Comparisons of Isolation Methods, Structural Features, and Bioactivities of the Polysaccharides from Three Common Panax Species: A Review of Recent Progress

Panax spp. (Araliaceae family) are widely used medicinal plants and they mainly include Panax ginseng C.A. Meyer, Panax quinquefolium L. (American ginseng), and Panax notoginseng (notoginseng). Polysaccharides are the main active ingredients in these plants and have demonstrated diverse pharmacological functions, but comparisons of isolation methods, structural features, and bioactivities of these polysaccharides have not yet been reported. This review summarizes recent advances associated with 112 polysaccharides from ginseng, 25 polysaccharides from American ginseng, and 36 polysaccharides from notoginseng and it compares the differences in extraction, purification, structural features, and bioactivities. Most studies focus on ginseng polysaccharides and comparisons are typically made with the polysaccharides from American ginseng and notoginseng. For the extraction, purification, and structural analysis, the processes are similar for the polysaccharides from the three Panax species. Previous studies determined that 55 polysaccharides from ginseng, 18 polysaccharides from American ginseng, and 9 polysaccharides from notoginseng exhibited anti-tumor activity, immunoregulatory effects, anti-oxidant activity, and other pharmacological functions, which are mediated by multiple signaling pathways, including mitogen-activated protein kinase, nuclear factor kappa B, or redox balance pathways. This review can provide new insights into the similarities and differences among the polysaccharides from the three Panax species, which can facilitate and guide further studies to explore the medicinal properties of the Araliaceae family used in traditional Chinese medicine.

Currently, most studies focus on the purification, structural analysis, and bioactivities of polysaccharides from the three above-mentioned different Panax species [29][30][31]. Ginseng polysaccharides have been deeply studied and recent findings have been extensively summarized [9,10]. Recently, the polysaccharides from two other species were examined, and most studies focus on the structural features and immune-stimulating effects of polysaccharides isolated from American ginseng and notoginseng [32,33]. The similarities and differences between these polysaccharides from Panax species and their effects on isolation and purification, structural characteristics, and biological functions remain unclear and have been not summarized. In this review, we summarize the methods of extraction and purification, structural characteristics, and the main biological activities of polysaccharides from three common ginseng species, which will provide new insights into the understanding of current research and future direction for these polysaccharides.
Most studies report that hot water extraction was used to isolate the polysaccharides from three Panax species, which is the most classical and convenient method for isolating water-soluble crude polysaccharide [29,46]. Briefly, the roots of Panax species were soaked in water overnight and boiled in water (2-4 times, 2-6 h each time) to obtain the supernatants, which were centrifuged at 5000 rpm for 30 min and concentrated under vacuum. The crude extracts were precipitated with cold ethanol at 4 • C for 24 h and deproteinated by the Sevag method to obtain crude polysaccharides [9]. Some reports showed that 80-95% ethanol was used to remove lipophilic compounds and increase purity before water extraction [36,47], which can increase the rate of extraction of polysaccharides from ginseng [48]. In fact, different extraction methods have been used to prevent the destruction of polysaccharide structures and bioactivities. EDTA extraction was used to extract pectin-type polysaccharides with high efficiency [42].
After water extraction, alkali extraction of acid polysaccharides with Na 2 CO 3 , 1 M KOH, or 4 M KOH is suitable when high temperature may degrade the activities of some polysaccharides [37,49]. Furthermore, enzyme-assisted extraction methods can destroy granular starch and enable a higher extraction yield of polysaccharides. Two enzymes, α-amylase and cellulase, were used to effectively extract ginseng polysaccharides with different structures and activities, compared with that of water extraction [39,40]. Microwaveassisted extraction is a novel, quick, and efficient method for extracting polysaccharides with higher bioactivities and a higher yield rate (41.6%) than that of hot water extraction (28.5%) [45]. For two other Panax species, water extraction has been used to isolate crude polysaccharides, and alkaline extraction [38,50,51], ethanol extraction [34,52], or ultrasonic extraction [43] have seldomly been used. Current studies demonstrate that the polysaccharide extraction rates for American ginseng and notoginseng by alkali or methanol ranged from 1.8% to 2.8% [51]. Based on these findings, we conclude that different polysaccharides from three species can be isolated with different reagents, enzymes, or equipment to avoid the disadvantages of extraction processes and obtain high-yield and high-activity polysaccharides. Importantly, a combined method might be a more optimal strategy for extracting polysaccharides with different structures and bioactivities.

