Extraction, purification, characterization and antidiabetic mechanisms of plant polysaccharides: a critical review

Hot water extraction

Hot water extraction is the most extensively employed method for polysaccharide extraction due to its simplicity, convenience, cost-effectiveness, and environmental friendliness. In this method, hot water is employed to break the cell wall through lysis, facilitating the release of intracellular polysaccharide molecules into the solvent [26]. Nevertheless, the conditions and parameters for hot water extraction may vary depending on the plant material and the specific polysaccharides targeted for extraction. Optimization of the extraction process is imperative to attain the maximum yield and quality of the extracted polysaccharides. The water temperature typically ranges from 60 to 100 °C, and the extraction time typically falls between 30 min and 3 h [27]. Following the extraction process, the mixture of water and plant material undergoes separation. Commonly used methods for separation include filtration or centrifugation to isolate the insoluble plant residue from the aqueous extract containing the extracted polysaccharides. To concentrate the aqueous extract containing the extracted polysaccharides, techniques such as evaporation or freeze-drying may be employed to eliminate excess water.

Alkali extraction

Alkali extraction stands as another method frequently employed for extracting polysaccharides from plant materials. In this extraction method, the plant material undergoes treatment with an alkaline solution, typically sodium hydroxide (NaOH) or potassium hydroxide (KOH), to disintegrate the cell wall structure and liberate the polysaccharides [28]. The principle underlying this method is that the hydroxide ions (OH⁻) from the alkali disrupt the hydrogen bonding in polysaccharides, resulting in the liberation of polysaccharides into the solvent. Extraction is conducted for a specified duration using alkali at a designated temperature with a specified material-liquid ratio.

An alkaline solution is prepared by dissolving a specific concentration of NaOH or KOH in water. The concentration of the alkali solution may vary typically around 0.15–0.3 mol/L [29]. The mixture is typically maintained at an elevated temperature, often in the range of 60–90 °C, for a specific duration, around 30 min to 4 h, to allow for efficient extraction [30]. Compared to hot water extraction, alkali extraction requires additional steps for neutralizing the mixture. The alkaline extract is typically neutralized by adding an acid, such as hydrochloric acid (HCl), to adjust the pH to a more neutral range. This step is crucial to prevent the degradation of polysaccharides. Neutralization may result in the precipitation of polysaccharides, facilitating their separation from the liquid.

Acid extraction

The acid extraction method effectively disintegrates the cell wall structure of plant materials, releasing polysaccharides into the extraction solution. This method is particularly suitable for extracting pectin. However, acid extraction may lead to some degradation or modification of the extracted polysaccharides due to the harsh acidic conditions employed. The acid method operates on the principle of acid penetration of cell fractions, where H⁺ interferes with the hydrogen bonds in polysaccharides, releasing them into the solvent. An acid solution is prepared by dissolving a specific concentration of hydrochloric acid or sulfuric acid in water. The concentration of the acid solution may vary depending on the plant material and the desired extraction efficiency. The mixture is heated and stirred to facilitate the extraction process. It is typically maintained at an elevated temperature, often in the range of 60–90 °C, for a specified duration to enable efficient extraction [31]. Various researchers have employed this method to isolate polysaccharides with different time-temperature combinations. Similar to alkali extraction, the acid extract also requires a neutralization step. This is achieved by adding a base, such as sodium hydroxide (NaOH) or ammonium hydroxide, to adjust the pH to a more neutral range. Neutralization is crucial to neutralize the acidity and prevent degradation of the polysaccharides. This may result in the precipitation of polysaccharides, facilitating their separation from the liquid. Liu et al. [32] reported a polysaccharide yield of 36.0 % with a molecular mass of 17.3 kDa using this method. In comparison to hot water and alkali extraction methods, the acid extraction method produced polysaccharides with a high molecular mass and iron-chelating ability.

