**6. Medicinal application of native and chemically modified forms of β-(1**→ **6)(1**→**3)-D-glucan**

Approximately 2000 research and review papers covering β-(1→6)(1→3)-D-glucan bioactivity and its medicinal applications have been published since the 1960's, and the majority of the literature has been reviewed (Bohn & BeMiller, 1995; Kogan, 2000; Zekovic et al., 2005; Vetvicka, 2011; http://www.betaglucan.org; and for patented applications see Laroche & Michaud, 2007). Many early assumptions have been confirmed by more rigorous studies, and many others have been disproven. Many of the studies that initially showed promise utilized poorly purified and not sufficiently standardized samples of yeast cell wall glucan (Jaehrig et al., 2007; Vetvicka & Vetvickova, 2007). Highly purified, insoluble, wholeglucan particles (**WGP)** with the size 2-4 µm (Yan et al., 2005), or soluble, yeast poly-(1→6) β-D-glucopyranosyl-(1→3)-β-D-glucopyranose (**PGG**) with molecular weight ~150 kDa (Gawronski et al., 1999) is presently used in animal and human studies. Glucan samples used in earlier studies could contain significant quantities of (also) bioactive impurities like yeast mannoproteins or α-glucans. The existence of a correlation between the β-(1→6)(1→3)- D-glucan structure and its bioactivity is well established having been confirmed by multiple animal and human studies (Novak & Vetvicka, 2009). The WGP-glucan fate after oral administration and PGG-glucan after intravenous administration has been studied in animals (Vetvicka & Vetvickova, 2008). Mammalian cells lack β-glucan, which is present in the cell wall of many infectious microbes. Thus, mammalian macrophages, which are present "on guard" in the cells lining the digestive track, recognize and bind purified βglucan, but not the whole yeast cells (which have mannoproteins on the cell surface), via the dectin-1 receptor, which is a glucan-sensing receptor expressed on dectin a transmembrane protein. The macrophages transport β-glucan molecules to the spleen, bone marrow and lymph nodes where they are disintegrated into smaller, soluble, β-glucan oligosaccharides that are released back into the bloodstream. The circulating monocytes, macrophages, neutrophils, natural killer and dendritic cells (Chan et al., 2009) which possesses β-glucan recognizing receptors, including: toll like receptor 2 (TLR-2, Underhill et al., 1999), dectin-1 (Brown et al., 2007), complement receptor 3 (CR-3) (Ross et al., 1987) and lactosyl ceramide receptor, bind to the β-glucan molecules triggering the non-specific-innate immune response, including phagocytosis and production of proinflammatory factors (Qi et al., 2011). This mechanism leads to the elimination of infectious agents (Chan et al., 2009). Cancer cells are recognized by the host immune system, but antibody response is too weak to destroy them. When humanized, anti-cancer monoclonal antibodies are used in cancer therapy the treatments are not uniformly effective. Combined antitumor monoclonal antibodies (mAb's) and β-glucan therapy yields much better results with fewer adverse side effects (Salvador et al., 2008; Liu et al., 2009). The mechanism of action is only partially understood with neutrophils and macrophages playing a role in the process of killing cancer cells (Liu et al., 2009). Strengthening immunity to various diseases (Hofer & Pospisil, 2011) and combating those that are already active in humans and animals are the main applications of yeast β-glucan. In Eastern culinary tradition, eating mushrooms (which contain large quantities of soluble β-glucan in their fruiting bodies) has long been recognized as healthy, and four pharmaceutical preparations, based on mushroom extracts, have been registered as drugs in Japan (Hyodo et al., 2005). In the Western world several clinical trials (Babineu et al., 1994; Weitberg, 2008; Spruijt et al., 2010) proved the beneficial properties of WGP and PGG glucans, which are still waiting to be registered as drugs (Vetvicka, 2011; Lehtovaara & Gu, 2011). The following therapeutic activities of yeast β-

58 The Complex World of Polysaccharides

(Slovakova et al., 1997).

**β-(1**→ **6)(1**→**3)-D-glucan** 

1999).

