Clinical and Physiological Perspectives of β-Glucans: The Past, Present, and Future

β-Glucans are a group of biologically-active fibers or polysaccharides from natural sources with proven medical significance. β-Glucans are known to have antitumor, anti-inflammatory, anti-obesity, anti-allergic, anti-osteoporotic, and immunomodulating activities. β-Glucans are natural bioactive compounds and can be taken orally, as a food supplement, or as part of a daily diet, and are considered safe to use. The medical significance and efficiency of β-glucans are confirmed in vitro, as well as using animal- and human-based clinical studies. However, systematic study on the clinical and physiological significance of β-glucans is scarce. In this review, we not only discuss the clinical and physiological importance of β-glucans, we also compare their biological activities through the existing in vitro and animal-based in vivo studies. This review provides extensive data on the clinical study of β-glucans.


Introduction
β-Glucans are groups of dietary fibers or polysaccharides composed of D-glucose monomers, linked by 1,3; 1,4 or 1,6 β-glycosidic bonds (Figure 1), and are naturally found in the cell wall of bacteria, fungi, algae, and higher crops, such as cereals. Highly-pure β-glucans are enzymatically extracted from the cell wall of yeast, fungi, seaweed, or grain seeds [1][2][3]. The biological and physiochemical properties of β-glucans strongly differ, depending on the source of extraction [4,5]. The degrees of purification, as well as the extraction method, also influence the physiological activity of β-glucans [6]. β-Glucans are generally divided into soluble and insoluble β-glucans, based on physiological properties [7]. In general, insoluble fibers decrease intestinal transit time as well as increase fecal bulk and the excretion of bile acids. However, soluble fibers slow glucose absorption and increase the total transit time by delaying gastric emptying [8]. Gel forming β-glucans are generally considered to be soluble β-glucans, including linear β-glucans (i.e., laminarin), high-molecular branched β-glucans (i.e., schizophyllan, grifolan, and scleroglucan), and chemically-modified particular β-glucans (i.e., phosphorylated or sulfonated β-glucans). However, most of these particular β-glucans are insoluble, such as yeast β-glucans [9]. The members of the first group are usually soluble in alkalies [10]. β-Glucans are a comparatively economical milling byproduct with proven health benefits. They are primarily isolated from the cell walls of yeast, fungi, and cereals, and the contents of β-glucans strongly depend on the environmental conditions [12][13][14][15]. Among cereals, the highest content of βglucans per 100 g dry weight of barley and oat has been reported as 20 and 8 g, respectively. Other cereals also contain β-glucans, but in much lower amounts, as sorghum (6.2 g), rye (2.7 g), maize (1.7 g), triticale (1.2 g), wheat (1.0 g), durum wheat (0.6 g), and rice (0.13 g) [16]. Other sources of β-glucans include yeasts, such as Saccharomyces cerevisiae, mushrooms, such as Maitake and Shiitake, and seaweeds, such as Laminaria sp. [17,18]. The major β-glucans of clinical significance, their structures, and sources are listed in Table 1.
The health benefits of β-glucans have been extensively documented over the past two decades. βglucans are allowed in several countries, including the United States of America, Canada, Finland, Sweden, China, Japan, and Korea, as potent immunological activators [19,20]. β-Glucans are used as a disease-preventing agent, as well as a part of anticancer or anti-inflammatory therapy. Among soluble fibers, β-glucans are the most commonly-consumed immunomodulator with strong anticancer, insulin resistance, anti-hypertension, and anti-obesity effects. β-Glucans are believed to stimulate the immune system, modulating humoral and cellular immunity, and thereby have beneficial effects in fighting infectious diseases, such as bacterial, viral, fungal, and parasitic diseases [21][22][23][24].
β-Glucans have proven characteristics in lowering blood total cholesterol and blood lipid profiles, as well as in maintaining body weight [25][26][27]. Kogan et al. demonstrated the potent inhibitory activity of β-glucans on lipid peroxidation, as well as the synergistic effects of β-glucans as antioxidant, antigenotoxic, and antimutagenic activities [28]. Daou and Zhang demonstrated the immune-stimulating activity of oat β-glucans by activating macrophages and increasing the amounts of immunoglobulin [29]. Murphy et al. reviewed the immune modulating effects of β-glucans and their subsequent benefits on infectious diseases and cancer [30]. Ooi and Liu reviewed the immunomodulating and anticancer effects of β-glucans from mushrooms, as well as the relationship of their structures and antitumor activities [31]. Antitumor mushroom polysaccharides, such as lentinan, schizophyllan, and krestin, have large markets in East Asian countries, including Japan, and Korea [31].
Jesenak et al. reviewed the impact of β-glucans on the treatment of allergic diseases [32], as well as the role of β-glucans in the management and prevention of respiratory tract infections [33]. Khoury et al. reviewed the capability of β-glucans in the prevention and treatment of metabolic syndrome, their underlying mechanisms of action, and their potential in food applications [34]. Chen and Raymond reported that β-glucans can mediate diabetes mellitus by controlling blood glucose levels and hypertension [35]. β-Glucans can reduce the risk factors associated with diabetes mellitus and benefit diabetes therapy. In addition, β-glucans can promote wound healing and alleviate ischemic heart injury. Hou et al. evaluated the effects of β-glucans on invasive fungal diseases [36]; they reported that a β-glucan assay is a useful screening tool with high sensitivity and specificity for discriminating between patients with and without invasive fungal diseases. β-Glucans are a comparatively economical milling byproduct with proven health benefits. They are primarily isolated from the cell walls of yeast, fungi, and cereals, and the contents of β-glucans strongly depend on the environmental conditions [12][13][14][15]. Among cereals, the highest content of β-glucans per 100 g dry weight of barley and oat has been reported as 20 and 8 g, respectively.
The health benefits of β-glucans have been extensively documented over the past two decades. β-glucans are allowed in several countries, including the United States of America, Canada, Finland, Sweden, China, Japan, and Korea, as potent immunological activators [19,20]. β-Glucans are used as a disease-preventing agent, as well as a part of anticancer or anti-inflammatory therapy. Among soluble fibers, β-glucans are the most commonly-consumed immunomodulator with strong anticancer, insulin resistance, anti-hypertension, and anti-obesity effects. β-Glucans are believed to stimulate the immune system, modulating humoral and cellular immunity, and thereby have beneficial effects in fighting infectious diseases, such as bacterial, viral, fungal, and parasitic diseases [21][22][23][24].
β-Glucans have proven characteristics in lowering blood total cholesterol and blood lipid profiles, as well as in maintaining body weight [25][26][27]. Kogan et al. demonstrated the potent inhibitory activity of β-glucans on lipid peroxidation, as well as the synergistic effects of β-glucans as antioxidant, antigenotoxic, and antimutagenic activities [28]. Daou and Zhang demonstrated the immune-stimulating activity of oat β-glucans by activating macrophages and increasing the amounts of immunoglobulin [29]. Murphy et al. reviewed the immune modulating effects of β-glucans and their subsequent benefits on infectious diseases and cancer [30]. Ooi and Liu reviewed the immunomodulating and anticancer effects of β-glucans from mushrooms, as well as the relationship of their structures and antitumor activities [31]. Antitumor mushroom polysaccharides, such as lentinan, schizophyllan, and krestin, have large markets in East Asian countries, including Japan, and Korea [31].
Jesenak et al. reviewed the impact of β-glucans on the treatment of allergic diseases [32], as well as the role of β-glucans in the management and prevention of respiratory tract infections [33]. Khoury et al. reviewed the capability of β-glucans in the prevention and treatment of metabolic syndrome, their underlying mechanisms of action, and their potential in food applications [34]. Chen and Raymond reported that β-glucans can mediate diabetes mellitus by controlling blood glucose levels and hypertension [35]. β-Glucans can reduce the risk factors associated with diabetes mellitus and benefit diabetes therapy. In addition, β-glucans can promote wound healing and alleviate ischemic heart injury. Hou et al. evaluated the effects of β-glucans on invasive fungal diseases [36]; they reported that a β-glucan assay is a useful screening tool with high sensitivity and specificity for discriminating between patients with and without invasive fungal diseases.
No adverse human effects have been reported following the consumption β-glucans, mainly from oat or barley [37]. The medical significance and effectiveness of β-glucans as antimicrobial, anticancer, anti-diabetic, and anti-hyperchloresterolemic polysaccharides have been reviewed [20,29,38]. However, systematic study of the clinical and physiological significance of β-glucans is scarce. Hence, there is a need to critically review the clinical and physiological aspects of β-glucans. In this review, we, not only discuss the clinical and physiological significance of β-glucans from selected studies, but we also compare with existing in vitro and animal-based in vivo studies. Our review provides extensive data on the clinical aspects of β-glucans. Table 1. Common bioactive β-glucans, their structure, and sources.

β-Glucan
Abbreviation Source Structure Reference

Antitumor Effects of β-Glucans
The antitumor effects of β-glucans, extracted from different sources, have been extensively studied in vitro, as well as in animal-based in vivo studies; however, human-based clinical trials have rarely been reported. The antitumor effects of β-glucans are been listed in Tables 2-4 and are described below.