Separation and Purification
After ethanol precipitation and deproteinization, crude polysaccharides are purified and fractionated by column chromatography and membrane separation technology [53]. Column chromatography methods, including gel column chromatography and ion-exchange chromatography, are commonly used to purify the polysaccharides from three Panax species [39,47,54]. Gel column chromatography, which includes dextran-and agarose-gel columns, acts as a molecular sieve and separates polysaccharide molecules according to their size and shape [39,55]. As previously reported, the polysaccharides from ginseng were purified using Sephadex G-25 or G-75 columns [39,55]. A study showed that four polysaccharides from notoginseng were purified using gel filtration chromatography [56]. For ion-exchange chromatographic separation, an anion-exchange column packed with diethylaminoethyl (DEAE)-cellulose, DEAE-dextran gel, or DEAE-agarose gel has been the most used to separate ginseng polysaccharides. These materials possess different advantages, including large adsorption capacity, strong stability, fast elution, and weak protein binding [53].
To be specific, the crude polysaccharides from ginseng can be purified on a DEAE Sepharose Fast Flow column, Sephadex G-75 [39], Sephadex G-100 [52], Sepharose CL-6B [34], or Sephacryl S-200 column [34], which are eluted with distilled water and different concentrations of stepwise NaCl solution. The alkali-extractable polysaccharides from North American ginseng have been purified with a DEAE Sepharose Fast Flow column [38] or DEAE-Sepharose CL-6B column [57]. After extraction with 1 M KOH, the polysaccharide fraction for notoginseng was applied to a DEAE-Sepharose CL-6B column, followed by gel-permeation chromatography for purification [58]. Further purification to remove oligosaccharides can be accomplished using a dialysis membrane (3.5 × 10 3 Da or 1 × 10 3 Da) based on the concentration difference [30,39,59,60]. In addition, ultrafiltration mainly separates starch and protein from the polysaccharide fractions of the three species, and this is suitable for the large-scale purification of polysaccharides [61].
Collectively, combined purification methods have been used to obtain different fractions of the polysaccharides from three common Panax species, according to experimental aims. To analyze the structures and activities of polysaccharides of three species, polysaccharide fractions should be commonly extracted using hot water or alkali, then purification should proceed using column chromatography and membrane separation technology to obtain target polysaccharides. A procedural comparison of the extraction and purification techniques for different polysaccharides from three Panax species is shown in Figure 1.

Structural Characteristics
The polysaccharides from the three species are natural polymers of more than 10 monosaccharides consisting of linear or branched carbohydrate chains joined by glycosidic linkages [62]. Owing to the complexity and diversity, chemical methods (acid hydrolysis, periodate oxidation, and Smith degradation) and physical methods (nuclear magnetic resonance spectroscopy and mass spectrometry) are used to analyze polysaccharide composition characteristics and primary structures, including monosaccharide composition, the sequence and linkage of sugar groups, and anomeric carbon or sugar ring configuration [9,10,40]. Currently, enzyme-linked immunoassay, electron scanning microscopy, and circular dichroism have been used to determine advanced structures, which is a challenge in the research area of polysaccharide structure [57]. Neutral polysaccharides are the main components of ginseng polysaccharides and acid polysaccharides are a small portion and include pectin-containing rhamnose (Rha) and homogalacturonic acid [63]. Ginseng pectin, an acid polysaccharide mixture, is comprehensively studied to explore its composition and structure, which contains galactose (Gal), galacturonic acid (GalA), arabinose (Ara), and Rha [34,64]. Polysaccharide composition and structure are analyzed by a series of methods and techniques, but it is difficult to identify

Structural Characteristics
The polysaccharides from the three species are natural polymers of more than 10 monosaccharides consisting of linear or branched carbohydrate chains joined by glycosidic linkages [62]. Owing to the complexity and diversity, chemical methods (acid hydrolysis, periodate oxidation, and Smith degradation) and physical methods (nuclear magnetic resonance spectroscopy and mass spectrometry) are used to analyze polysaccharide composition characteristics and primary structures, including monosaccharide composition, the sequence and linkage of sugar groups, and anomeric carbon or sugar ring configuration [9,10,40]. Currently, enzyme-linked immunoassay, electron scanning microscopy, and circular dichroism have been used to determine advanced structures, which is a challenge in the research area of polysaccharide structure [57]. Neutral polysaccharides are the main components of ginseng polysaccharides and acid polysaccharides are a small portion and include pectin-containing rhamnose (Rha) and homogalacturonic acid [63]. Ginseng pectin, an acid polysaccharide mixture, is comprehensively studied to explore its composition and structure, which contains galactose (Gal), galacturonic acid (GalA), arabinose (Ara), and Rha [34,64]. Polysaccharide composition and structure are analyzed by a series of methods and techniques, but it is difficult to identify advanced structures such as hydrogen bonding of polysaccharide main chains, repeating sequences of sugar chains, and non-covalent bonding of polymer chains.
