Trends in Food Science & Technology 18 (2007) 4e19

Review Antitumor

Introduction polysaccharides from In Asian countries like China and Japan, mushrooms such as lingzhi (Ganoderma lucidum), shiitake (Lentinus edodes), and yiner (Tremella fuciformis) that have been col- mushrooms: a review lected, cultivated and used for hundreds of years, are being evaluated as edible and medicinal resources. Most tradi- on their isolation tional knowledge about the mushroom as a food and medic- inal agent comes from these species. Traditionally, mushroom has been defined as a fleshy, aerial umbrella- process, structural shaped, fruiting body of macrofungi, and has been consumed by Asian people for over two thousand years because of the pleasant flavor and texture (Miles & Chang, characteristics and 1997; Wasser, 1997). In the literature, it is widely accepted that ‘‘mushroom’’ is: a macro-fungus with a distinctive antitumor activity fruiting body that is large enough to be seen by the naked eye and to be picked up by hand (Chang & Miles, 1992). a a, The macrofungi with distinctive fruiting bodies com- M. Zhang , S.W. Cui *, monly occurred in fungi of the class of Basidiomycetes, P.C.K. Cheungb and Q. Wanga and sometimes in the class of Ascomycetes. Although truffle (genus tuber) and fruiting bodies of morel (both of them aFood research program, Agriculture and Agri-Food belong to the class Ascomycetes) have been reported for Canada, 93 Stone Road West, Guelph, their artificial cultivation, no Ascomycetes mushroom has Ontario, Canada, NIG 5C9 (Tel.: D1 519 780 8028; been successfully commercially cultivated (Hawksworth, fax: D1 519 829 2600; e-mail: [email protected]) 1998). Therefore, all the mushroom species mentioned in bThe Department of Biology, The Chinese University of this article belong to the class of Basidiomycetes. The bio- Hong Kong, Shatin, NT, HK, China activity of Basidiomycetes mushrooms was confirmed by Lucas for the first time in 1957 (Lucas, 1957). Lucas iso- lated a substance from Boletus edulis that has a significant Mushrooms have been valued as edible and medicinal re- inhibitory effect against Sarcoma S-180 tumor cells. Carry- sources, and antitumor substances have been identified in ing out an extensive study in 1966, Gregory isolated the many mushroom species. Polysaccharides are the best known active substances from fruiting bodies of more than 200 and most potent mushroom-derived substances with antitumor Basidiomycetes mushroom species, and from 7000 culture and immunomodulating properties. Although the isolation media produced by applying submerge fermentation to process, structural characterization and antitumor activity of the correspondent mushroom types (Gregory, 1966). The mushroom polysaccharides have been extensively investigated antitumor assays of these active substances were applied in the past three decades, the relationship between the antitu- to three rodent animal models and revealed that the mor activity and the chemical composition as well as the high polysaccharides isolated from 22 mushroom species and order structure of their active components is still not well 50 culture media displayed an inhibitory effect on tumor established. These studies are still in progress in many labora- cells, including Sarcoma S-180, adenocarcinoma 755, and tories, and the role of polysaccharides as antitumor agent is leukemia L-1210. Since then, scientists have subsequently especially under intense debate. The purpose of the present re- isolated antitumor mushroom polysaccharides from view is to summarize the available information, and to reflect G. lucidum (Miyazaki & Nishijima, 1981; Mizuno, 1997), the current status of this research area with a view for future Poria cocos (Kanayma, Togami, Adachi, Fukai, & direction. Okumoto, 1986), L. edodes (Chihara, 1969; Chihara, Hamuro, Maeda, Arai, & Fukuoka, 1970; Hobbs, 2000), * Corresponding author. Coriolas versicolor (Hiroshi & Takeda, 1993), Grifola 0924-2244/$ - see front matter Ó 2006 Published by Elsevier Ltd. doi:10.1016/j.tifs.2006.07.013 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 5 frondosa (Cun et al., 1994; Mizuno, Ohsawa, Hagiwara, & substances from mushroom material with 80% ethanol, fol- Kuboyama, 1986; Mizuno & Zhuang, 1995) and Auricu- lowed by three successive extractions with water (100 C, laria auricular-judae (Ukai et al., 1982, 1983). Table 1 lists 3 h), 2% ammonium oxalate (100 C, 6 h), and 5% sodium the source, type and bioactivities of some distinctive fungal hydroxide (80 C, 6 h). The extraction with hot water polysaccharides with their demonstrated activities. Bioac- yielded water-soluble polysaccharides, and the extraction tive polysaccharides can be isolated from mycelium, the with alkali solution yielded the water-insoluble ones. The fruiting body, and sclerotium, which represent three differ- extraction method can be varied based on the structure ent forms of a macrofungi in the life cycle. The 28 species and water-solubility of polysaccharides, but the basic rule listed in Table 1 have been extensively studied in the past is to break the cell wall from outer layer