Antitumor Effects of β-Glucans-In Vitro Studies
The immunostimulatory effects of the extracellular and intracellular polysaccharide fractions of Ganoderma lucidum strain MZKI G97 were tested for the induction of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) synthesis in primary cultures of human peripheral blood mononuclear cells, isolated from a buffy coat [113]. The TNF-α-inducing activity of G. lucidum fractions showed potential for use as a supporting therapy in cancer patients receiving chemotherapy and/or radiotherapy. The mechanisms of action of β-glucans from Saccharomyces cerevisiae, as an antigenotoxic, and anticlastogenic agent, as well as its capacity to preserve cell viability, were demonstrated by Oliveira et al. [114]; the study was carried out in the CHO-xrs5 and CHO-k1 cell lines. The tested doses of β-glucan (5-40 g/mL) did not show clastogenic effects; however, a chemoprotective effect was observed in CHO-k1 cell lines, whereas the yeast-derived β-glucan did not show a protective effect after treatment in repair-deficient CHO-xrs5 cell lines, which supports the involvement of bioantimutagenesis. Neither a genotoxic nor an antigenotoxic effect were observed in the CHO-k1 cell lines, however, yeast-derived β-glucan preserved cell viability in both cell lines.
The biological activities of β-glucans differ in terms of their sources and structures. Chan et al. compared the immunological effects of β-glucans from mushroom and barley, and the response of human dendritic cells to the isolated glucans [115]. β-glucans from different sources showed different immune potencies and effects on human immune cells, including dendritic cells. Yeast-derived particulate β-glucan (p-β-glucan) has the ability to activate macrophages and dendritic cells via the dectin-1 pathway [87]. Activated dendritic cells, by p-β-glucan, promoted Th1 and cytotoxic T-lymphocyte priming and differentiation. In an animal-based model and in vitro studies, yeast-derived p-β-glucan revealed significant antitumor immune responses. Yeast-derived p-β-glucan, alone, had no therapeutic effect, but significantly augmented antitumor monoclonal antibody-mediated therapeutic efficiency via the complement activation pathway. Qi et al. reported that the yeast-derived soluble p-β-glucan could be used as an adjuvant in antibody-mediated tumor therapy [87].
The immunostimulatory and antitumor activities of β-glucans (IS-2) purified from mutated S. cerevisiae were investigated by Yoon et al. [88]. IS-2 significantly inhibited lung metastasis in B16-BL6 melanoma and colon 26-M3.1 carcinoma cells, as well as in CDF1 mice. The survival time of tumor-bearing mice was prolonged when pretreated with IS-2, two days before tumor inoculation. IS-2 enhanced splenocyte proliferating activity during in vitro cytotoxicity analysis and produced various cytokines, such s IL-12, IFN-γ, and IL-1β. IS-2 also induced the antitumor activity of the peritoneal macrophages against colon 26-M3.1 cells and supported natural killer cell cytotoxicity against Yac-1 tumor cells. IS-2 β-glucan inhibited tumor metastasis by activating natural killer cells and macrophages.
The association of Th17-inducing activities with notch ligand expression was studied by Higashi et al. [116]. A mixed lymphocyte reaction was induced by co-culturing human monoclonal-dendritic cells (Mo-DCs) with HLA-DR-non shared allogeneic CD4+ naive T cells and curdlan as an adjuvant. The expression of notch ligand in THP-1 cells and Mo-DCs were evaluated using the enzyme-linked immunosorbent assay (ELISA), as well as RT-PCR, for the presence of interleukins (IL-17, IL-5), and IFN-γ. Curdlan induced DC-mediated Th17 differentiation and upregulated Jagged1 mRNA expression in THP-1 and Mo-DCs. Higashi et al. reported that bacterial β-glucans (curdlan) have the ability to induce human DC-mediated Th17 polarization, which shows the tumor suppressing activities of curdlan [116].    The antitumor activities of yeast-, and fungi-derived insoluble β-glucans have been widely recognized, but the insolubility of these compounds creates several problems, especially in topical formulations. Furthermore, the high-molecular weight and high-viscosity of oat-derived β-glucans restrict their application. Choromanska et al. studied the antitumor activities of low-molecular-weight 1,3;1,4-β-glucan derived from oat in normal and cancerous cells [117]. The low-molecular-weight β-glucan from oat significantly decreased cancer cell viability, while, for normal cells, it was non-toxic. This study showed the strong antitumor potential of low-molecular-weight β-glucan from oat, which showed no toxicity to normal cells.

Antitumor Effects of β-Glucans-Animal Studies
The antitumor activities and physicochemical properties of a fungal β-glucan (OL-2), isolated from Omphalia lapidescens, were examined by Ohno et al. [122]. OL-2 displayed sharp signals on a nuclear magnetic resonance spectrum and a significant change in λ max to a longer wavelength, which was observed using Congo red assay. OL-2 showed significant antitumor activity against the ascites form of Sarcoma-180 and MH-134. However, it showed no or low antitumor activity against the solid form of Sarcoma-180. Saito et al. reported that the antitumor activity of OL-2 is related to β-linked branching and it showed the potent antitumor effect of OL-2 against the solid form of Sarcoma-180 in imprinting control region (ICR) mice [123]. The water-soluble β-glucans (H-3-B, S-H-3-B) isolated from a hot-water extract of Cryptoporus volvatus also showed significant antitumor activities against Sarcoma-180 tumor [65]. Leung et al. investigated the antitumor activities of an alkaline-soluble β-glucan, isolated from the cell wall of Flammulina velutipes against Sarcoma-180 mice [118]. β-glucan was non-toxic to a brine shrimp assay. It did not show antitumor activity in vitro, but, when injected into mice, it triggered proliferation of splenic lymphocytes and showed vascular dilation and a hemorrhage response.
The antitumor effects of polysaccharide fractions prepared from hot water extracts of an edible mushroom, Sparasis crispa, against the solid form of Sarcoma-180 in ICR mice were studied by Ohno et al. [124]. In another study, Ohno and colleagues measured the antitumor activity of polysaccharide fractions from hot water extracts of Agaricus blazei against the solid form of Sarcoma-180 tumor in ICR mice [125]. The S. crispa fractions showed enhanced hematopoietic response to cyclophosphamide-induced leukopenic response in mice. Ebina and Fujimiya studied the antitumor effects of extracts from A. blazei Murill in a double-grafted tumor system comprising BALB/c mice and Meth-A tumor cells [126]. Intratumoral administration of ethanol, water-ethanol, and ammonium oxalate soluble fractions showed inhibited tumor growth of Meth-A tumor. Significant macrophage chemotactic factor activity, as well as serum levels of immunosuppressive acidic protein, were observed in the culture media from the tumor tissues.
Antitumor monoclonal antibodies (mAb) are known for their applicability in tumor therapy. mAb binds to the tumor and activates components by coating tumors with inactivated C3b (iC3b). Hong et al. investigated the effects of yeast β-glucan as an adjuvant for antitumor mAb therapy in C3 or CR3 deficient mice [119]. The yeast β-glucan degraded in the bone marrow into smaller soluble β-glucan fragments that were taken up by CR3 of the marginated granulocytes, and these β-glucans inhibited the growth of iC3b-opsonized tumor cells. Driscoll et al. compared the therapeutic efficacy of various sources of β-glucans [127]. The yeast β-glucan, in combination with anti-tumor mAb, resulted in significantly smaller tumor sizes and revealed an enhanced long-term survival compared to mAb alone or β-glucan extracts from other sources, such as mushrooms. Cytokine production was markedly decreased in dendritic cells (DCs), and MyD88-deficient macrophages, but not in CR3-deficient mice.
The yeast β-glucan demonstrated much stronger adjuvant activity compared to mushroom β-glucan extracts in tumor therapy.
The chemoprevention of tumors is linked with the induction of xenobiotic metabolizing enzymes. Since the cytochromes are associated with metabolizing certain pro-carcinogens to their ultimate forms, the prevention of cancer can be achieved by down regulation of cytochromes via food-grade additives. Okamoto et al. reported that lentinan suppressed hepatic CYPIAs expression through the production of tumor necrosis factor-α and caused an increase in the DNA-binding activity of nuclear factor-κB [128]. Zhang et al. estimated the antitumor effects of polysaccharides (L-FV-IB) isolated from water extracts of Lentinus edodes against the solid form of Sarcoma-180 tumor [120]. L-FV-IB also showed significant antitumor activities in both in vitro and in vivo studies. Vetvicka and Yvin investigated the antitumor effects of phycarine isolated from Laminaria digitate [121]. Phycarine significantly stimulated phagocytic activity, as well as potentiated the synthesis and release of TNF-α, IL-6, and IL-1. In addition, it increased NK cell-mediated killing of tumor cells under both in vitro and in vivo studies.