Briefly, the differences and similarities among these three common Panax species polysaccharides are listed as follows. Similarities: Most of the three common Panax species polysaccharides are extracted by hot water extraction. Some monosaccharides exist in all three Panax species polysaccharides, such as Ara, Gal, GalA, Glc, GlcA, Man, and Rha. RG-II and 1→4 glycosidic linkages as linear backbone are extensively found in three common Panax species. In addition, the activity research of three common Panax species polysaccharides focuses on antitumor activity, immunomodulatory activity antioxidative activity. Differences: Nowadays, the ginseng polysaccharides that have been reported could be extracted by enzyme, EDTA, microwave, ultrasonication, and other new methods. While American ginseng and notoginseng polysaccharides only recently began to use new extraction methods. The molecular weights of three common Panax ginseng species polysaccharides are different, which may be related to the extraction and purification methods. The structure of ginseng polysaccharide is complex and diverse, while American ginseng and Panax notoginseng are rather simple. In addition to anti-tumor activity, immunomodulatory, and anti-oxidative activity, ginseng polysaccharide also has antihyperglycemic activity and anti-fatigue activity, and prolongs life. Panax notoginseng can protect the liver. The activity of American ginseng is mainly immunomodulatory and other active polysaccharides are less studied. Otherwise, there is no relevant study comparing the efficacy of these three common Panax species polysaccharides, at present.

Antitumor Activity
Many studies have shown that polysaccharides from the Araliaceae family, especially ginseng polysaccharides, exhibit anti-tumor activity in cell and animal models. In peritoneal macrophage and leukemia cell models, ginseng polysaccharide (GPS) stimulated macrophages to increase the levels of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, and nitric oxide (NO) production against leukemia [88]. In malondialdehyde (MDA)-MB-231 breast cancer cells, GPS activated p65-IKZF1 signaling and apoptosis to inhibit cell proliferation [89]. In HCT-116 and HT-29 human colon cancer cells, ginseng berry polysaccharide extract (GBPE) and its purified fragment, GBPP, significantly inhibited IL-8 secretion and Th1 and Treg cell differentiation to suppress cell growth [90]. In B16-BL6 melanoma, GBPP exhibited a function similar to that observed in colon cancer [18]. Similarly, NFP, which is a polysaccharide from Korean red ginseng, inhibited melanoma cell metastasis to the lung, which may have resulted due to its immunity-enhancing effect [91]. Furthermore, a polysaccharide from ginseng leaves, GS-P, promoted macrophages and natural killer (NK) cells to exhibit anti-metastatic activity against colon cancer [92].
A neutral polysaccharide, WGPN, exhibited functions similar to those of GPS and, when combined with 5-fluorouracil, a synergistic effect was observed, which indicates that it has potential as an adjuvant that can be used against sarcoma tumors [93]. An acidic polysaccharide, WGPA, and its fraction, WGPA-3-HG, inhibited HT-29 colon cancer cell proliferation and caused G 2 /M phase arrest [94]. Moreover, temperature-modified WGPA-3-HG (MWGPA-3-HG) increased the percentages of S and G 2 /M phase cells and induced apoptosis by activating caspase-3 [94]. In gastric cancer cells, PGPW1 regulated Twist expression to block metastasis [36]. In a mouse model of Lewis lung carcinoma, two ginseng polysaccharides (GP and GFP1) significantly increased the ratio of CD4 + /CD8 + T lymphocytes and promoted NK cytolytic activity, which shows that they possess a satisfactory immunomodulatory effect against lung cancer [95,96]. In addition, serum analysis demonstrated that GPS increased Th1 cytokines (INF-γ and IL-2) and decreased Th2 cytokines (IL-4 and IL-5) in 96 patients with non-small-cell lung cancer [97]. A new report demonstrated that ginseng polysaccharides altered the gut microbiota and the kynurenine/tryptophan ratio to potentiate the anti-tumor effect of anti-PD-1/PD-L1 immunotherapy [98].