to the inner layer thirty years. Among them, several polysaccharides and with mild-to-strong extraction conditions (pH and polysaccharide conjugates have been commercialized for temperature). the clinical treatment of patients undergoing anticancer Extracted polysaccharides can be further purified using therapy. They are schizophyllan, lentinan, grifolan, krestin a combination of techniques, such as ethanol precipitation, (polysaccharideepeptide complex) and PSK (polysaccharidee fractional precipitation, acidic precipitation with acetic protein complex). acid, ion-exchange chromatography, gel filtration, and The natural antitumor polysaccharides isolated from affinity chromatography. The ethanol precipitation excludes mushroom include acidic and neutral ones with different the impurities from the polysaccharides. The separation of types of glycosidic linkages, while some are bound to acidic and neutral polysaccharides can be achieved by protein or peptide residues such as polysaccharideeprotein anion-exchange chromatography on a DEAE-cellulose col- or epeptide complexes (Cun et al., 1994; Jong, Birmingham, umn. The neutral polysaccharide in the mixture is first & Pai, 1991; Mizuno & Zhuang, 1995). In addition to the pri- eluted by an appropriate running buffer; the acidic polysac- mary structure, a higher structure of polysaccharides, such as charide is then eluted at a higher salt concentration. Neutral chain conformation, also plays an important role in their polysaccharides can be further separated into a-glucans antitumor activities (Wasser, 2002). Most polysaccharides (adsorbed fraction) and b-glucans (non-adsorbed fraction) have remained classified as nonspecific bioactive substances with the help of gel filtration and affinity chromatography. because their exact mode of action was unknown, and chain Affinity chromatography is a process of bioselective conformation of their active components was undefined. adsorption and subsequent recovery of a compound from This article reviews recent work in this field with em- an immobilized ligand. This process now allows for the phasis on the structure-bioactivity relationship, the extrac- highly specific and efficient purification of some carbohy- tion process, structural features, physical properties, drates. The Sigma-Aldrich Co. has developed several car- antitumor activities, and cellular mechanism. bohydrate-binding matrices which have high specific affinity for many diverse glycoproteins and carbohydrates Extraction and purification procedures (http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/ Mushroom polysaccharides exist as a structural compo- Proteomics_and_Protein_Expr_/Protein_Analysis/Chroma- nent of fungal cell wall. Fungal cell wall is composed of tography/Affinity_Chromatography.html). L2507 and two major types of polysaccharides: one is a rigid fibrillar L5147 are used for the purification of both O-linked glyco- of chitin (or cellulose), the other one is a matrix-like b- proteins, and those glycoproteins containing a-D-galactose. glucan, a-glucan and glycoproteins (Ruiz-Herrera, 1956). Specificity of L8775 is directed to the nonreducing end of Schizophyllan, a water-soluble 1/6 branched b-(1/3)- the terminal a-D-mannosyl residue of glycoconjugates. glucan loosely attached to the outer layer of the cell wall, L4018 has an affinity for terminal a-D-mannosyl and a-D- is secreted to the extracellular matrix (exopolysaccharides glucosyl residues, and is used for the separation of a- and or extracellular polysaccharides). These water-soluble glu- b-glucans in combination with gel filtration chromatogra- cans fill the outer layer resisting the external pressure. phy. The polysaccharide with a broad polydispersity can The inner-layer polysaccharide of schizophyllan is alkali- be fractionated by stepwise precipitation or preparative insoluble and remains insoluble even in 40% KOH at gel permeation chromatography, yielding polysaccharides 100 C. It has been shown that the inner-layer polysaccha- with different molecular weights and low polydispersity. ride of the schizophyllum commune is a glucanechitin It should be noted that the particular fractionation proce- complex because the chitinase-treated polysaccharide was dure scheme in each case depends on the polysaccharide found to be a water-soluble glucan (Sietsman & Wessels, composition of the original material, involving their molec- 1979; Wessels & Sietsman, 1979). ular weight, branching degree and pattern of branches. Selection of an extraction method depends on the cell wall structure. Hot water extraction has been a popular ap- Structural and physical properties proach. Mizuno (Mizuno, 1996) developed reliable proce- Structural features and analytical techniques dures for successful extraction of polysaccharides from Polysaccharides with strong antitumor action differ fruiting bodies or cultured mycelia. In general, the extrac- greatly in their chemical structures. Antitumor activity is tion method involves elimination of low molecular exhibited by a wide range of glycans extending from 6 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19