Enzymatic extraction

The enzymatic extraction principle involves the enzymatic degradation of the cell wall structure, resulting in the release of intracellular molecules into the solvent. Enzymes play a crucial role in breaking down the glycosidic bonds within the polysaccharides, leading to hydrolysis and the formation of smaller oligosaccharides or monosaccharides [30]. This process facilitates the release of polysaccharides from the plant material into the extraction solution. Various enzymes, such as cellulase, complex protease, xylanase, α-amylase, pentosan complex enzyme, flavorase, and neutral protease, have been employed for the extraction of polysaccharides 33], [34], [35], [36], [37. The choice of suitable hydrolytic enzymes markedly influences the extraction efficiency. Furthermore, the pH and time-temperature combinations during enzymatic hydrolysis also impact the rate of enzyme reactions [37]. Crude polysaccharides were further degraded by H₂O₂ and vitamin C to obtain polysaccharides of different molecular masses. Compared with the above methods, this method has less off-target degradation and high extraction efficiency of high molecular mass polysaccharides. Nevertheless, the selection of appropriate enzymes and optimization of influencing factors should be considered to enhance extraction efficiency and achieve the desired molecular mass of the extracted polysaccharides.

Microwave-assisted extraction

The microwave-assisted method utilizes microwave radiation in the presence of a suitable solvent to heat the solvent, enabling it to come into contact with the sample. This process aids in the distribution of analytes from the sample matrix into the solvent, thereby facilitating the extraction process. Common solvents for this method include water, aqueous alcohol solutions, and organic solvents. Shang et al. [38] found that deep eutectic solvents, specifically choline chloride and 1,4-butanediol, were effective for polysaccharide extraction from the bladder-wrack. Similarly, Silva et al. [39] used water as a solvent for polysaccharide extraction from Spirulina platensis, following the principles of green chemistry. Lin et al. [40] optimized polysaccharide extraction from Lentinus edodes through an aqueous two-phase system, employing ethanol and ammonium sulfate. In a similar vein, Cheng et al. [41] devised a technique for the concurrent extraction and purification of polysaccharides from Gentiana scabra Bunge, employing an ethanol-salt aqueous two-phase system. These studies demonstrate the potential of microwave-assisted extraction for polysaccharides, with a range of solvents being effective. By employing this method, it is crucial to optimize the extraction conditions to achieve the maximum yield. Typically, the optimized conditions of this method yield better results compared to other extraction methods [42].

In summary, several auxiliary extraction methods hold the potential to increase polysaccharide yield, decrease extraction time, and reduce energy consumption in the extraction process. A recent development involves using ionic liquids for polysaccharide extraction at low temperatures and in a short time [43]. As this innovative method is not widely adopted, further studies can explore its potential in the future.

Molecular weight distribution determination

The determination of polysaccharide purity is a necessary step to ensure homogeneity. The molecular weight distribution of polysaccharides can indicate whether the purified polysaccharide components are uniform or pure. Good homogeneity is a prerequisite and foundation for the subsequent process of structure identification. Gel chromatography is presently the most commonly employed method for evaluating the purity and homogeneity of polysaccharides [61, 62]. For instance, high-performance gel permeation chromatography, utilizing columns of different specifications, can be employed to assess the purity and homogeneity of polysaccharides across a range of molecular weight gradients [63].

Monosaccharide composition determination

The determination of monosaccharide composition holds paramount significance in anticipating the core structure of the targeted polysaccharide backbone and scrutinizing its physicochemical properties. Widely employed techniques for the analysis of polysaccharide monosaccharide composition encompass pre-column derivatization in conjunction with high-performance liquid chromatography analysis and capillary electrophoresis [64]. Additionally, high-performance anion exchange chromatography has emerged as a new technology for polysaccharide sample analysis in recent years [65, 66]. The principle involves the elution of monosaccharides and oligosaccharides hydrolyzed from polysaccharides under conditions with a pH>12 [67]. Polysaccharides undergo exchange separation on a high-performance anion exchange resin, with detection carried out using a pulsed amperometric detector. This method enables the direct analysis of hydrolyzed products without the need for derivatization, making it increasingly adopted in polysaccharide analysis.