Antitumor Effects of β-Glucans-Clinical Studies
The therapeutic efficacy of fungal 1,3;1,6-β-glucan in patients with cancer, as well as in adjunctive therapy in patients receiving chemotherapy to suppress hematopoiesis, was studied by Weitberg et al. [129]. In this study, patients with advanced malignancies receiving chemotherapy were given β-glucan preparation and were monitored for tolerability and effect on hematopoiesis. The study showed that fungal β-glucan was well tolerated in cancer patients receiving chemotherapy, and it might have a beneficial effect on hematopoiesis in cancer patients. Ostadrahimi et al. studied the effect of yeast β-glucan on the quality of life in women with breast cancer undergoing chemotherapy [130]. The study was conducted on women with breast carcinoma and the patients were given 10-mg capsules of commercial yeast β-glucan, daily, for 21 days. The findings of these studies suggested that β-glucans might be useful as an adjuvant during chemotherapy to improve the quality of life of patients with breast cancer.

Immunomodulating Effects of β-Glucans
β-Glucans possess strong immune-modulatory activities, which have been proved by in vitro, as well as by animal-and human-based clinical trials. The immunomodulating effects of β-glucans are listed in Tables 5-7 and are described below.

Immunomodulating Effects of β-Glucans-In Vitro Studies
The immunomodulatory activities of yeast-derived cerevan on rat thymocytes were evaluated by Sandula et al. [90]. Cerevan showed higher stimulation indices compared to zymosan (a known immunomodulating β-glucan). Wakshull et al. investigated the immune-modulating and antimicrobial activity of yeast-derived β-glucan (PGG-glucan) in different human and murine cell line models [131]. PGG-glucan significantly enhanced the oxidative burst response of blood leukocytes and increased leukocyte microbial activity. The study shows that PGG-glucan is beneficial in enhancing neutrophil antimicrobial effects.
The effect of Ganoderma lucidum-derived polysaccharide-glucan (PS-G) on human monocyte-derived dendritic cells (DCs) was studied by Lin et al. [132]. It enhanced cell-surface expression of human DCs, leukocyte antigen, and interleukin. In addition, PS-G resulted in enhanced T cell-stimulatory capacity and increased secretion of interferons and interleukin-10. This study suggested that PS-G has the potential to regulate immune responses by regulating the activation and maturation of immature dendritic cells. Chaung et al. also reported that a synthetic particulate-β-glucan (p-β-glucan) significantly enhanced cell activity and phagocytosis in porcine alveolar and dendritic cells [133]. Table 5. Immunomodulating effects of β-glucans-in vitro study.

β-Glucan
Cell Line Analysis Results Reference Yeast p-β-glucan (Cerevan) Wistar rat thymocytes HPGPC, Mitogenic, and co-mitogenic activity assay Higher stimulation indices of immunomodulatory activity. Whole blood chemiluminescence assay, Microbicidal assay, Measurement of cytokine secretion from whole blood, 3 H-PGG-Glucan binding assay, Flow cytometry, and Electrophoretic mobility shift assay Induced activation of NF-κB-Like nuclear transcription factor in purified human neutrophils, and enhanced neutrophil anti-microbial function. [131] PS-G DC from PBMC, and CD14 + Determination of cytokine levels, RT-PCR, Flow cytometry analysis, Western blot, Allogeneic MLR, EMS, and IKK activity assay Increased activation and maturation of immature DC, suggesting a potential regulation of immune response. [132] Yeast p-β-glucan (synthetic glucan) Porcine alveolar macrophages and bone hematopoietic cell-derived dendritic cells MTT assay, ELISA, RACE PCR, and Phagocytic activity Enhanced cell activity and phagocytosis, and complex collaborating interaction between dectin-1 and TLRs. [133] Barley [134] Algal β-glucan Murine splenic cells from BALB/c mice NMR, Immunomodulatory activity assay, Immunofluorescence staining assay, and FACSCanto II flow cytometry Increased activation of CD19 + B lymphocytes. [135] Polysaccharide glucan fractions Spleen cells from female C3H/He mice, and Bone marrow cells from C57BL/6 mice Mitogenic activity assay, and CSF-inducing activity assay T-4-N and T-5-N fraction showed mitogenic and CSF-inducing activities. [49] Yeast β-glucan (WGP) Mouse intestinal tumor cell line Colon26 produced in BALB/c mice ELISA, and Tumor-protective effect assay Stimulation of cytokines such as IL-2, IFN-γ, and TNF-α. [136] Bacterial β-glucan  Oat β-glucan 6-weeks old female C57BL/6 mice ELISA, and ELISPOT assay Higher levels of total serum immunoglobulins and antigens against Eimeria vermiformis infection. [142] Yeast β-glucan (WGP) 6-weeks old female BALB/c mice Anthrax-protective prophylactic effect and tumor-protective effect assay Significant effect as a prophylactic treatment to reduce the mortality of anthrax infection. [136] SSG-glucan 6-week old, female inbred, specific pathogen-free NIH/OlaHsd mice Bacterial β-glucan 4-weeks old male BALB/c and ICR mice IFN-γ assay of PBMCs, and Antitumor activity assay Induced IFN-γ and cytokines in spleens and thymus of mice. Enhanced cytotoxic and antitumor activity. [95] β-glucan from different sources 8-week old female BALB/c mice Changes in blood glucose and blood cholesterol levels, and Phagocytosis of HEMA particles Significant stimulation of IL-2 production and phagocytosis of peripheral blood leukocytes. Lowered blood sugar and cholesterol levels. [6] Phycarine 6-10 weeks old female BALB/c and C57B1/6J mice, and male and female pups Apoptosis, Absorption, and Phagocytosis activity assay Significant stimulation of phagocytosis, Strong influence on experimentally induced leucopenia, could be used to cure gastrointestinal diseases.
[103]  Decreased URTI symptoms, and increased mental/physical energy levels. [163] Yeast β-Glucan (Glucan #300) 8-12 years old, male and female children with chronic respiratory problems A randomized, double-blind, placebo-controlled trial. Changes in levels of lysozyme, albumin, and CRP in saliva Increased changes in production of lysozyme and CRP. Improvement in the general condition and stimulated mucosal immunity. [164] Yeast β-glucan (Glucan #300) 8-12 years old children with chronic respiratory problems A randomized, double-blind, placebo-controlled trial. Measurement of levels of IgA, IgG, and IgM A significant increase in production of salivary immunoglobulins, and improvement in the mucosal immunity. [165] Yeast β-glucan (Glucan #300)   Chanput et al. compared the immunological aspects of β-glucans from different sources (oat, barley, and mushroom) on phorbol myristate acetate differentiated THP-1 macrophages [134]. All the tested β-glucans slightly upregulated inflammation-related gene expression, but the expression intensity and patterns differed. It was concluded that β-glucans from different sources show varying levels of immunomodulatory properties. Bobadilla et al. investigated the immunostimulatory properties of seaweed-derived water-soluble β-1,3/1,6-β-glucan on mouse cells [135]. The algae-derived β-glucan showed no adverse effects on the survival of cells and showed an increase in activated CD19 + B lymphocytes.