Table 1. Source, type and bioactivity of some macrofungal polysaccharides

Fungi source References Polysaccharide source Type Main bioactivity

Pleurotus Zhang, Cheung, & Sclerotium, b-D-glucan Hepato-protective, anti-breast cancer tuber-regium Zhang, 2001; Zhang, mycelium Chiu, Cheung, & Ooi, 2006; Zhang, Zhang, & Cheung, 2003 Ganoderma Miyazaki & Nishijima, Fruiting body, Heteroglycan, mannoglucan, Hyperglycemia, immunomodulating, lucidum 1981; Mizuno, 1997 culture broth glycopeptide antitumor, antioxidative, anti-decrepitude Auricularia Ukai et al., 1983; Fruiting body Glucan Hyperglycemia, immunomodulating, auricula Ukai et al., 1982 antitumor, antiflammatory, antiradiative Schizophyllum Yamamoto, 1981 Mycelium Glucan, schizophyllana Antitumor commune Hericum Kawagishi, Ando, & Fruiting body, Heteroglycan, hetero- Hyperglycemia, immunomodulating, erinaceus Mizuno, 1990; Mizuno, mycelium glycanpeptide antitumor 1992; Mizuno, 1998 Lentinus Chihara, 1969; Culture broth, Mannoglucan, polysaccharidee Immunomodulating, antitumor, edodes Chihara et al., 1970; fruiting body protein complex, antiviral Hobbs, 2000 glucan, lentinana Sclerotinia Palleschi, Bocchinfuso, Sclerotium Glucan, scleroglucan (SSG)a Antitumor sclerotiorum Coviello, & Alhaique, 2005 Polystictus Cui & Chisti, 2003 Fruiting body, Heteroglycan, glycopeptide, Immunomodulating, antitumor, versicolar culture broth, krestin (PSK)a antiradiative, hyperglycemia, mycelium antiflammatory Grifola Cun et al., 1994; Fruiting body Proteoglycan, glucan, Immunomodulating, antitumor, frondosa Zhuang et al., 1994; galatomannan, heteroglycan, antiviral, hepatoprotective Zhuang, Mizuno, Ito, grifolana Shimura, & Sumiya, 1993 Inonotus Kim et al., 2005 Fruiting body, Glucan Antitumor, immunomodulating obliquus mycelium Agaricus Mizuno, 1992; Fruiting body, Glucan, heteroglycan, glucan Antitumor blazei Mizuno, 1998 mycelium protein, Glucomannaneprotein complex Flammulina Zeng, 1990 Fruiting body, Glucaneprotein complex, Antitumor, antiflammatory, antiviral, velutipes mycelium glycoprotein immunomodulating Ganoderma Nakashima, Umeda, & Fruiting body Glucan Antitumor applanatum Kanada, 1979 Polypours Yang et al., 2004 Mycelium Glucan Antitumor, immunomodulating umbellatus Clitopilus Liang, Miao, & Fruiting body Glucan Antitumor caespitosus Zhang, 1996 Pleurotus Wang, Hu, Fruiting body Galactomannan Antitumor citrinopileatus Liang, & Yeh, 2005 Trametes Zhang, 1995 Mycelium Proteoglycan Immunomodulating, robiniophila hepatoprotective, anticancer Tremella Huang, 1982 Fruiting body, Heteroglycan Hyperlipidemia, hyperglycemia, fuciformis mycelium, culture immunomodulating, antitumor, broth anti-decrepitude, anti-thrombus Tremella Liu, Xie, Su, Han, & Fruiting body, Heteroglycan Immunomodulating, hyperglycemia aurantialba Liu, 2003 mycelium Pleurotus Solomko, 1992 Fruiting body Glycoprotein Antitumor, hyperglycemia, ostreatus antioxidant Morchella Duncan et al., 2002 Fruiting body Heteroglycan Hyperglycemia, antitumor esculenta Omphalia Saito, Nishijima, Ohno, Fruiting body Glucan Antiflammatory, immunomodulating lapidescens Yadomae, & Miyazaki, 1992 Phellinus Kim, Choi, Fruiting body Glucan Antitumor linteus Lee, & Park, 2004 Armillariella Kiho, Shiose, Mycelium Heteroglycan Antitumor tabescens Nagai, & Ukai, 1992 Dictyophora Hara et al., 1991 Fruiting body Heteroglycan, Antitumor, hyperlipidemia indusiata mannan, glucan M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 7