2001) yields high purity (98.5% β-glucan, <0.1% mannan, 0.4% α-glucan, 0.3% protein, 0.2% chitin) microparticulate β-glucan with reduced molecular weight (from ~1-3 MDa to ~150 kDa) that is much more easily absorbed by the digestive tract and shows improved activity compared with food-grade products containing only ~65% β-glucan. Even further hydrolysis produces soluble yeast β-glucan (Jamas et al., 1998; Lee et al., 2001) that still retains most of the particular β-glucan bioactivities (Janusz et al., 1986; Wakshull et al.,

Yeast *Saccharomyces cerevisiae*, its cell wall and products of its fractionation are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA, 1997), and they can be legally used as food ingredients but not as food additives. The European Food Safety Authority (EFSA) issued an opinion that yeast β-glucans are a "safe food ingredient" (EFSA, 2011) that can be used as a "food supplement" up to 375 mg/day and in foods for "particular nutritional uses" at dose levels up to 600 mg/day. (The uses of yeast cell wall as an animal

Food-grade yeast β-glucans such as BetaRight® and WGP® (Biothera, Inc.) are used as ingredients in baked foods, beverages, ceral, yogurt, fruit juices, chocolate and as food thickeners in salad dressings, ice cream, mayonnaise, sauces and cheese. The majority of these applications have been patented (Zechner-Krpan et al., 2009; Thammakiti et al., 2004) and a critical review of 300 patented applications is available (Laroche & Michaud, 2007). Yeast β-glucans improve food rheological properties, gelling, water and oil-holding properties, without impacting its taste or odor (Petravic-Tominac et al., 2011). Beta-glucans also add health benefits (Laroche & Michaud, 2007) like antioxidative, bacteriostatic and immunostimmulating activities. Cosmetic products used in skin treatment contain yeast βglucans as moisturizing and moisture-retaining components that also provide a proper moistening feeling. Because of its emulsion-stabilizing effects, pleasant texture and antioxidant activity yeast β-glucans can prevent skin injuries caused by solar radiation and therefore are used in sun-screens, oils and gels (Michiko & Yutaka, 2007). Deodorants containing yeast β-glucans have proved to be useful in oral preparations, mouthwashes and diapers (Michiko et al., 2005). Acid-treated cell walls (AYC) can be used as new binders in pharmaceutical formulations and, when mixed with traditional fillers like hydroxypropylcellulose or polyvinylpyrrolidone, yield harder pills with very short (~2 min) dissolution times (Yusa et al., 2002). Its adhesive and biological properties can be also utilized in producing coating for surgical instruments (Klein, 2003) and in the manufacture of packaging for the food industry (Cope, 1987). Its antibacterial and antiviral properties have found application in the control of plant pests (Kitagawa, 2007) and viral invasions

**6. Medicinal application of native and chemically modified forms of** 

Approximately 2000 research and review papers covering β-(1→6)(1→3)-D-glucan bioactivity and its medicinal applications have been published since the 1960's, and the

feed ingredient were discussed in section 4 of this chapter).

glucan are well-established in treating the following conditions: post-surgical infections, hospital pneumonia, acute renal failure (Koc et al., 2011), pressure ulcers (bed sores), wound healing (surgical and as result of injury, Spruijt et al., 2010) and burns caused by heat, UV or X-ray radiation. As an adjuvant yeast β-glucans alleviate stress and improves the mAb's plus PGG anticancer treatment for breast, colorectal, colon, leukemia, lung, ovarian and skin cancers, chemotherapy and radiotherapy; antireumatic drug therapy (Sener et al., 2006), and antifungal therapies. They stimulate bone marrow healing and bone repair (due to injuries), which increases red blood cell count and neutrophil production. Many of these activities were studied in the 1990s and 2000s and have been reviewed. Studies since 2010 have been referred to herein.