Infrared spectroscopic analysis

Currently, infrared spectroscopy is widely used for determining polysaccharide structures. Infrared spectroscopy serves as a valuable tool for offering preliminary insights into the functional groups inherent in polysaccharides and evaluating the purity of such compounds [68]. This method is characterized by its simplicity in operation and a brief experimental cycle. However, due to the significant overlap of spectral peaks in infrared spectroscopy, it is insufficient for determining the functional group structures of polysaccharides on its own. It is often necessary to combine it with other methods for further elucidation [69].

Methylation analysis

Methylation analysis can be employed to determine the sequence of monosaccharide residues, the types of monosaccharide residues, and the bonding patterns in polysaccharides [70]. The commonly used methylation method involves first fully methylating the polysaccharide, freeze-drying the product, and then adding trifluoroacetic acid for complete hydrolysis. Subsequently, the hydrolyzed products are acetylated after reduction with borohydride, resulting in partially methylated alditol acetates (PMAA). Gas chromatography-mass spectrometry is utilized for the analysis of PMAA, and the results are compared with spectra in the University of Georgia PMAA Spectral Database to acquire information about the composition and linkage patterns of the residues [71].

Nuclear magnetic resonance analysis

Nuclear magnetic resonance (NMR) stands as a crucial method in the structural analysis of polysaccharides. One-dimensional spectra, including ¹H-NMR and ¹³C-NMR spectra, serve as initial tools for determining the type of monosaccharide, the number of sugar residues, the configuration of glycosidic bonds, and other relevant information. Additionally, two-dimensional NMR spectra encompasses homonuclear shift correlated spectra (COSY, COSY-45, DQF-COSY, TQF-COSY, TOCSY, NOESY) and carbon-hydrogen correlated spectra (HSQC, HMBC) can further elucidate the connection mode of sugar residues in polysaccharides and determine the structure of repeating sugar units [72]. For instance, in ¹H-NMR spectra of polysaccharides, terminal substrate signals appear between 4.5 and 5.5 ppm, while ¹³C-NMR spectra of polysaccharides typically show most peaks between 60 and 110 ppm [73]. Moreover, ¹³C, fully decoupled with integral terminal signals in NMR spectra is used to detect polysaccharide residue types, and HMBC is employed to bind hydrogen protons to distant carbon for structural skeleton analysis.

The advancement of biotechnology has led to the gradual application of technologies such as lectin identification and fluorescent labeling in the structural analysis of polysaccharides [35]. Given the intricate nature of polysaccharide structures, the selection of appropriate analytical methods is imperative, contingent upon the specific characteristics of the structures under investigation.

Promotes insulin secretion and inhibits islet cell apoptosis

Insulin, a hormone synthesized and released by pancreatic β-cells and complemented by the secretion of glucagon from pancreatic α-cells, holds a central role in preserving blood glucose equilibrium and regulating glucose homeostasis. This is accomplished by facilitating the uptake and utilization of glucose in peripheral target tissues, including the liver, muscles, and adipose tissue. Insulin further supports hepatic glycogen synthesis and improves sugar metabolism, consequently reducing postprandial blood glucose levels [81]. Plant polysaccharides contribute to blood glucose reduction by safeguarding and enhancing the activity of β-cells, inhibiting apoptosis of pancreatic cells, and effectively promoting insulin secretion.