Immunomodulating Effects of β-Glucans-Animal Studies
The mitogenic and colony-stimulating factor (CSF)-inducing activity of β-glucan polysaccharide fractions, extracted from Dictyophora indusiata FISCH, were reported by Hara et al. [49]. The β-glucan polysaccharide fraction (T-4-N) showed mitogenic and CSF-inducing activity. Sakurai et al. investigated the effect of SSG-glucan isolated from Sclerotinia sclerotiorum on alveolar macrophage activities of CDF1 mice [137]. SSG-glucan, at a concentration of 80 mg/kg, showed increased lysosomal enzyme activity of alveolar macrophages. An increase in phagocytic activity and interleukin-1 production was also observed. The study proved that SSG-glucan can activate murine alveolar macrophage, both quantitatively, as well as qualitatively.
Di Luzio et al. investigated the immune-stimulating effect of yeast-derived β-glucan in A/J and C57BL/6 mice [138]. Significant reductions in the growth of syngeneic anaplastic mammary carcinoma and melanoma B16, and increased survival rates of mice with subcutaneous tumor implants, were observed. Yeast β-glucan decreased renal necrosis in Staphylococcus aureus-challenged mice. Yeast-derived soluble β-glucan showed significant antitumor and anti-staphylococcal activity.
Reynolds et al. investigated host-resistance to infectious-disease response of yeast β-glucan in mice, rats, and healthy cynomolgus male and female monkeys (Macaca fascicularis) [139]. The pre-infection administration of yeast β-glucan significantly enhanced the survival of mice against Venezuelan equine encephalomyelitis (VEE) or Raft valley fever virus and Pseudomonas pseudomallei. However, post-infection administration did not enhance the survival of mice.
Similarly, pre-infection intravenous administration of β-glucan significantly increased resistance to virulent Francisella tularensis. A combined dose of yeast β-glucan and the VEE vaccine showed higher resistance to homologous virus challenge. A similar effect of combined doses was observed in cynomolgus monkeys. This study reported the adjuvant effect of yeast β-glucan in treating infectious diseases.
Hotta et al. investigated the antiviral effects of fungal polysaccharide schizophyllan in mice infected with the lethal Sendai virus [140]. Both oral and intraperitoneal administrations of schizophyllan were effective against the Sendai virus, and significantly inhibited virus infection and spread in lungs. Furthermore, schizophyllan accelerated protective immunity when administered together with low doses of a live Sendai virus vaccine. This study suggested the protective and adjuvant immune response of schizophyllan when administered together with a live Sendai virus vaccine. Kaiser and Kernodle reported the enhanced infection-preventing activity of PGG-glucan when used together with antibiotics (Cefazolin) in guinea pigs inoculated with Staphylococcus bacteria [141]. Guinea pigs receiving PGG-glucan and Cefazolin showed increased infection-prevention compared to those receiving Cefazolin or PGG-glucan alone. This study showed the synergistic effects of PGG-glucan and Cefazolin in preventing staphylococcal wound infection.
The immunostimulatory effects of oat-derived β-glucan formulations on Eimeria vermiformis disease resistance in C57BL/6 mice were studied by Yun et al. [142]. The administration of oat-derived β-glucan resulted in reduced fecal oocyst shedding, higher levels of total serum immunoglobulins and antigen-specific immunoglobulins compared to the non-treated control. Oat-derived β-glucan upregulated immune response with enhanced resistance to Eimeria coccidiosis in mice. Yeast β-glucan (WGP), as a prophylactic treatment, significantly reduced mortality due to anthrax infection, as well as inhibited the growth of cancer cells in mice [136]. Hetland et al. investigated the antimicrobial effects of SSG-glucan isolated from Sclerotinia sclerotiorum in six-weeks old female inbred, specific-pathogen-free NIH/OlaHsd mice [143]. SSG-glucan showed a significant dose-dependent protective effect against Streptococcus pneumoniae type 4 and 6B. The study showed that SSG-glucan can be used in the treatment of pneumococcal infection in mice.
Hasegawa et al. studied the immunomodulatory effects of β-glucan formulations of extracts from Sparassis scispa [144]. SC-glucans showed reduced tumor size of Sarcoma-180 tumor and prolonged survival of mice. In addition, blood IgE levels, and the scratching index, were decreased, while human natural killer cell cytotoxicity was enhanced. The SC-glucan in this study promoted a shift in the Th1/Th2 balance towards Th1-dominant immunity. Methotrexate is widely used in the treatment of malignant tumors and rheumatic disorders. However, its efficiency is often limited by severe side effects and toxic sequelae. Sener et al. investigated the protective effects of yeast β-glucan in methotrexate-induced toxicity [145]. The application of yeast β-glucan eradicated the depletion of tissue glutathione, and inhibited an increase in tissue malondialdehyde, myeloperoxidase activity, and collagen contents and suppressed tissue damage. In addition, yeast β-glucan inhibited leukocyte apoptosis and cell death. This study showed that yeast β-glucan may be beneficial in alleviating leukocyte apoptosis and oxidative tissue injury.
Shim et al. investigated the immunostimulating activities of β-glucans, isolated from Agrobacterium sp., on various cancer cell lines, as well in ICR mice [95]. An induction effect of IFN-γ and cytokines, as well as the adjuvant effect on antibody production, were observed. Vetvicka and Vetvickova investigated the immunological and pharmacological effects of β-glucans from different sources, including yeast, fungi, and cereals, in eight-week old female BALB/c mice [6]. The yeast-derived β-glucan (glucan #300) significantly stimulated the production of IL-2 by mouse splenocytes and phagocytosis of peripheral blood leukocytes. Furthermore, yeast-derived β-glucan significantly lowered blood sugar and cholesterol levels in mice. The remaining tested β-glucans showed marginal immunological activity. This study strongly supported the concept that the immunological effects of β-glucans depend on the source and method of extraction.
Vetvicka and colleagues studied the immunostimulatory effects of algae-derived β-glucans (phycarine) [103]. Phycarine showed significant stimulation of phagocytosis in peripheral blood cells and helped in Lewis lung carcinoma chemotherapy. The strong immunostimulatory effects of phycarine on experimentally-induced leucopenia were observed. A majority of phycarine was detected in the gastrointestinal tract, thus supporting the feasibility of using it in the treatment of gastrointestinal diseases. Later, Vetvicka's group compared the immune-stimulating effects of β-glucans from different sources. Yeast-derived β-glucan (Betamune) significantly stimulated phagocytosis and showed an increase in the synthesis and release of interleukins (IL-1, 2, 4, 6, 8, and 13), as well as tumor necrosis factor-α [146]. Vetvicka and Vancikova compared the stress-related immunosuppressing effects of β-glucans, extracted from different sources, in mice [147]. All the tested β-glucans showed the capability of inhibiting cold-stress-related inhibition; one of the β-glucan fractions (glucan #300) was able to retain phagocytosis at a normal level. In addition, glucan #300 inhibited the increase in stress-related corticosterone and retained IL-6 and IL-12 levels above those of the control.
McCormack et al. investigated the chemoimmunostimulatory properties of lentinan, extracted from Lentula edodes, in male BN/RijHsd rats [148]. A significant increase in weight gains, monocytes, blood cells, circulatory cytotoxic T-cells, and a reduction in anti-inflammatory cytokines IL-4, IL-6, and IL-10 were observed. The combined effects of lentinan in acute myeloid leukemia chemotherapy with cytarabine and idarubicin enhanced the average survival of rats. Mallick et al. explored the immunomodulatory properties of polysaccharide glucan formulations, prepared using the hot-alkaline extracts of Astraeus hygrometricus [149]. A significant increase in the production of interleukin-1 and nitric oxide, as well an increase in phagocytotic potential, were observed. The extracted polysaccharide glucan also showed increased activation of natural killer cells and the proliferation of splenocytes. This study showed no toxic effects on the tested organisms, which suggests that the extracted polysaccharide glucan from A. hygrometricus could be effectively used as an immunomodulatory agent. Sugiyama et al. investigated the suppressive effects of paramylon, extracted from Euglena gracilis Z., on the development of atopic dermatitis-like skin lesions in NC/Nga mice [150]. Paramylon significantly inhibited the development of atopic dermatitis-like skin lesions with no adverse effects on weight loss. This study suggested that paramylon could be used as an alternative therapy to treat atopic dermatitis. Chang et al. investigated the immunopharmacological effects of β-glucan isolated from Paenibacillus polymyxa JB115 on 5-6-week-old Sprague-Dawley rats [151]. The isolated β-glucan showed a significant increase in red blood cell count, white blood cell count, hemoglobin, and thrombocytes, in male rats, whereas, no marked changes were observed in female rats. No adverse effects on general condition, growth, behavior, water, and feed consumption were observed. The β-glucan isolated from P. polymyxa demonstrated no toxic effects in rats, suggesting the strong immunostimulatory effect of P. polymyxa derived β-glucans.
The influence of a purified β-glucan preparation on canine atopy in dogs was studied by Beynen et al. [152]. The effects of β-glucan were studied in a double-blind, placebo-controlled trial. The dogs received 800 ppm of β-glucan, daily, for a period of eight weeks and the clinical signs (scaling, redness, thickness, itching, and stripping of skin) of atopic dermatitis were evaluated. The β-glucan-fed dogs showed significant improvements in the overall index of improvement of atopic dermatitis compared to the control. Hong et al. investigated the exercise-induced stress response of β-glucan on the expression of oncogenes (c-Jun, c-Fos) in male Sprague Dawley rats [153]. An enhanced expression of c-Jun, and c-Fos in the dentate gyrus, dorsal raphe and hypothalamus of rats, after exhaustive treadmill running, was observed. An increase in exhaustion time and suppression of exercise-induced increments of oncogene expression shows that β-glucan administration exerted an alleviating effect on exercise-induced stress in rats. These animal-based studies strongly suggested that β-glucans could serve as a good candidate as an immune-modulating agent.