For American ginseng polysaccharides, only one study showed that ginseng polysaccharide nanoparticles (GPS-NPs) inhibited oxidative damage and skin cancer induced by UVB exposure [82]. Three studies were reported that tested notoginseng polysaccharides against pancreatic and hepatic cancers. In a BxPC-3 pancreatic cancer xenograft model, an arabinogalactan polysaccharide (RN1) from notoginseng flowers inhibited microvessel formation and migration by the inhibition of BMP2/Smad1/5/8/Id1 signaling [86]. In H22 cells and a tumor-bearing mouse model, PPN, a notoginseng polysaccharide, activated CD4 + T-cells and elevated serum IL-2 to inhibit liver cancer growth and prolong survival [99]. Furthermore, a neutral notoginseng polysaccharide, NPPN, was combined with CTX and significantly inhibited H22 tumor growth via myelosuppression [100]. The mode of action of different ginseng polysaccharides is mainly that they regulate immune cytokines to inhibit tumor progression. Because there are few studies that have reported that the polysaccharides from the other two species exhibit anti-cancer activities, this could be a potential research direction.

Immunomodulatory Activity
Many studies have shown that different ginseng polysaccharides possess immunomodulatory functions against numerous immune-related diseases. Two ginseng polysaccharides named FGWP-CA and GPS increased the NK cell activity induced by CTX and played a role in immune regulation [40,101]. In an autoimmune encephalomyelitis mouse model, APG, which is an acidic polysaccharide from ginseng, promoted Treg cell generation through Foxp3 activation and the production of inflammatory cytokines, such as interferon-γ (IFN-γ), IL-1β, and IL-17 [102]. This might also modulate the infiltration of CD4 + T cells and CD11b + macrophages into the spinal cord [103]. In different cell models, four ginseng polysaccharides (GPNE-I, WGPA-2-RG, ginsan, and PS-NPs) enhanced lymphocyte proliferation, macrophage phagocytosis, and dendritic cell maturation by regulating various cytokine levels and NO production [20,23,35,64,104]. Several studies reported that RGAP, an acidic polysaccharide from Korean red ginseng, activated the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB)/AP-1 signaling pathways and augmented the production of IL-6, IL-12, TNF-α, and NO in macrophages from a female BALB/c mouse model [49,105]. Moreover, RGAP increased the numbers of T cells, B cells, macrophages, and IgM antibody-forming cells to enhance macrophage phagocytosis activity [106] and increase the number of plaqueforming cells [107]. RGP-AP-I and RG-CW-EZ-CP are polysaccharides that exhibit functions and molecular mechanisms that are similar to those of RGAP [22,41]. Furthermore, a neutral ginseng polysaccharide (NGP) stimulated the maturation of bone marrow dendritic cells through upregulation of MHC class II, CD80, and CD86 [108]. In addition, PGP-SL and GMP are two polysaccharides that exhibited immunopotentiation effects by enhancing the Ca 2+ /calcineurin/NFAT signaling pathway in spleen lymphocytes [109] and reactive oxygen intermediates in peritoneal macrophages [110], respectively. In weaned piglets induced with lipopolysaccharide (LPS), GPS regulated the TLR4/MyD88-NF-κB pathway to reduce immunological stress [111].

Anti-Oxidative Activity
At present, there is a greater number of reports on the anti-oxidant activity of ginseng polysaccharides than that of reports on American ginseng and notoginseng polysaccharides.