Table 1.(continued) Fungi source References Polysaccharide source Type Main bioactivity Peziza Mimura, Ohno, Fruiting body Proteoglycan, glucan Immunomodulating, antitumor vericulosa Suzuki, & Yadomae, 1985 Tricholoma Wang, Ooi, Ng, Fruiting body Glucan Antitumor mongolium Chiu, & Chang, 1996 Cordyceps sp Hsu, Shiao, Hsieh, & Fruiting body, Glucan, heteroglycan Antitumor, immunomodulating, Chang, 2002 mycelium, culture antitumor, heperglycemia broth a Commercially developed polysaccharide products. homopolymers to highly complex heteropolymers (Ooi & Several methods for this purpose are listed in Table 3.In Liu, 1999). A wide range of antitumor or immuno-stimulat- the early 1990s, high-performance anion-exchange chroma- ing polysaccharides of different chemical structures from tography with pulsed amperometric detection (HPAEC- higher Basidiomycetes mushrooms have been investigated, PAD) was developed to supplement traditional methods and the main types are listed in Table 2. Monosaccharide because this process does not require monosaccharide de- types of these antitumor polysaccharides contain glucose, rivatization. In the mid 1990s, fluorescent derivatives of galactose, mannose, xylose, arabinose, fucose, ribose monosaccharides were produced using reductive amination. and glucuronic acid. In some mushroom species, poly- This technique became popular in conjunction with the ap- saccharides are bound with proteins or peptides as a plication of reverse-phase HPLC with on-line fluorescent polysaccharideeprotein or epeptide complex which detection, or gel eletrophoresis or high-performance capil- showed higher potent antitumor activity (Cui & Chisti, lary electrophoresis. Tagging polysaccharides with a fluo- 2003). In addition to the well-known antitumor 1/3- rescent compound has resulted in an increase in detection b-glucans, a wide range of biologically active glucans sensitivity that extends into the fentomole range. with other structures are described in Table 2. These glu- The positions of glycosidic linkages can be analyzed by cans are linear or branched molecules having a backbone enzyme digestion, methylation analysis and NMR spectros- composed of a-orb-linked glucose units, and some of copy. The exoglycosidic digestion method is limited to them contain side chains that are attached at different posi- a few enzymes of high specificity. In methylation analysis, tions. Heteroglucan side chains contain glucuronic acid, polysaccharides are first converted to partially methylated xylose, galactose, mannose, arabinose, or ribose, and may acetyl alditol and then analyzed. Resulting chromato- have different combinations. Another large group of bioac- graphic peaks are identified by a combination of their reten- tive polysaccharides is called heteroglycans which are clas- tion times and their electron impactemass spectrometry sified as galactans, fucans, xylans, and mannans by (EI-MS) fragmentation patterns. This type of analysis individual sugar components in the backbone. Heterogly- indicates which residues are terminal and how each mono- can side chains may contain arabinose, mannose, fucose, saccharide is substituted, as well as the occurrence of galactose, xylose, glucuronic acid, and a glucose moiety branching points. Methylation analysis also allows the as a main component, or in various combinations. determination of the ring size for each monosaccharide. The structure of naturally occurring glycans is so However, methylation analysis does not provide informa- diversified that it is difficult to define a universal protocol tion on the sequence of constituent residues and the type for their analysis. The building blocks (derived from sugar of anomeric configurations (i.e. a or b). residues) of complex polysaccharides exhibit very similar The anomerity (a or b) of each sugar residue can be structures, but their differentiation (diversified linkage determined by NMR spectroscopy. By now there are quite style) is more inclusive than that of amino acids. The pri- a few review papers and books concerning structural anal- mary structure of a polysaccharide is defined by monosac- ysis of complex carbohydrates (Cui, 2005; Vliegelhart & charide composition, configuration of glycosidic linkages, Dorland, 1983). If a sufficient quantity of polysaccharides position of glycosidic linkages, sequence of monosaccha- (5e20 mg) is available, the anomericity of a particular rides, as well as the nature, number and location of ap- monosaccharide residue is more reliably determined by pended non-carbohydrate groups. The analytical methods 1H NMR spectroscopy. The anomeric resonances appear used to determine primary structures of polysaccharides in a clear region in the spectrum and show characteristic are described in Table 3 (Varki et al., 1999). Monosaccha- doublets with a splitting that is significantly larger for ride analysis provides precise molar ratios of individual b anomers than for a anomers. NMR spectroscopy is the sugars, and may suggest the presence of specific oligosac- only method that has the potential for full structural charac- charide classes such as N- or O-glycans. Monosaccharide terization of a polysaccharide, with little or no assistance composition analysis involves cleavage of all glycosidic from other methods. Complete structural elucidation re- linkages, fractionation of the resulting monosaccharides, quires the full assignment of both the 1H and 13C NMR and detection and quantification of each monosaccharide. spectra of the oligosaccharide. Full assignment of 1H and 8 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19