Immunomodulating Effects of β-Glucans-Clinical Studies
The immune-modulatory effects of PGG-glucan in high-risk patients, undergoing major abdominal or thoracic surgery, were studied by Babineau et al. [154]. An international multicenter, randomized, double-blind, placebo-controlled study showed a dose-dependent response of PGG-glucan against postoperative infections. β-Glucans are suggested to play an important role in the development of respiratory and organic dust-related diseases. The anti-fungal immunostimulatory response of β-1,3 poly-glucose in patients infected with Paracoccidioides brasiliensis was studied by Meira et al. [155]. A significant reduction in erythrocyte sedimentation rate and serum antibody levels were observed; however, higher serum levels of tumor necrosis factor were observed. In addition, patients in post-operation treatment groups showed a positive reaction in the phytohemagglutinin skin test, as well an increase in CD4 + T lymphocytes. This study suggests that patients who received β-glucans showed stronger and more favorable responses to therapy.
Holck et al. studied the potential of yeast-and fungi-derived β-glucans to improve immune response to inflammatory and allergic diseases [156]. Twenty-eight-to fifty-six-year-old healthy volunteers, and patients allergic to house dust mites, were treated with curdlan, laminarin, scleroglucan, and pustulan. All the tested β-glucans showed enhancement of anti-immunoglobulin E (IgE) mediated histamine release. Sarinho et al. studied the immunomodulatory effects of yeast-derived β-glucans against asthma and other allergic diseases in 6-12-year-old children suffering from mild to moderate asthma [157]. A significant increase in serum IL-10 levels, and a reduction in asthma responses was observed in children administrated with β-glucans. These studies showed that β-glucans could be used for understanding, and treatment of, allergic and inflammatory diseases.
Juvonen et al. studied the effects of a modified oat bran beverage, containing β-glucans, on satiety-related gastrointestinal hormone responses in normal healthy male and female volunteers [158]. A greater postprandial increase in satiety, plasma glucose, cholecystokinin, insulin, and glucagons, such as peptide 1 and peptide YY, and a greater decrease in postprandial ghrelin were observed. The viscosity differences in oat bran beverage containing β-glucans strongly influenced short-term gut hormone responses and modulated postprandial satiety-related physiology. Carpenter et al. investigated the post-exercise immunosuppressive response of yeast-derived whole glucan particles (WGP) in male and female volunteers [159]. The supplementation of WGP-glucan significantly enhanced CD14 + and CD14 + /CD16 + , LPS-stimulated production of interferon-γ, and IL-2, IL-4, and IL-5. The study suggested that WGP-glucan has post-exercise immunoprotective abilities.
Lee et al. studied the immunomodulating effects of nutrients enriched with β-glucans in critically ill patients with pulmonary disease and trauma [160]. In a randomized, double-blind, placebo-controlled study, β-glucan-enriched fractions showed an increase in natural killer cell activities and serum albumin. However, a decrease in high sensitivity C-reactive protein was observed. In this study, nutrients enriched with β-glucans showed a beneficial effect on natural killer cell activity, which indicated that β-glucans can serve as an attractive candidate to stimulate protective immunity.
Talbott and Talbott investigated the effects of yeast β-glucan (Wellmure, WGP) supplement on mood state and upper-respiratory tract infection (URTI) symptoms in 18-53-year-old marathon runners [161]. The Talbott group later studied the effects of the same β-glucan (WGP) in 18-65-year-old moderate to highly-stressed men and women [162]. In another study, they reported the effects of β-glucans (WGP) on URTI symptoms and psychological well-being in women with moderate levels of psychological stress [163]. During these studies, a significant reduction in URTI symptoms, increased vigor, and overall health, as well as decreased fatigue, confusion, and tension were observed. These studies showed that a dietary supplement of Wellmure (WGP) could help to reduce upper respiratory tract infections and could maintain immune protection against daily stresses.
Lower and upper respiratory tract infections are common in children. β-Glucans have been proven as natural immunomodulators for the prevention and treatment of various respiratory-related disorders. Vetvicka et al. investigated the effects of yeast-derived β-glucans (glucan #300) in 8-12-year-old, male and female children with chronic respiratory problems [164]. A significant increase in changes in the production of CRP and lysozymes in children and a greater improvement in the general health condition of children was observed. Vetvicka and colleagues reported an increased production of salivary IgM, IgA, and IgG, as well as strongly-stimulated immunity in β-glucan (glucan #300)-fed children with chronic respiratory disorders [165]. Later, in another study, they reported a significant increase in exhaled nitric oxide and physical endurance from orally-administrated β-glucan in children with chronic respiratory disorders [166].
Richter et al. reported the effects of oral administration of yeast β-glucan in 8-12-year-old children with chronic respiratory disorders [167]. A significant increase in the production of CRP, lysozyme, and calprotectin was observed in children supplemented with β-glucan. In another study, Richter et al. reported a strong reduction of salivary cortisol and cotinine levels in yeast β-glucan-supplemented children [168]. A significant reduction in clinical problems of children affected with chronic respiratory disorders, and an increased physical endurance, was observed. Richter and colleagues later reported the effects of oral supplementation of yeast β-glucan on the physical activity and immune status of children with respiratory disorders [169]. A significant difference between male and female children in terms of physical endurance was observed. Additionally, a significant reduction in exhaled nitric oxide levels and a stabilization of salivary IgA levels were observed. These studies showed that short-term administration of yeast β-glucan (glucan #300) could stimulate mucosal immunity and regulate the energetic metabolism in children with chronic respiratory problems.
Jesenak et al. evaluated the effects of Imunoglukan P4H ® syrup on recurrent respiratory infections in children [170]. The administration of Imunoglukan P4H ® syrup in an open clinical trial showed a 50% reduction in the frequency of recurrent respiratory infections in children with no adverse effects. Jesenak and group, again, reported the effectiveness of Imunoglukan P4H ® syrup on the prevention of respiratory infections in children [171]. A double-blind, placebo-controlled, randomized, multicenter study showed the preventive effect of Imunoglukan P4H ® syrup in children with respiratory problems. A significant reduction in the frequency of flu and flu-like diseases, as well as respiratory tract infections, were observed. Imunoglukan P4H ® syrup significantly modulated cellular and humoral immunity. In another study, the Jesenak group reported the significant immunomodulating and anti-allergic effect of pleuran, isolated from Pleurotus ostreatus [172]. A significant reduction in peripheral blood eosinophilia and stabilized levels of total IgE in serum, in children with recurrent respiratory infections, were observed. Later, this same group investigated the immunomodulatory and anti-inflammatory activity of an Imunoglukan P4H ® cream, containing β-glucans (pleuran), in patients suffering from atopic dermatitis [173]. The topical application of Imunoglukan P4H ® showed significant improvements in both subjective and objective symptoms of atopic dermatitis and a significant decline in disease severity; exacerbation was observed. These studies suggest the potential of pleuran-containing creams and syrup (Imunoglukan P4H ® ) as a supportive complementary therapy for atopic dermatitis and respiratory disorders in children.
Grau et al. reported the immunomodulatory activity of β-glucans in children with respiratory tract infections [174]. A significant reduction in the average number of respiratory tract infection episodes and occurrence risk, as well as respiratory disorders, such as the common cold, otitis, laryngitis, pharyngitis, and bronchitis, were observed. Pasnik et al. also investigated the immunomodulatory effects of Imunoglukan P4H ® syrup, containing pleuran, on recurrent respiratory tract infections in children [175]. Similar to the above-mentioned studies, the Imunoglukan P4H ® syrup showed a significant reduction in the total number of respiratory tract infections during treatment. Additionally, a significant reduction in respiratory infections, such as otitis, flu, bronchitis, and laryngitis, was observed. Interesting, the syrup was well tolerated in children and no serious adverse effects were observed. These studies showed that β-glucans (pleuran) from P. ostreatus can be helpful in reducing respiratory tract infections in children with chronic respiratory disorders.

Bone Regeneration/Bone Injury Healing Effects of β-Glucans
The antiosteoporotic, bone healing, and bone regeneration effects of β-glucans have been extensively studied using in vitro, as well as animal-and human-based clinical trials. The antiosteoporotic effects of β-glucans are listed in Tables 8-10 and are described below.

Bone Regeneration/Bone Injury Healing Effects of β-Glucans-In Vitro Studies
The hematopoietic response of PGG-glucan on human bone marrow mononuclear cells was investigated by Turnbull et al. [176]. PGG-glucans significantly enhanced the bone marrow mononuclear myeloid colony formation factor. This study demonstrated that PGG-glucans can effectively enhance human hematopoietic activity on myeloid progenitors, independent of SCF, IL-3 or secondary cytokines.
Previously, we investigated the effects of a mixture of polycan and calcium lactate gluconate (polycalcium) on osteoporosis in murine osteoclast progenitor cells (RAW264.7) [177]. Osteoblast proliferation was stimulated, which prevented RANKL-induced osteoclast differentiation of RAW264.7 cells. In another study, the wound healing properties of β-glucans, isolated from Aureobasidium pullulans, were studied in human fetal dermal fibroblast cell lines [178]. β-Glucans mediated transforming growth factor (TGF-β1) showed increased procollagen production and a significantly-decreased optical density value at 570 nm. In addition, fibroblast proliferation and migration into wound defects were suppressed in a dose-dependent manner. These studies revealed the wound healing properties of β-glucans and suggested that it could be used as an anti-osteoporotic agent to accelerate bone formation and to inhibit bone resorption.
Przekora et al. studied the biomedical capabilities of chitosan/hydroxyapatite (chit/HA) and novel chitosan/bacterial β-glucan/hydroxyapatite (chit/glu/HA) materials as scaffolds for bone regeneration using human fetal osteoblast cell line [179]. The chit/HA scaffold showed better mechanical properties than chit/glu/HA; however, chit/glu/HA showed improved porosity, flexibility, and significantly higher water uptake capability. In addition, chit/glu/HA showed favorable osteoblast survival, proliferation and spreading. Except for poor mechanical properties, chit/glu/HA showed a better biomedical potential. Biocompatibility of scaffolds, cytotoxicity, and osteoblast proliferation rate, Porosity using computed microtomography analysis and mechanical properties by compression test Improved flexibility and porosity, significant higher water uptake capability, favorable osteoblast survival, proliferation, and spreading, but poor mechanical properties. [179] BMMC: Bone marrow mononuclear cells; CD: Cluster of differentiation; ELISA: Enzyme-linked immunosorbent assay; hOBs: Human primary osteoblasts; RANKL: Receptor activator of nuclear factor ligand; RP-HPLC: Reverse phase-High performance liquid chromatography.    DPYR: Deoxypyridinoline; OSC: Osteocalcium; BALP: Bone-specific alkaline phosphatase; Ca: Calcium; P: Phosphorus; CTx: C-telopeptide of collagen cross-links; NTx: N-telopeptide of collagen cross-link.

