63][64]. Studies have investigated the antioxidant activity of LBPs that extract with hot water and the protective effect of LBPs against tissue oxidative injury, with results showing that LBPs exhibit a good antioxidant activity and a protective effect against skin oxidative injury [65]. The antioxidant effects of LBPs extracted by hot water have been explored via an in vivo model, showing that LBP treatments can significant increase the serum levels of SOD and GSH-Px and significantly decrease MDA contents [66][67][68][69]. The antioxidant function of LBPs shows that LBPs can significantly enhance macrophage NO, phagocytic capacity, and acid phosphatase, and exhibit good antioxidant activities in vitro [70]. LBPs can significantly increase cell viability that decreases by LPS and regulate oxidative stress by inhibiting caspase-3 activation and ROS levels in vitro [71]. Studies have investigated the effects of LBPs on stressed RPE cells and have shown that LBPs can decrease ROS levels via free radical scavenging and downstream gene function to prevent ROS-induced apoptosis [72]. Antioxidant enzyme activities, GSH levels, and MDA levels in rats fed with a high-fat diet and LBPs decreased when compared with those in the control group (p < 0.01) [73]. The antioxidant activity and mechanisms of LBPs are shown in Table 3.

Immune Regulation
Immune regulation is an important function of LBPs in people's health. Previous studies have shown that LBPs exhibit an immune-modulating function in target dendritic cells, macrophages, Tand B-lymphocytes, and natural killer (NK) cells [79]. The immune regulation of LBPs is an important function that has been studied widely in previous studies.
Dendritic cells are used to study the effects of LBPs, and results have shown that LBPs can induce the phenotypic and functional maturation of DCs via Notch signaling and promote the cytotoxicity of DC-mediated CTLs [80]. In nutritive additives, LBPs can be used as an additive in the growth of broilers, indicating that LBPs may possess the clinical efficacy for growth promotion and immunomodulation and can be used as an alternative to nutritive additive in broilers [81]. The nanoliposome technique is used to load LBPs and study their function, showing that LBPs can significantly promote splenocyte proliferation, increase the amount of CD4(+) to CD8(+) T cells, and promote the cytokine secretion of macrophages [82]. The extract of L. barbarum exhibits a significant immunomodulatory activity through the promotion effects of nitric oxide and cytokines in RAW264.7 cells [83]. The efficacies of sulfated LBPs on immune enhancements in cultured chicken are high [84]. In summary, the immune regulation and mechanism of LBPs are shown in Table 4. Table 4. Immune regulation activity and mechanisms of LBPs.

Enhanced macrophage endocytic and phagocytic capacities in vivo
Activate transcription factors NFAT, AP-1, prompt CD25 expression, induce IL-2 and IFN-gamma gene transcription and protein secretion In vitro [85] Regulation of immune cells Maintain high levels of T cells, prevent the increase of Tregs, promote infiltration of CD8+ T cells In vivo [86] Induce the phenotypic and functional maturation of DCs Upregulate the expression of Notch and Jagged and Notch targets Hes1 and Hes5 In vitro [80] Promote the proliferation of spleen cells Increase secretion of INF-alpha and IL-6, mRNA expression of iNOS, IL-beta and IL-6 through activating phosphorylation of ERIC, JNK, p38 and p65 In vitro [87] Increased immune organ indexes Promote blood B and T lymphocyte proliferation In vivo [81] Improve immune responses Stimulate CD4(+) and CD8(+) T cell proliferation In vitro [88] Enhance the immune activity Enhance PCV2-specific IgG antibody responses, promote Th1 cytokines (IFN-gamma and TNF-alpha) and Th2 cytokine (IL-4) secretion In vitro [82] Enhance the immune activity Inhibit cell proliferation, retard cell cycle growth, and promote apoptosis In vitro, In vivo [89]

Antitumor Activity of LBPs
The antitumor activity of natural products isolated from plants have been reported [90][91][92]. Previous studies have shown that more than 100 polysaccharides exhibit good anticancer activity via in vitro studies and in vivo animal models [93].
As natural products, LBPs exhibit potential antitumor activity [94]. Studies have shown that the anticancer activity of LBPs occurs because of their effects on cancer tissue or cancer cells. The exposure to hot water extracts of Lycium barbarum for 24 h made cell viability reduce to 15.31% of hepatocellular carcinoma cells, as reported in a previous study [95]. Colorectal cancer is one of the most common cancers worldwide, with a study showing that the treatment of LBP (5000 mg/L) can decrease cell viability of SW480 and Caco-2 cells to 10% after 5 days of treatment, and that the treatment with LBP resulted in a dose-dependent increase in the distribution of cells in the G0/G1 phase [96]. For gastric cancer cells, LBPs can inhibit the proliferation of these cells and arrest the cell cycle at the G0/G1 phase, suggesting that LBPs are candidate anticancer agents [97]. The inhibitory effect of LBPs on the growth of glioma in rats and the underlying mechanism have been explored, and results have shown that the mechanism may be related to the regulation of the blood-brain barrier and to the promotion of CD[8] + T cell invasion in the brain [98]. Studies on the effects of LBPs on the viability, cell cycle, and apoptosis of human hepatoma cells have demonstrated that LBPs can inhibit cell growth, arrest the cell cycle in the S phase, and induce apoptosis, suggesting the antiproliferative activity of LBPs by inducing cell cycle arrest and increasing intracellular calcium in the apoptotic system [99]. The anticancer activity of LBPs is mainly due to the inhibited growth of cells, arrested cell cycle, and induced cell apoptosis. The antitumor activities of LBPs on different cancer cells are shown in Table 5. LBPs exhibit a good antitumor activity on various cancer cells.  [84,86] These results show that LBPs can decrease the viability of cancer cells and have inhibitory effects on cancer cells, indicating that LBPs can be used as a candidate anticancer agent in cancer treatment.