Table 2. Chemical structures of antitumor mushroom polysaccharides

Polysaccharide Linkages and types Mushroom resources Main chain Branch Mw 5 Homoglucans (1/3)-b-D-glucan Lentinan from Lentinus (1/3)-b-D-glucan (1/6)-b-5Â 10 with 1e6 branches edodes (Mizuno, 1997) Schizophyllan from (1/3)-b-D-glucan (1/6)-b- Schizophyllum commune (Yamamoto, 1981) 5 Grifola from Grifola frondosa (1/3)-b-D-glucan (1/6)-b-5Â 10 (Zhuang et al., 1994) Scleroglucan from (1/3)-b-D-glucan (1/6)-b- Sclerotium sclerotia (Palleschi et al., 2005) 5 An alkali-soluble glucan from (1/3)-b-D-glucan (1/6)-b-2Â 10 Pleurotus tuber-regium (Zhang et al., 2003) Linear (1/3)-b- Auricularia auricula (1/3)-b-D-glucan dd D-glucan (Ukai et al., 1983) Lyophyllum decastes (Ukawa, Ito, & Hisamatsu, 2000) Linear (1/6)- Armillariella tabescents (1/6)-b-D-glucan dd b-glucan (Kiho et al., 1992) 5 (1/3)-b-D- Pachyman from Poria (1/3)-b-D-glucan (1/2)-b-or 1 Â 10 glucan with 1e2 cocos (Kanayma et al., 1986) (1/6)-b- or 1e6 branches (1/3)-a-glucan Armillariella tabescens (1/3)-a-glucan dd (Ukawa et al., 2000) Linear a-(1-3)-glucan from Amanita muscaria (Kiho et al., 1992) (1/4)-a-; (1/6)-a-glucan (1/4)-a- d (1/6)-a-glucan (1/4)-a-; (1/6)- Agricus blazei (Mizuno, (1/6)-b-D-glucan (1/4)-a- d b-glucan 1992; Mizuno, 1998) (1/6)-b-; (1/3)-a-glucan (1/6)-b- d (1/3)-a-glucan Heteroglucans (1/3)-b- Ganoderma lucidum (1/3)-b- Glucuronic acid 5.3 Â 104 glucuronoglucan (Mizuno, 1998) glucuronoglucan Xyloglucan Grifola frondosa (Mizuno & Glucan Xylose d Zhuang, 1995; Zhuang et al., 1994) Polyporus confluens d (Sugiyama et al., 1992) Pleurotus pulmonarius d (Wasser, 2002) Arabinoglucan Ganoderma tsugae Glucan Arabinose d (Wang et al., 1993) Riboglucan Agricus blazei (Mizuno, Glucan Ribose d 1992; Mizuno, 1998) Flammulina velutipes (Zeng, 1990) Galactomannoglucan Hohenbuehelia serotina Glucan Galactose, and d (Ma, Mizuno, & Ito, 1991) mannose Leucopaxillus giganteus (Wasser, 2002) Galactoxyloglucan Hericium erinaceus Glucan Galactose and d (Kawagishi, Kanao et al., xylose 1990; Mizuno, 1992; Mizuno, 1998) Mannoxyloglucan Grifolan frondosa (Cun et al., Glucan Mannose and xylose d 1994; Mizuno & Zhuang, 1995; Zhuang et al., 1994) Xylogalactoglucan Inonotus obliquus Glucan Xylose, galactose d (Kim et al., 2005) M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 9