Bone Regeneration/Bone Injury Healing Effects of β-Glucans-Animal Studies
The inhibitory response of barley β-glucans on chromosomal aberrations in bone marrow and spermatogonial cells of CD1 mice was investigated by Tohamy et al. [180]. A significant reduction in a total number of cells with structural chromosomal aberrations was observed. This study showed that barley β-glucans have a significant role in reducing genotoxicity induced by anti-neoplastic drugs during cancer chemotherapy. Previously, we determined the optimum composition of polycan and calcium lactate-gluconate complex with optimum activity in ovariectomy-induced osteoporotic rats [181]. A significant decrease in ovariectomy-induced osteoporotic changes was observed at a ratio of 10:90. The continuous administration of polycal preserved bone mass and strength. In another study, we investigated the synergistic effects of polycalcium (polycan + calcium lactate gluconate) in an osteoarthritis rat model [182]. Osteoarthritis-related changes in rats were inhibited after continuous administration of polycal for 28 consecutive days, and anti-osteoarthritis effects, including induction of chondrocyte proliferation, were observed.
Park et al. investigated the inhibitory activities of polycal (polycan + calcium lactate gluconate) on ligation induced-experimental periodontitis and related alveolar bone loss [183]. The continuous topical application of polycal significantly inhibited bacterial proliferation, periodontitis, and alveolar bone loss. These studies showed the beneficial synergistic effects of polycan and calcium lactate gluconate formulations against osteoarthritis. Borkowski et al. investigated the synergistic effects of carbonated hydroxyapatite (CHAP) granules and β-glucans as a filler for bone defects in New Zealand white rabbits [184]. Significant integration of implants with bone tissue, as well as no signs of graft rejection, were observed, and the biomaterial showed a stimulating effect on bone formation and mineralization. This study highlighted the potential application of β-glucans in bone tissue regeneration and as a filler in bone defects.
Ku et al. evaluated the effects of polycan isolated from Aureobasidium pullulans in different osteoporosis model rats [76]. A significant increase in bone mineral density of the femur, tibia, and L6, as well as an increase in calcium bioavailability and a decrease in calcium secretion in ovariectomy thyroparathyroidectomy rat models, were observed. This study showed that calcium bioavailability improved the properties of polycan in bones. Jung et al. also investigated the protective effects of polycan isolated from A. pullulans in ovariectomy-induced osteoporosis [77]. Polycan treatment inhibited ovariectomy-induced alterations in bone resorption and turnover in a dose-dependent manner; however, a significant increase in histomorphometrical indices of bone formation and serum expression levels of bALP were observed. Polycan preserved bone mass and strength and increased the rate of bone formation, supporting the idea that polycan can be used as an anti-osteoporosis agent.

Bone Regeneration/Bone Injury Healing Effects of β-Glucans-Clinical Studies
Previously, we reported on the significant antiosteoporotic effects of polycalcium in in vitro and animal-based studies [177,178,181,182]. We also estimated the antiosteoporotic effects of polycalcium in human-based clinical trials [185]. An open-label, single-center trial of polycalcium in 40-60-year-old healthy women demonstrated significant changes in urinary deoxypyridinoline levels, bone-specific alkaline phosphatase, serum osteocalcin, urinary cross-linked N-telopeptide of type-1 collagen, urinary cross-linked C-telopeptide of type-1 collagen, calcium, and phosphorus levels. Additionally, polycalcium, at a dose of 400 mg, showed significant efficiency for improving bone metabolism and was well tolerated.
The efficacy and safety of polycan on bone biochemical markers in healthy perimenopausal women were studied by Kim et al. [186]. A randomized, double-blind, placebo-controlled study showed significant changes in osteocalcium, deoxypyridinoline, and bone-specific alkaline phosphatase levels, and no significant difference in adverse event during the safety assessment test were observed. However, after four weeks of polycan treatment, no statistically-significant results on bone metabolism biomarkers were observed. Further studies in a large population and a longer treatment period are needed to confirm the findings.

Anti-Diabetic/Anti-Obesity Effects of β-Glucans
The anti-diabetic and anti-obesity effects of β-glucans have been largely studied in animal-and human-based clinical trials, but have not been reported using in vitro studies. The anti-diabetic and anti-obesity effects of β-glucans are listed in Tables 11 and 12, and are described below.

Anti-Diabetic/Anti-Obesity Effect of β-Glucans-In Vitro Studies
To the best of our knowledge, there are no available reports on in vitro studies of the anti-diabetic and/or anti-obesity effects of β-glucans.

Anti-Diabetic/Anti-Obesity Effect of β-Glucans-Animal Studies
The effects of lentinan from L. edodes and a polysaccharide glucan from A. blazei on cytochrome P450s (CYPs) expression in female BALB/c mice were investigated by Hashimoto et al. [187]. Lentinan and A. blazei-derived polysaccharide glucans both suppressed constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver. Neyrinck et al. studied the efficiency of fungal-derived chitin-glucan in modulating gut microbiota, as well as glucose and lipid metabolism, in high-fat-diet induced obese mice [188]. Significant cecal enlargement, with prominent changes in gut microbiota, was observed. In addition, chitin-glucan significantly decreased body weight gains, fat mass development, glucose intolerance, fasting hyperglycemia, hepatic triglyceride accumulation, and hypercholesterolemia. The chronic consumption of chitin-glucan positively affected the development of obesity and associated metabolic disorders by restoring the composition and/or activity of gut microbiota.
Lim et al. studied the anti-diabetic effects of polycan isolated from A. pullulans on high-fat-diet (HFD)-induced hyperlipemia and hepatic damage in hamsters [75]. Polycan did not cause a significant change in body weight and food consumption by hamsters. However, a significant reduction in serum levels of triglyceride, alanine aminotransferase, aspartate aminotransferase, total-, and LDL-cholesterol, as well as levels of arteriosclerosis, were observed. This study showed the favorable effects of polycan in protecting from liver damage, as well as in reducing HFD-induced hyperlipemia and associated arteriosclerosis. Sohn et al. examined the combined effects of β-glucan isolated from A. pullulans and Folium mori extract (BG-FM) for treating diabetes, complicated hepatopathy, and nephropathy using streptozotocin-induced diabetic rats [189]. The BG-FM formulations showed a significant reduction in hyperglycemia, hepatopathy, and nephropathy of body weight changes. The synergistic effects of the BG-FM complex at a 1:4 ratio was most effective in suppressing hyperglycemia and diabetic response.