Tang et al. [74] reported a significant improvement in glucose and lipid metabolism in T2DM rats through the administration of Astragalus polysaccharide (APS), a polysaccharide extracted from Astragalus. This beneficial effect is ascribed to the safeguarding and enhancement of β-cells, leading to an increase in insulin secretion. Additionally, APS activate the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This activation results in the upregulation of GLP-1receptors, pancreatic and duodenal homeobox 1 (PDX-1), and the anti-apoptotic protein B-lymphocyte tumor-2 (Bcl-2) [82]. Concurrently, it downregulates the expression of the pro-apoptotic protein Bcl2-associated X (Bax). These molecular actions collectively impede apoptosis in mouse pancreatic β-cells (β-TC6 cell line), ultimately accomplishing the goal of reducing blood glucose levels. Research indicates that GLP-1 plays a pivotal role in safeguarding pancreatic islet cells through diverse mechanisms, exerting a notable impact on T2DM. Li et al. [83] postulated that Polygonatum cyrtonema Hua polysaccharides (PCP) possess the capability to enhance GLP-1 secretion, thereby contributing to the amelioration of diabetes.

Enhances insulin sensitivity and reduces insulin resistance

IR encompasses a range of factors that diminish the body’s responsiveness to insulin. Augmenting insulin sensitivity constitutes a crucial pathway in the regulation of blood glucose balance [81]. Individuals afflicted by IR are predisposed to diverse metabolic disorders, including T2DM, hypertension, dyslipidemia, and so on. Moreover, IR is linked to elevated levels of inflammation and alterations in lipid metabolism [84]. Current research has established a positive correlation between the insulin resistance index and serum triglycerides (TG) and fasting blood glucose while demonstrating a negative correlation with high-density lipoprotein cholesterol (HDL-C) [85]. The regulation of TG and HDL-C levels has been identified as a means to ameliorate insulin resistance. Chen et al. [86] documented that polysaccharides derived from Polygonatum sibiricum predominantly elicit hypoglycemic effects through the activation of insulin receptor substrate-1 (IRS-1), PI3K, and glucose transporter 4 (GLUT4), concurrently inhibiting the c-Jun N-terminal kinase (JNK) signaling pathway. Research findings indicate that polysaccharides extracted from Chroogomphus rutilus induce hypoglycemic effects in leptin-deficient mice by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway, thereby enhancing autophagy. This cascade promotes the expression of peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase-1A, resulting in heightened lipolysis and subsequent inhibition of hepatic lipid deposition. Ultimately, these effects contribute to the enhancement of insulin sensitivity [75].

Regulates enzyme activities

α-Amylase and α-glucosidase are pivotal enzymes directly engaged in starch and glycogen metabolism, with their activity intricately linked to glucose release [87]. In individuals with T2DM, the onset of insulin resistance in body cells disrupts the insulin signaling pathway, impeding glucose uptake in tissues such as adipose and muscle. The accelerated degradation of glucose by α-amylase and α-glucosidase contributes to a rapid surge in postprandial blood glucose levels [88]. Consequently, a viable strategy for diabetes management involves competitively inhibiting key digestive enzymes (α-amylase and α-glucosidase) with natural products.

This inhibition decelerates the absorption and digestion of carbohydrates in the digestive tract, effectively mitigating postprandial hyperglycemia [87]. Polysaccharides extracted from South African seaweed, such as fucoidan, demonstrate a significant inhibitory effect on the activity of α-amylase and α-glucosidase, thereby efficiently controlling blood glucose and presenting substantial potential for the treatment of T2DM [89]. Shao et al. [76] synthesized carbon nanoparticles (CNPs) derived from burdock root polysaccharides using a hydrothermal method and illustrated that CNPs can inhibit α-glucosidase activity, consequently lowering blood glucose levels in mice induced with a high-fat diet and streptozotocin. These studies collectively indicate that the direct inhibition of α-amylase and α-glucosidase activities can suppress glucose release, diminish glucose absorption, and thereby ameliorate disorders in glucose metabolism.