Three ginseng polysaccharides, named ginseng-SDF, native ginseng polysaccharide, and ginseng polysaccharide, significantly scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, or superoxide anion radicals to exhibit their anti-oxidant activities [24,25,117]. Neutral and acidic polysaccharides from ginseng (WGNP and WGAP) are antioxidants that scavenged hydroxyl radicals and decreased reactive oxygen species (ROS) and lipid peroxidation in a Caenorhabditis elegans model [31]. In a streptozotocin-induced diabetic mouse model, neutral and acidic ginseng polysaccharides (WGPN and WGPA) exhibited anti-diabetic potential, which was mediated by the inhibition of anti-oxidative activity [35]. WGPA and its fraction, WGPA-A, inhibited oxidative stress by regulating the balance of oxidation and anti-oxidation, which resulted in an anti-fatigue effect [59,118]. AEP-2 is an alkali-extractable polysaccharide from American ginseng that exhibited higher values of Trolox equivalent and oxygen radical antioxidant capacities [38]. A polysaccharide from notoginseng (FPNP) decreased the amount of MDA and increased the activities of antioxidant enzymes, such as catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD), by activating the transforming growth factor-β (TGF-β)/Smad signaling pathway into H 2 O 2 -induced human dermal fibroblast cells [2]. Collectively, nine polysaccharides from three Panax species have been reported as natural antioxidants.

Other Biological Functions
In addition to the main functions above, other biological functions of different polysaccharides from ginseng and notoginseng have been reported in recent years. GPS promoted food intake in mice, which may be related to appetite-regulation peptides and circulating glucose levels [119]. In an ethanol-induced gastrically injured rat model, GPS increased anti-oxidant activity and suppressed inflammation [27]. In a diarrhea mouse model induced by antibiotics, a ginseng polysaccharide named WGP changed the gut microbiota composition and diversity and balanced metabolic processes to recover the mucosal structure [69]. Moreover, WGP prevented cisplatin-induced endoplasmic reticulum stress and cell death in renal cells by activating PERK-eIF2α-ATF4 signaling [120].
In a diabetic rat model, ginseng polysaccharide (GP) enhanced ginsenoside Rb1 biotransformation and this resulted in an anti-diabetic effect and protected against dextran sulphate sodium-induced colitis, which may be associated with the gut microbiota [121,122]. Similarly, ginseng polysaccharide APG protected the mouse small intestine from irradiation by inhibiting the p53-dependent and mitochondrial apoptosis pathways [123]. Furthermore, GP decreased lung viral titers and inhibited IL-6 to protect mice from H1NI influenza virus infection [124]. Additionally, an acidic polysaccharide fraction of ginseng (AP1) activated the reperfusion injury salvage kinase and endothelial nitric oxide synthase (eNOS)-dependent pathways to maintain mitochondrial function against myocardial hypoxia/reoxygenation injury [125].
For anxiety disorders, another acidic polysaccharide, WGPA, exhibited an anti-depressantlike effect by affecting social interactions and aggressive behaviors in mice [65]. For skin health, a byproduct polysaccharide from red ginseng was processed by enzyme-linked high pressure to produce ELHPP-RGBPs, which inhibited the AP-1/MMP-1 pathway and prevented solar ultraviolet-induced skin wrinkles and atopic dermatitis [126]. The notoginseng polysaccharides PNPS and PNPS-0.5M inhibited a caspase-3 cascade or regulated the alcohol dehydrogenase pathway to protect from cerebral ischemia/reperfusion injury or alcoholic liver damage, respectively [43,127]. These three species of polysaccharides exhibited anti-tumor, immunoregulatory, and anti-oxidation activities, as well as anti-diabetic, anti-fatigue, and anti-depression activity. The biological functions, detailed models, and molecular mechanisms of the polysaccharides from the three Panax species are listed in Table 5 and are shown in Figure 2. Table 5. Activities and molecular mechanisms of polysaccharides from ginseng, American ginseng, and notoginseng.