Anti-Diabetic/Anti-Obesity Effect of β-Glucans-Clinical Studies
Tappy et al. investigated the glycemic and insulinemic response of breakfast cereal, containing oat-derived β-glucans, in 49-57-year-old men and women suffering from non-insulin-dependent diabetes mellitus [190]. Oat β-glucan showed a dose-dependent increase in plasma glucose. Bourdon et al. investigated postprandial glycemic response of barley-derived β-glucan-containing meals in healthy men [191]. Plasma insulin, glucose, cholecystokinin, and triacylglycerol concentrations increased significantly in all test meals, but barley-containing meals showed a higher insulin response. Cavallero et al. also investigated the effects of barley-derived β-glucan on glycemic response in 20-27-year-old healthy, non-diabetic men and women [192]. A linear decrease in glycemic response and postprandial blood glucose levels were observed. The barley-containing meals appeared to stimulate reverse cholesterol transport, which may contribute to the cholesterol-lowering ability of barley.
Jenkins et al. investigated the effects of oat-derived β-glucans on glycemic response in 59-63-year-old men and women suffering from type 2 diabetes [193]. A randomized, open-labeled, crossover study with type 2 diabetic patients showed a significant reduction in glycemic index, but maintained palatability of the test food. Tapola et al. also investigated the postprandial glycemic response of oat bran containing β-glucans in 61-73-year-old patients suffering from type 2 diabetes [194]. In a randomized, controlled, repeated measure design, oat bran, high in β-glucans, showed a reduction in glycemic response, and actively decreased postprandial glycemic response in type 2 diabetic patients.
Biorklund et al. investigated the postprandial glycemic responses of beverages enriched with oat-or barley-derived β-glucans [195]. A single blind, controlled study was conducted in parallel groups of 18-70-year-old healthy men and women, with mildly elevated cholesterol concentrations. The oat-derived β-glucans significantly lowered total cholesterol, postprandial glucose, and insulin concentrations; whereas barley-derived β-glucans did not show significant responses. Mäkeläinen et al. investigated the physiological responses and the effects of oat-derived β-glucans on insulin and glycemic indeces in male and female volunteers [196]. Significant reductions in insulin and glycemic indeces were observed with the administration of oat β-glucans. Granfeldt et al. investigated the postprandial insulinemia and glycemic responses of a muesli product containing oat β-glucans [197]. The muesli product, containing 4 g of oat-derived β-glucans, significantly reduced insulin and glucose responses in healthy people.
Thonder and Henry investigated the glycemic response of food products (Chapati) fortified with barley β-glucans [198]. A randomized, single-blind, controlled, crossover study was conducted in 26-50-year-old healthy men and women. The study showed a significant reduction in glucose, and postprandial blood glucose concentrations, as well as glycemic index. Cugnet-Anceau et al. investigated the effects of diet formulations enriched with oat-derived β-glucans in 30-75-year-old type 2 diabetic men and women [199]. A parallel, randomized, double-blind, placebo-controlled study showed a significant reduction in triacylglycerides while no significant differences in fasting glucose, HbAa1c, apoB, and total-, LDL-, and HDL-cholesterol were observed. In this study, a low-dose of β-glucans did not improve the metabolic profile of type 2 diabetic patients. No significant change in body weight and food consumption, serum levels of AST, ALT, triglyceride, LDL-and total-cholesterol levels. Dose-dependent reduction of atherosclerosis with relatively good protective effects on liver damage. [75] Yeast β-glucan + Folium mori extract (BG-FM) STZ-induced diabetic rats Changes in blood glucose levels, body weight, liver, and kidney weight, and Serum BUN, AST, ALT levels Reduced hyperglycemic changes in the F. mori extract. Dose-dependent increase in anti-diabetic and hypoglycemic effect.   [191] Barley β-glucan 20-27 years old, healthy, non-diabetic men and women Sensory properties, proximate composition, and glycemic indices Dose-dependent decrease in glycemic content, and decreased postprandial glycemic index. [192] Oat β-glucan 59-63 years old, type 2 diabetic men and women, with a BMI range of 27-31 kg/m 2 Changes in blood glucose, total-, HDL-, and LDL-cholesterol, and triglyceride levels Reduced blood glucose levels, glycemic indices, and postprandial glycemia. [193] Oat Decreased glycemic, and postprandial glycemic response. [194] Oat β-glucan, Barley β-glucan Oat β-glucan showed reduced total-cholesterol, postprandial glucose, and insulin concentrations as well as improved lipid and glucose metabolism. [195] Oat β-glucan Healthy volunteer men and women Changes in insulin and glycemic response index, and Glucose tolerance test Reduced insulin and glycemic index. [196] Oat β-glucan Healthy volunteer men and women Blood insulin and glucose response Significant reduction in insulin and glucose responses in healthy people. [197] Barley β-glucan 26-50 years old, healthy men and women, with a BMI range of <30 kg/m 2 Changes in blood glucose contents, and GR, and GI response Significantly reduced postprandial blood glucose, and glycemic index. [198] Oat β-glucan 30-75 years old, diabetic men and women, with a BMI range of 20-35 kg/m 2 Changes in lipid profile, apo B, TAG, HbA1c, and fasting glucose concentrations A single daily ingestion of 3.5 g oat β-glucan showed no significant changes in lipid profile and HbA1c in type 2 diabetic subjects whereas, TAG significantly decreased. [199] NIDDM: Non-insulin dependent diabetes mellitus; BMI: Body mass index; HPLC: High performance liquid chromatography; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; GR: Glycemic response; GI: Glycemic index; TAG: Triacylglycerol; HbA1c: Glycosylated hemoglobin.

Cholesterol and Blood Pressure Lowering Effects of β-Glucans
The cholesterol and/or blood pressure lowering effects of β-glucans have been largely studied in human-based clinical trials, but are rarely reported using in vitro or animal-based in vivo studies. The cholesterol and blood pressure lowering effects of β-glucans are listed in Tables 13 and 14, and are described below.

Cholesterol and Blood Pressure Lowering Effects of β-Glucans-In Vitro Studies
To the best of our knowledge, there are no available reports of in vitro studies on the cholesterol and/or blood pressure lowering effect of β-glucans.

Cholesterol and Blood Pressure Lowering Effects of β-Glucans-Animal Studies
Vetvicka and Vetvickova investigated the effects of yeast-derived β-glucans on blood cholesterol levels and macrophage functionality in mice [200]. A significant dose-dependent decrease in plasma cholesterol and triglycerides was observed. Kusmiati and Dhewantata investigated the anti-cholesterolemic effects of yeast-derived β-glucans in Sprague-Dawley rats [201]. Yeast-derived β-glucans significantly reduced the total cholesterol in blood plasma and liver to a normal level. Additionally, a significant decrease in triglyceride and malondialdehyde levels were observed.

Cholesterol and Blood Pressure Lowering Effects of β-Glucans-Human Studies
The hypocholesterolemic response of oat-derived β-glucans in 30-65-year-old men and women with elevated LDL-cholesterol levels was investigated by Davidson et al. [202]. A significant dose-dependent reduction in LDL-cholesterol levels was observed, which supports the idea of independent hypochloesterolemic effect of oat-derived β-glucans. Bourdon investigated the cholesterol-lowering response of barley β-glucans in healthy men [191]. β-glucan-containing meals showed a significant increase in plasma insulin, glucose, cholecystokinin, and triacylglycerol levels. The consumption of meals enriched with barley β-glucans appeared to stimulate reverse cholesterol transport, which showed the cholesterol-lowering ability of barley β-glucans. Nicolosi et al. studied the cholesterol-lowering effects of yeast-derived β-glucans in hypercholesterolemic, obese men [203]. Yeast-derived β-glucan consumption significantly reduced plasma total-, LDL-, and HDL-cholesterol concentrations; however, changes in triacylglycerol concentrations were non-significant.
Lovergrove studied the effects of low doses of oat bran, containing β-glucans in mild-to moderate-hyperlipidemic volunteers [204]. A randomized, double-blind, parallel study was conducted in 30-70-year-old men and women with slightly-elevated LDL-cholesterol levels. A significant decline in HDL-cholesterol concentrations was observed. However, the low-dosage of β-glucans (3 g/day) showed no significant reduction in plasma total cholesterol, LDL-cholesterol, insulin, and glucose concentrations, which shows that oat β-glucans, at a low dose, are not sufficient to lower cholesterol. Jenkins investigated the effects of oat-derived β-glucans in reducing serum lipid risk factors for cardiovascular disease in hyperlipidemic adults [205]. A significant reduction in total-, total:HDL-, LDL:HDL-cholesterol and apolipoprotein B:A-I, as well as a reduction in cardiovascular disease risk, were observed in the high-fiber fed group.
Keogh et al. investigated the effects of barley-derived β-glucan formulations for reducing cardiovascular disease risk factors in 18-65-year-old, mildly-hyperlipidemic men [206]. No significant changes in total-, LDL-, and HDL-cholesterol, fasting glucose, triacylglycerol or postprandial glucose were observed. Kerckhoffs et al. investigated the effects of oat-derived β-glucans on serum lipoproteins in 18-65-year-old, mildly-hypercholesterolemic men and women [207]. A minor decrease in LDL-cholesterol and a total:HDL-cholesterol ratio was observed in a β-glucan-rich drink-fed group. However, triacylglycerol and HDL-concentrations showed non-significant differences.   Behall et al. investigated the cardiovascular disease risk factor reducing activity of barley-derived β-glucans in 28-62-year-old, moderately-hypercholesterolemic men [25]. Significant reductions in totaland LDL-cholesterol, and triacylglycerol concentrations, were observed. However, HDL-cholesterol concentrations increased. Behall and colleagues, later, compared different sources of soluble dietary fiber, including barley-derived β-glucans, and their effect in reducing cardiovascular disease risk factors in 38-53-year-old, mildly-hypercholesterolemic men and women [26]. A significant reduction in total cholesterol, as well as LDL particle size, was observed in diets containing barley-derived β-glucans; HDL-cholesterol and triacylglycerol concentrations showed non-significant differences. These studies suggested that cardiovascular disease risk factors might be reduced by consuming foods rich in higher concentrations of soluble fiber.
He et al. investigated the blood pressure reducing effects of oat dietary fiber (β-glucans) [208]. A randomized, double-blind, placebo-controlled study was conducted on 30-65-year-old volunteers with elevated blood pressure or stage-1 hypertension. Maki et al. studied the effects of foods rich in oat-derived β-glucans on carbohydrate homeostasis, blood pressure, and oxidative stress [209]. A randomized, double-blind, controlled study was conducted on >40-year-old men and women with elevated blood pressure. Significant changes in insulin were observed while blood pressure response and biomarkers of oxidative stress showed non-significant differences. The oat-dietary fiber intake showed a positive effect in lowering systolic as well as diastolic blood pressure, suggesting that oat-derived β-glucans-rich dietary fibers could help to lower blood pressure.
Queenan et al. studied the physiological effects of oat-derived β-glucans on cardiovascular disease in 22-65-year-old hypercholesterolemic men and women [109]. Oat-derived β-glucan concentrate consumption (6 g/day) significantly reduced total-and LDL-cholesterol concentrations. Shimizu et al. investigated the effects of rice diet substitution with barley-derived β-glucans on the reduction of cholesterol and visceral fat areas in 30-60-year-old hypercholesterolemic Japanese men [210]. A randomized, double-blind, placebo-controlled study showed a significant reduction in serum total-and LDL-cholesterol concentrations, as well in visceral fat areas. Liatis et al. investigated the effects of oat-derived β-glucan-enriched bread consumption on glucose homeostasis and lipid profiles in 50-70-year-old patients with type 2 diabetes [27]. A randomized, double-blind study showed a significant reduction in total-and LDL-cholesterol, insulin resistance, and fasting plasma insulin levels. The consumption of oat-derived β-glucans-enriched bread showed improvements in insulin resistance and lipid profiles in patients with type 2 diabetes.