Table 2.(continued)

Polysaccharide Linkages and types Mushroom resources Main chain Branch Mw Heterogalactan Glucogalactan Ganoderma teugae Galactan Glucose d (Peng, Zhang, Zeng, & Kennedy, 2005) Arabinogalactan Pleurotus citrinopileatus Galactan Arabinose d (Wang et al., 2005) Fucogalactan Sarcodon aspratus (Mizuno Galactan Fucose d et al., 2000) Mannogalactan Pleurotus pulmonarius Galactan Mannose d (Wasser, 2002) Fucomannogalactan Grifola frondosa Galactose Fucose mannose d (Cun et al., 1994; Mizuno & Zhuang, 1995; Zhuang et al., 1994) Other Xylan Hericium erinaceus Xylan dd heteroglycans (Kawagishi, Kanao et al., 1990; Mizuno, 1992; Mizuno, 1998) Glucoxylan Xylan Glucose d Mannogalactofucan Grifola frondosa (Cun et al., Fucan Mannose and d 1994; Mizuno & Zhuang, galactose 1995; Zhuang et al., 1994) Mannoglucoxylan Hericium erinaceus Xylose Mannose, glucose d (Kawagishi, Kanao et al., 1990; Mizuno, 1992; Mizuno, 1998) (1/3)-a-mannan Dictyophora indusiata (1/3)-a-mannan dd (Hara et al., 1991) Glucomannan Mannan Glucose d (1/2)-b-; (1/3)- Agricus blazei (1/3)-b-linked (1/2)-b-glucan d b-glucomannan (Kawagishi, Kanao mannose et al., 1990; Mizuno, 1992; Mizuno, 1998) Galactoglucomannan Mannan Galactose and d glucose Polysaccharidee Polysaccharidee Coriolus versicolor a-1,4 and b-1,3 Peptide mainly 1 Â 105 protein/peptide peptide complex (Cui & Chisti, 2003) glucoside linkage; consists of aspartic complexes containing arabinose, and glutamic acids rhmanose, but no fucose Polysaccharidee a-1,4 and b-1,3 Peptide mainly 1 Â 105 protein complex glucoside linkage; consists of aspartic containing fucose, and glutamic acids but no arabinose and rhmanose