Some of the negative side effects that have been observed in response to prolonged or large doses of β-glucan preparations are the consequences of their mode of action as well as their physical and chemical properties. Their therapeutic activity is based upon immune system mobilization and the production of monocytes, macrophages, neutrophils, natural killer and dendritic cells which fight and destroy pathogens. However, prolonged pro-inflammatory changes in cells can cause the development of autoimmune diseases. The human body is missing enzymes that can hydrolyze β-glucan polysaccharides and for their degradation uses less efficient oxidative pathways. This insufficiency extends the cellular half-life of WGP and PPG glucans and can lead to the formation of granulomas in the liver, causing inflammation that can result in liver cirrhosis. Therefore the use of soluble and relatively low molecular weight PGG glucan is recommended. The well documented β-glucan abilities to lower blood sugar and blood pressure justify further study aimed at producing a glucan material with molecular weight lower than that of PGG glucan to preclude accumulation in granulomas, when used as a long-term food additive. The next stage of development of less toxic, soluble β-glucan preparations should include its size fractionation to produce more standardized material for medicinal applications. The majority of *in vivo* studies on β-glucan medicinal applications were done using rodent models, therefore it is easy to foresee veterinary applications of this food/feed ingredient to improve immune systems, facilitate burn and wound healing, stimulate post-trauma bone repair and help fight cancers.

## **Author details**

Stefan Kwiatkowski *Alltech Inc. Nicholasville KY, USA* 

Stefan Edgar Kwiatkowski *University of Kentucky, USA* 

### **Acknowledgement**

The author expresses deep gratitude to Dr. Pearse T. Lyons, President, and Dr. Karl Dawson, CSO, Alltech Inc. for their continuing support and inspiration.

#### **7. References**

60 The Complex World of Polysaccharides

referred to herein.

**Author details** 

Stefan Kwiatkowski

*Alltech Inc. Nicholasville KY, USA* 

Stefan Edgar Kwiatkowski *University of Kentucky, USA* 

**Acknowledgement** 

glucan are well-established in treating the following conditions: post-surgical infections, hospital pneumonia, acute renal failure (Koc et al., 2011), pressure ulcers (bed sores), wound healing (surgical and as result of injury, Spruijt et al., 2010) and burns caused by heat, UV or X-ray radiation. As an adjuvant yeast β-glucans alleviate stress and improves the mAb's plus PGG anticancer treatment for breast, colorectal, colon, leukemia, lung, ovarian and skin cancers, chemotherapy and radiotherapy; antireumatic drug therapy (Sener et al., 2006), and antifungal therapies. They stimulate bone marrow healing and bone repair (due to injuries), which increases red blood cell count and neutrophil production. Many of these activities were studied in the 1990s and 2000s and have been reviewed. Studies since 2010 have been

Some of the negative side effects that have been observed in response to prolonged or large doses of β-glucan preparations are the consequences of their mode of action as well as their physical and chemical properties. Their therapeutic activity is based upon immune system mobilization and the production of monocytes, macrophages, neutrophils, natural killer and dendritic cells which fight and destroy pathogens. However, prolonged pro-inflammatory changes in cells can cause the development of autoimmune diseases. The human body is missing enzymes that can hydrolyze β-glucan polysaccharides and for their degradation uses less efficient oxidative pathways. This insufficiency extends the cellular half-life of WGP and PPG glucans and can lead to the formation of granulomas in the liver, causing inflammation that can result in liver cirrhosis. Therefore the use of soluble and relatively low molecular weight PGG glucan is recommended. The well documented β-glucan abilities to lower blood sugar and blood pressure justify further study aimed at producing a glucan material with molecular weight lower than that of PGG glucan to preclude accumulation in granulomas, when used as a long-term food additive. The next stage of development of less toxic, soluble β-glucan preparations should include its size fractionation to produce more standardized material for medicinal applications. The majority of *in vivo* studies on β-glucan medicinal applications were done using rodent models, therefore it is easy to foresee veterinary applications of this food/feed ingredient to improve immune systems, facilitate

burn and wound healing, stimulate post-trauma bone repair and help fight cancers.

The author expresses deep gratitude to Dr. Pearse T. Lyons, President, and Dr. Karl

Dawson, CSO, Alltech Inc. for their continuing support and inspiration.


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