Antigenotoxic/Antimutagenic/Antioxidative Effects of β-Glucans
The antigenotoxic, antimutagenic, and/or antioxidative effects of β-glucans have been largely studied using in vitro and animal-based in vivo studies, but are rarely reported in human-based clinical trials. The antigenotoxic, antimutagenic or antioxidative effects of β-glucans are listed in Tables 15 and 16, and are described below.

Antigenotoxic/Antimutagenic/Antioxidative Effects of β-Glucans-In Vitro Studies
The antigenotoxic effects of yeast-derived β-glucans and fungus-derived β-glucan-chitin complex in V79 hamster lung cells were investigated by Slamenova et al. [211]. The comet assay exhibited a significant reduction in genotoxicity, indicating the protective effect of the tested β-glucans against oxidative damage to DNA caused by scavenging of singlet oxygen or OH radicals. Krizkova et al. investigated the antigenotoxic and antioxidant activity of yeast-derived mannan and mannan conjugates with microbial enzyme penicillin G acylase and human serum albumin in the unicellular flagellate, Euglena gracilis [212]. The tested β-glucan formulations showed significant protective antigenotoxic and antioxidative activities against acridine orange and ofloxacin. Oliveira et al. investigated the antimutagenic activity of barley-derived β-glucans, using a micronucleus assay in HTC and CHO-k1 cell lines [213]. β-Glucans showed a significant chemoprotective activity against methylmethane sulfonate-induced DNA damage in CHO-k1 cells. The study showed that barley-derived β-glucans have bioantimutagenic and desmutagenic activities.
Angeli et al. investigated the genotoxic and antigenotoxic effects of β-glucans isolated from A. brazei Murrill in human peripheral lymphocytes [214]. A significant dose-dependent protective effect against DNA damage induced by H 2 O 2 and trp-P-2 was observed. Later, Angeli and colleagues investigated the clastogenic and anti-clastogenic effects of barley-derived β-glucans in CHO-k1 and HTC cells [215]. The barley-derived β-glucans showed a significant anti-clastogenic effect against methylmethane sulfonate and 2-aminoanthracene induced DNA damage in the tested cell lines. In another study, Angeli's group investigated the chemoprotective effects of β-glucans isolated from A. blazei in human hepatoma cell lines [216]. The mushroom (A. blazei)-derived β-glucan did not show mutagenic or genotoxic effects, but showed a chemoprotective effect against DNA damage caused by B[a]P. This study suggested that the mushroom-derived β-glucan might modulate cell metabolism. Xia et al. investigated the antioxidative activities of chrysolaminarin from Odontella aurita [101]. Chrysolaminarin exhibited a significant hydroxyl radical scavenging activity, but was less effective in reducing the 1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging activity.

Antigenotoxic/Antimutagenic/Antioxidative Effects of β-Glucans-Animal Studies
Yamamoto et al. investigated the antimetastatic and antiangiogenic effects of mushroom-derived β-glucans (SBG), extracted from Sparassis crispa, in ICR and C57BL/6J mice [74]. SBG suppressed B16-F10 cell-induced angiogenesis in female ICR mice; in addition, vascular endothelial growth factor induced neovascularization in female C57BL/6J mice. In addition, it suppressed the growth and number of metastatic tumor foci in the lungs of female C57BL/6J mice. This study showed the suppressive effect of SBG against tumor growth and metastasis in lung, as well as the inhibition of tumor-induced angiogenesis. Erkol et al. investigated the effects of yeast-derived β-glucans on oxidative damage to liver during obstructive jaundice in Wistar albino rats [217]. A significant reduction in levels of alanine and aspartate aminotransferases, lactate dehydrogenase, gamma-glutamyl transpeptidase in serum, and levels of lipid peroxide and malondialdehyde in the liver were observed. However, significantly greater glutathione and superoxide dismutase levels were observed in groups administrated yeast-derived β-glucans. This study showed the phagocytotic and antioxidative activities of yeast-derived β-glucans in reducing liver damage and oxidative stress in obstructive jaundice.
For the last decade, the increasing trend of using irradiation-emitting devices has been observed in medical, industrial, domestic, and military applications. Ceyhan et al. investigated the effects of electromagnetic radiations on the antioxidant status of skin, and the possible protective effects of yeast-derived β-glucans against oxidative injury in Wistar albino rats [218]. β-Glucans significantly reversed the elevation of malondialdehyde levels and superoxide dismutase activity caused by irradiation exposure. In addition, yeast-derived β-glucans slightly enhanced catalase activity and prevented the depletion of glutathione peroxidase activity, caused by electromagnetic radiation. Pillai and Devi investigated the radioprotective effect of mushroom β-glucans, extracted from Ganoderma lucidum, in young swiss albino mice [219]. A significant increase in post-irradiation mouse survival, and a reduction in the number of aberrant cells was observed. These studies demonstrated the antioxidative, and radioprotective activities of yeast-derived β-glucans, through which β-glucans could ameliorate the oxidative skin injury caused by electromagnetic radiation.

Antigenotoxic/Antimutagenic/Antioxidative Effects of β-Glucans-Clinical Studies
To the best of our knowledge, there are no reports available on clinical studies of antigenotoxic/antimutagenic, and/or antioxidative effects of β-glucans.  Suppression of growth and number of metastatic tumor foci in lung. Anti-angiogenic and anti-metastatic effect. [74] β-glucan Wistar albino rats SOD, MPO, MDA, LPO, and GSH activity analyses A significant reduction in AST, ALT, LDH, GGT, MPO, LPO, and MDA levels and greater levels of GSH and SOD. [217] Yeast β-glucan Male Wistar albino young, healthy rats Antioxidant activities (SOD, GSH-Px, CAT, MDA) Significantly reversed elevation of MDA levels and reduction in SOD activities. Slightly enhanced activity of CAT and prevented depletion of GSH-Px activity caused by EMR, and higher antioxidant activities. [218] Fungal β-glucan 6-8 weeks old swiss albino mice

Conclusions and Future Perspectives
The use of fibers and polysaccharides from mushrooms, yeasts, and cereals have been widely documented, along with their observed antitumor, anti-microbial, anti-allergic, as well as immune-modulating effects, in addition to the cardiovascular disease risk-reducing activities, commonly attributed to their bioactive compound, β-glucans. β-Glucans are natural bioactive compounds and can be taken orally as a food supplement or as part of a daily diet and are considered safe to use. When polysaccharide glucans are included in a meal, the rate of carbohydrate and lipid absorption slows down, ultimately modifying the alimentary hormone and lipid responses. The crude extracts or purified β-glucans have been clinically used as part of therapy for cancer and other infectious diseases. In 1997, the US Food and Drug Administration registered oat bran (3 g β-glucan/day dosage) as the first cholesterol-reducing food. Furthermore, β-glucans mobilized murine progenitor cells from bone marrow as well as enhanced murine hematopoietic recovery following bone marrow injury.
β-Glucans possess strong immune-modulatory and anti-osteoporotic activities, which have been proved by in vitro and animal-and human-based clinical trials. The anti-diabetic and/or anti-obesity effects of β-glucans have been largely studied in animal-and human-based clinical trials, but have not been reported in in vitro studies. The cholesterol and/or blood pressure lowering effects of β-glucans have been largely studied in human-based clinical trials, but are rarely reported through in vitro or animal-based in vivo studies. The antitumor, antigenotoxic, antimutagenic and/or antioxidative effects of β-glucans have been widely studied using in vitro and animal-based in vivo studies, but human-based clinical trials are rarely reported. The medical significance and effectiveness of β-glucans, as antimicrobial, anticancer, anti-diabetic and anti-hyperchloresterolemic polysaccharides, have been reviewed [20,29,38]. However, systematic study of the clinical and physiological significance of β-glucans is scarce. In this review, we discussed the medical significance of β-glucans through in vitro, as well as through animal-and human-based clinical studies.