13C NMR spectra can be accomplished by using a combina- The structure characterization of glycans in a typical tion of two-dimensional (2D) NMR techniques consisting glycoprotein has been investigated in recent years (Verbert, of correlated spectroscopy (COSY) and total correlated 1995). A polysaccharideeprotein complex presents one or spectroscopy (TOCSY) for 1H, and then heteronuclear more diffuse bands when it is separated by sodium dodecyl single-quantum coherence (HSQC) for 13C. 2D hetero- sulfateepolyacrylamide gel electrophoresis and then elec- nuclear multi-bond correlation (HMBC) is the key experiment troblotted onto polyvinylidene difluoride (PVDF) mem- for sequence determination. However, HMBC is not a very branes; this results from heterogeneity in the carbohydrate sensitive technique so that a relatively large amount of sam- moiety. Some polysaccharideeproteins or epeptides of ple is needed for the 2D 1H13C NMR experiments. In the very high molecular weight do not enter ordinary gels, or cases where there is not enough sample for this experiment, they migrate as smears. In such a situation, agarose gels polysaccharide sequencing relies exclusively on 2D or combination of polyacrylamideeagarose gels can be 1H NMR spectroscopy, using through-space effects (nuclear used. Visualized by protein staining reagents (recently overhauser effect, NOEs) as a source of evidence for linking many analytical options to verify a positive carbohydrate positions and sequence. staining reaction are available depending on the type and 10 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19

Table 3. Common methods for primary structure analysis (modified from reference (Varki et al., 1999, chap. 38))

Primary structure features Methods Derivatization Information obtained

Monosaccharide composition GLCeFID Complete derivatization Type, quantity and D (nature and number of Developed in to produce volatile compounds; or L configuration of monosaccharide, including early 1960s complete derivatization with monosaccharide absolute configuration of chiral aglycone D or L, and ring size of GLCeMS Complete derivatization to Type and quantity of pyranose or furanose) Developed in 1970s produce volatile compounds monosaccharides HPLC Pre- and post-column Type and quantity of Developed in 1980s derivatization: fluoresecent monosaccharides tagging of reducing end HPAEC-PAD Not needed Type and quantity of Developed in early 1990s monosaccharides Configuration (a, b) IR Not needed Configuration of a or b of anomeric carbon NMR Not needed Configuration of a or b Positions of glycosidic Exoglycosidase digestion Fluorescent tag may Presence of residues in linkages with specific enzymes (limited to need to be introduced a or b linkages to specific a few enzymes of high specificity) at reducing end positions of the underlying with proper on-line detectors saccharides such as HPLC, HPCE MS Complete derivatization Linkage type and position might to produce volatile compounds be inferred from specific fragmentations across sugar rings NMR Not needed Anomericity of each monosaccharide residue obtained from the chemical shift and coupling constant of H-1 linkage positions deduced from 2-D HMBC experiment Sequence NMR Not necessary Sequence may be inferred by comparison with standards; sequence deduced from 2-D HMBC experiment quantity of sample), an appropriate piece of PVDF mem- monosaccharides with respect to each other, and flexibility brane is subjected to sequential hydrolysis with mild acid of the spatial structure which is defined by dihedral angles, (0.2M trifluoroacetic acid, TFA), 2M TFA and finally 6M torsion angles around glycosidic bonds, inter-atomic dis- HCl at 100 C for 24 h to release acidic sugars, neutral tances and dynamic parameters, respectively. For most sugars and amino acids, respectively. Complete removal mushroom polysaccharides, the secondary- and higher- of N- and O-glycans can also be achieved by using order structures in solution are not readily defined, due to enzymes and chemical treatments such as hydrazinolysis, their inherent flexibility. Characterization of polysaccharide b-elimination, or hydrogen fluoride treatment (Ausubel dynamics by both experimental and theoretical systems et al., 1996; Jackson & Gallagher, 1997; Townsend & remains an area of active research. With the development Hotchkiss, 1997). The high sensitivity of current instrumen- of high resolution instrumental processes, such as various tation facilitates on-line composition analysis of sequential light scattering techniques (i.e. light scattering, x-ray and hydrolysates released by treating polysaccharideeprotein neutron scattering), x-ray diffraction analysis, small-angle complexes what are blotted onto polyvinylidene difluoride neutron scattering (SANS), atomic force microscopy (PVDF) membranes with acids (Zdebska & Koscielak, (AFM) and high resolution NMR, it is possible to study the 1999). This method has the benefit of assuring that the conformation and 3D structure of a polysaccharide at the mo- hydrolytes are easily recovered, and leaving any peptide lecular level. By using molecular mechanics and computer- or protein bound on the membrane. assisted energy minimization methods, it is possible to The extent to which the primary structure of a polysac- simulate and visualize the 3D structure of polysaccharides. charide has been assessed depends on the available tech- niques. The contribution from a variety of disciplines will Conformation and chain rigidity be required to advance such technologies. To put together Based on the molecular parameters obtained from laser all the pieces of information for the primary structure will light scatterings (LLS) and SANS, Rees and his coworkers be an interesting challenge for the carbohydrate chemists. (Rees, 1969; Rees & Scott, 1969, 1971) simulated linear and branched pyranosic glucans and delineated their most Conformational properties and analytical methods probable conformations. Fig. 1 summarizes the conforma- The conformational aspects of a polysaccharide include tions of glucan with various glycosidic linkages. 1/4-b- conformation of each monosaccharide, orientation of glucan and 1/3-a-glucan have the similar extended and M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 11