Conclusions and Future Perspective
This review summarized recent advances associated with 112 polysaccharides from ginseng, 25 polysaccharides from American ginseng, and 36 polysaccharides from notoginseng and compared the differences in extraction, purification, and structural features. Most studies focused on ginseng polysaccharides and, if comparisons were made, the polysaccharides used were from American ginseng and notoginseng. For the extraction, purification, and structural analysis of polysaccharides, the processes were similar for all three Panax species. Generally, ginseng (4-5-year-old, crushed and passed through 60 or 80 mesh sieves, dried at 60 • C and stored in the freezer), American ginseng (4-5-year-old, ground and passed through a 40-mesh, 60-mesh or 80-mesh sieve and dried at room temperature), notoginseng (dried at 60 • C for 24 h, ground and passed through a 60-mesh sieve and stored in a desiccator at room temperature). The greatest number of articles has been written on ginseng polysaccharides, followed by American ginseng and Panax notoginseng. They possess anti-tumor activity, immunoregulatory effects, anti-oxidant activity, and other pharmacological functions, which are mediated by multiple signaling pathways, including the MAPK, NF-κB, or redox balance pathways ( Figure 3). rification, and structural analysis of polysaccharides, the processes were similar for all three Panax species. Generally, ginseng (4-5-year-old, crushed and passed through 60 or 80 mesh sieves, dried at 60 °C and stored in the freezer), American ginseng (4-5-year-old, ground and passed through a 40-mesh, 60-mesh or 80-mesh sieve and dried at room temperature), notoginseng (dried at 60 °C for 24 h, ground and passed through a 60-mesh sieve and stored in a desiccator at room temperature). The greatest number of articles has been written on ginseng polysaccharides, followed by American ginseng and Panax notoginseng. They possess anti-tumor activity, immunoregulatory effects, anti-oxidant activity, and other pharmacological functions, which are mediated by multiple signaling pathways, including the MAPK, NF-κB, or redox balance pathways (Figure 3). Seven important aspects should be further considered based on the recent findings for these polysaccharides from the three Panax species. (1) The structural characteristics and biological activities of the polysaccharides from American ginseng and notoginseng should be deeply investigated. (2) As shown in a recent report [128], new approaches, such as two-dimensional attenuated total reflection Fourier transform infrared spectroscopy based on a gradient heating program, should be developed to discriminate and identify the structural features of Panax polysaccharides. (3) The differences in biological activity might be related to functional groups, branching, and conformational characteristics of the polysaccharides, which could be a future direction for exploring the relationship  Seven important aspects should be further considered based on the recent findings for these polysaccharides from the three Panax species. (1) The structural characteristics and biological activities of the polysaccharides from American ginseng and notoginseng should be deeply investigated. (2) As shown in a recent report [128], new approaches, such as twodimensional attenuated total reflection Fourier transform infrared spectroscopy based on a gradient heating program, should be developed to discriminate and identify the structural features of Panax polysaccharides. (3) The differences in biological activity might be related to functional groups, branching, and conformational characteristics of the polysaccharides, which could be a future direction for exploring the relationship between structure and activity. (4) The polysaccharides can accelerate the microbial metabolism of ginsenoside Rb1, suggesting the potential roles of the polysaccharides on the gut microbiota [114,121,122]. Studies on the effects of the polysaccharides on ginsenoside absorption in vivo should be strengthened in future. (5) The polysaccharides from American ginseng suspension culture [78,79] and ginseng polysaccharide nanoparticles have been heavily researched in recent studies [82,104,116] and these can provide new opportunities for further development of the polysaccharides with obvious pharmacological properties. (6) Currently, only ginseng polysaccharides have been evaluated to explore their efficacy and safety in healthy volunteers and patients with non-small cell lung cancer [97,129]. Clinical trials using the polysaccharides from three Panax species should be performed to determine their efficacies. (7) At present, we found that the articles on ginseng polysaccharides include experiments with ginseng or American ginseng, which are mostly grown in Jilin Province, China (34.8% and 50%, respectively). Most of the studies on Panax notoginseng originated from Yunnan Province, China (52%). Therefore, it was rare that articles mentioned information describing the cultivation period, storage conditions, or dryness; thus, we were not able to determine the relevant links between different conditions for different ginseng species. We suggest that a greater number of studies should focus on these Panax spp., because it could be meaningful to use Panax herbs of different ages and compare their effects on the isolation, purification, and activity of polysaccharides. Collectively, this review can provide new insights into the similarities and differences of the polysaccharides from three Panax species, which can facilitate and guide further studies to explore the properties of the Araliaceae family used in traditional Chinese medicine.