Disruption of glucose metabolism stands as a primary contributor to the onset of T2DM. The hydrolysis of starch by α-amylase results in the production of glucose, which, in turn, can lead to elevated blood sugar levels [90]. Essential enzymes in sugar metabolism, including hexokinase (HK), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PD), and pyruvate kinase (PK), play critical roles in this process. HK and GK serve as key catalysts in sugar metabolism by converting glucose to glucose-6-phosphate, facilitating the synthesis of liver glycogen. G6PD is a crucial enzyme in the pentose phosphate pathway, catalyzing the conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone [91]. PK acts as a pivotal rate-limiting enzyme in sugar glycolysis. Substantial evidence indicates that individuals with T2DM exhibit diminished glucokinase activity, resulting in an imbalance in blood sugar homeostasis. Plant polysaccharides have the capacity to enhance sugar metabolism by regulating the activity of pertinent enzymes, thereby exerting control over blood sugar levels. Studies have revealed that mulberry leaf polysaccharides significantly enhance the liver lipid metabolism and sugar metabolism function in diabetic rats. This improvement is achieved by elevating liver glycogen content and enhancing liver GK activity, consequently ameliorating insulin resistance [92].

Inhibition of inflammatory factor expression

Research suggests that plant polysaccharides can exert anti-inflammatory effects by inhibiting the expression of inflammatory factors. This action prevents damage to β-cell function, ultimately leading to a reduction in blood glucose levels [93]. Qiao et al. [94] found that the mechanism by which Schisandra acidic polysaccharides improve insulin resistance involves the inhibition of inflammation. This encompasses a decrease in the levels of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, C-reactive protein, and nuclear factor kappa-B (NF-κB), along with a reduction in their mRNA expression in liver tissue. It also entails the inhibition of the expression of phosphorylated JNK and NF-κB proteins, coupled with a substantial increase in the expression of phosphorylated IRS-1, phosphorylated PI3K, and phosphorylated AKT.

Improves antioxidant stress capacity

Diabetes patients generate a large amount of reactive oxygen species in the body, and β-cells have low antioxidant stress capacity, leading to β-cell damage, abnormal metabolism of lipids and glucose, and prolonged high-fat diet exacerbating the burden on the antioxidant system. Zhang et al. [77] reported that polysaccharides from Codonopsis lanceolata improved high-fat diet-induced insulin resistance by activating antioxidant signaling pathways. This improvement was evidenced by the repair of damaged AKT phosphorylation and high phosphorylation at Ser307 of IRS-1, a significant reduction in malondialdehyde levels, and an enhanced ratio of reduced glutathione to oxidized glutathione. Moreover, it elevated the expression of antioxidant enzymes and activated the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Oat β-glucan complex was shown to improve diabetes by enhancing the body’s antioxidant capacity, scavenging free radicals, and preventing and reducing the occurrence of diabetes complications [95]. Coarse polysaccharides from Dendrobium officinale Kimura enhanced antioxidant capacity in the liver and pancreas of mice in a model of hyperuricemia, repairing oxidative damage to these organs [96]. In in vitro and animal experiments utilizing crude and purified polysaccharides from Huidouba, Chen et al. [97] demonstrated that these polysaccharides could effectively reduce blood glucose levels. This reduction was attributed to their ability to combat oxidative stress, inhibit α-glucosidase activity, and improve insulin resistance. Furthermore, their findings revealed that low molecular weight purified polysaccharides exhibited markedly greater blood glucose-lowering activity compared to crude polysaccharides. This observation suggests that the structure of polysaccharides can influence their activity, and the process of purification has the potential to enhance both the antioxidant and blood glucose-lowering activities of polysaccharides.

Modulates related signaling pathways

Insulin exerts its influence by interacting with insulin receptors on the cell membrane, predominantly regulating cellular physiological activities through the PI3K/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. Elevating the expression of crucial molecules within the PI3K/AKT pathway, including mRNA and relevant signal proteins, proves instrumental in enhancing β-cell function and maintaining the regulation of blood glucose levels [42]. Su et al. [79] reported that the potential mechanism driving the hypoglycemic effect of Aconitum coreanum polysaccharides in high-fat diet-induced diabetic mice includes heightened insulin sensitivity, improved glucose tolerance, suppression of inflammatory factor expression in serum and insulin-targeted tissues, and inhibition of the activation of the NF-κB cellular pathway. This leads to a reduction in the serine phosphorylation of IRS-1, subsequently restoring glucose utilization through the PI3K/AKT signaling pathway. In the in vivo hypoglycemic test of Hericium erinaceus polysaccharide, it was found that its hypoglycemic mechanism actively mediates glycogen synthesis primarily through the PI3K/AKT signaling pathway [98].