Neuroprotective Effects of LBPs
Natural products isolated from plants exhibit certain biological functions and have been extensively investigated because of their efficiency and biosafety. As such, they have been widely used to treat diseases. Plant extracts have been utilized to treat various functions of central nervous systems [102].
Polysaccharides are effective compounds from neurobiologically active plants, and many studies have demonstrated their beneficial effects, including neurological disorders [102,103].
The neuroprotective effects of LBPs on ischemic injury is mainly through the signaling pathways of NR2A activation and NR2B inhibition, and LBPs can be used to treat ischemic stroke [104]. A mouse experiment model of the neuroprotective effects of LBPs have shown that LBPs may exert neuroprotective effects and help prevent neurodegenerative diseases [105]. In modern society, visual impairments and blindness cause heavy damage on people's health. LBP treatment can significantly weaken these injuries with enhanced endogenous autophagy in the body [106]. The neuroprotective effects and molecular mechanisms of LBPs are shown in Table 6.

Other Biological Activities
The antioxidant activity, antitumor properties, immune regulation, and neuroprotective effects of LBPs were explored in in vitro and in vivo models. Besides this, LBPs also have other biological activities, such as protecting the liver from hepatotoxicity [122], alleviating dry-eye disease [123], eliciting antidiabetic effects [124], increasing cell abilities, decreasing cell morphologic impairment, protecting against ultraviolet-induced damage [122], and alleviating CCl4-induced liver fibrosis [125]. As bioactive constituents, LBPs exhibit various biological functions and show potential benefits to people's health. The other biological activities and mechanism of LBPs are summarized in Table 7.

Conclusions
In China, goji berries are a traditional medicinal herb that have been used for thousand years to cure disease and improve the function of the liver, kidney, and lungs. More recently, western countries have also started cultivating Lycium barbarum L. plants, whose fruits are consumed fresh or dried. Previous reports have showed that L. barbarum fruit exhibits a wide array of pharmacological activities. This beneficial L. barbarum component consists of a complex mixture of glycoconjugates-these being LBPs-with a molecular weight range of 10-2300 kDa, water solubility, and containing a carbohydrate portion (≥90%), represented by highly-branched polysaccharides. The LBP glycan backbones have been found to be mainly represented by α-(1→4)-galA, α-(1→6)-glc, β-(1→3)-galp (typical of arabinogalactan proteins), and β-(1→6)-galp. Other structures, less representative, are α-(1→5)-ara and β-(1→4)-galp, with different branching and terminal sites. The extraction methods of LBPs, including water extraction, enzyme-assisted extraction, microwave-assisted extraction, and ultrasonic-assisted extraction, produce different effects on the quality of LBPs. The biological functions of LBPs indicate that LBPs exhibits antioxidant, immunomodulation, antitumor, neuroprotection, and hepatoprotection.
LBPs are the main active substances in L. barbarum fruits and are involved in various biological functions. LBPs have great potential health benefits for further use in nutraceutical and pharmaceutical fields. The biological function of LBPs has been explored using in vitro and in vivo models but not in the human body. However, the relationship between a high-order LBP structure and bioactivities has yet to be further explored. Novel omics technologies, such as proteomics, metabolomics, and genomics, are effective in investigating the biological function of LBPs. Future works should focus on the high-order structures of LBPs, their biological function in the human body, and the relationship between their structure and bioactivity to enhance our understanding of the functional effects of LBPs. Current extraction methods give low yields and other weaknesses in the extraction of LBPs. Novel environmentally friendly and high-yielding extraction methods of LBPs should be further developed. The extraction methods and structural and biological functions of LBPs were summarized in this review to provide a useful bibliography for further investigations and applications of LBPs in medicine and food.
Author Contributions: X.T., T.L., and Z.M. conceived the idea; X.T. and T.L. write the draft; Y.L., F.Z., and G.D. edited the manuscript; all authors read and approved the final manuscript.

Funding:
We gratefully acknowledge financial support from National Natural Science Foundation of China (31560477), ministry of science and technology project (KY201501005), Gansu province Science Technology funding plan through Project (17YF1WA166) and Natural Science Foundation of Gansu province (18JR3RA371).

Conflicts of Interest:
The author(s) declare that they have no conflicts of interest to disclose.

LBPs
Lycium barbarum polysaccharides WEM water extraction method EAM enzyme-assisted extraction method MAM microwave-assisted extraction method UAM ultrasonic-assisted extraction method SEC size exclusive chromatography ESI-MS electrospray ionization mass spectrometry GC-MS gas chromatography-mass spectrometry NMR nuclear magnetic resonance HPGPC high performance gel permeation chromatography