The MAPK transduction pathway encompasses the extracellular signal-regulated kinase 1/2 (ERK1/2), JNK1/2/3, p38, and ERK5 pathways, which collectively regulate a spectrum of cellular activities, including cell proliferation, differentiation, apoptosis, inflammation, and immunity [99]. It has been observed that various cytokines and stress stimuli can induce β-cell apoptosis through one or more MAPK signaling pathways. Therefore, plant polysaccharides have the potential to inhibit β-cell apoptosis by modulating the MAPK signaling pathway. For instance, spurge polysaccharides have been shown to up-regulate the phosphorylation levels of p38, NF-κB, p65, and ERK1/2 proteins in mouse macrophages. This activation stimulates the MAPK and NF-κB signaling pathways, leading to improved immunity and inhibition of diabetes mellitus [100].

Protein kinase C (PKC) functions as a multifunctional enzyme implicated in gene expression and regulation. Under high glucose conditions, PKC is activated as an inducer, subsequently triggering the activation of the NF-κB pathway. This activation, in turn, leads to inflammation and abnormal lipid metabolism in diabetic rats [101]. Li et al. [102] reported that Arctium lappa L. polysaccharides regulate lipid metabolism in diabetic rats through the PKC/NF-κB pathway, resulting in reduced levels of total cholesterol, triglycerides (TG), and low-density lipoprotein cholesterol. Nrf2 plays a crucial role in anti-oxidative stress and anti-inflammatory processes, and it has the capacity to inhibit insulin resistance. The deficiency of Nrf2 has been associated with pancreatic β-cell injury. Radix pseudostellariae polysaccharides have been shown to improve IR by activating the Nrf2 signaling pathway. This activation helps attenuate oxidative stress, inhibit hepatic inflammatory signaling, and enhance the abundance of beneficial intestinal flora [103].

Regulates intestinal flora

Various studies have highlighted that the gut microbiota can impact the body’s metabolism through pathways such as bile acid metabolism, short-chain fatty acid metabolism, and inflammatory responses [104]. Imbalances in the gut microbiota are linked to insulin resistance, and plant polysaccharides can contribute to the regulation of the gut microbiota, thereby reducing blood sugar levels. For instance, Apocynum venetum leaves polysaccharides have been shown to reverse gut microbiota imbalance in diabetic mice. This is achieved by increasing the abundance of Odoribacter, Anaerotruncus, Faecalibaculum, and Muribaculum genera while decreasing the abundance of Coprococcus, Klebsiella, and Allobaculum genera [80]. Shang et al. [105] fed diabetic mice with varying doses of purified Cordyceps militaris polysaccharides (CBPS-II) and confirmed that CBPS-II regulates the energy metabolism, gut microbiota, and lipid metabolism disorders associated with diabetes. Various serum metabolites have been identified as potential biomarkers, offering valuable insights for the early diagnosis and treatment of diabetes. Wang et al. [106] discovered that oral administration of Crataegus pinnatifida polysaccharides in spontaneous T2DM mice has hypoglycemic and hypolipidemic effects. The mechanism primarily entails a noteworthy downregulation of the elevated expression levels of G6PD, fatty acid synthase, acetyl-CoA carboxylase 1, sterol regulatory element-binding protein-1c, and peroxisome proliferator-activated receptor γ mRNA associated with gluconeogenesis and lipid synthesis. Consequently, this leads to a reduction in the Firmicutes/Bacteroidetes ratio in the intestines of diabetic mice, an augmentation in the abundance of beneficial bacteria, and a decline in the abundance of harmful bacteria.