-(1-2)-D-glucan characteristic ratio (CN), persistence length (q) and config- Rigid and crumpled conformation uration entropy (DSc) to describe chain rigidity. The molec- ular parameters of some glucans are listed in Table 4 for -(1-3)-D-glucan Extended and ribbon-like conformation quantitative comparison of their extension and flexibility. According to the q value (a larger q stands for higher chain

-(1-4)-D-glucan rigidity), the order of chain rigidity of glucans is as follows: Flexible and helical conformation (1/4)-b- > (1/3)-a- > (1/4)-a- > (1/3)-b- > (1/6)- a- > (1/6)-b-. Such a conclusion is consistent with that of -(1-2)-D-glucan Rees (Rees, 1969). Rigid and crumpled conformation Helical conformation -(1-3)-D-glucan The helical conformation is an important structure that Flexible and helical conformation was found in antitumor mushroom polysaccharides. Six mushroom polysaccharides that have been used for treatment of certain cancers adopt triple helical conforma- -(1-4)-D-glucan Extended and ribbon-like conformation tion in solution. They are schizophyllan (Kashiwagi, Nori- suye, & Fujita, 1981; Sato, Norisuye, & Fujita, 1983; -(1-6)-D-glucan Flexible conformation Yanaki, Norisuye, & Fujita, 1980), scleroglucan (Sato et al., 1983), lentinan (Saito, Yoshioka, Yakoi, & Yamada, Fig. 1. Regular a- and b-glucan conformations, deduced from computer 1990), curdlan (Saito et al., 1990), cinerean (Gawronski, outputs. Modified from Rees and coworkers (Rees & Scott, 1971). Aguirre, Conrad, Springer, & Stahmann, 1996; Gawronski, Conrad, Springer, & Stahmann, 1996; Gawronski et al., 1997) and (1/3)-b-D-xylan (Itou, Teramoto, Matsuo, & Suga, 1986). A linear water-soluble (1/3)-b-D-glucan ribbon-like conformation; 1/3 b-glucan and 1/4 a- isolated from Auricularia auricula that existed as a single glucan have the flexible and helical conformation; 1/2 helical chain in solution also showed very strong antitumor b-glucan and 1/2 a-glucan have rigid and crumpled con- activity (Zhang & Yang, 1995). Since helical conformation formation. 1/6 b- and 1/6 a-glucans are quite different has been considered a very important factor by playing from glucans in the other linkages because they have many a significant role in the biological recognition within cells, possible conformations. This is because residues connected a basic understanding of the conformation and conforma- through the 1/6 linkage are separated by three bonds tion transition is essential for medicinal applications. It rather than two, so that its freedom of rotation is much has been shown that schizophyllan, which has the primary higher than that of other linkages. Therefore, the outstand- structure of (1/3)-b-D-glucan with one 1/6 branches for ing property of 1/6 linkage is its flexibility. Most of Rees’ every three b-(1/3)-glucopyranosides, adopts a triple he- (Rees, 1969; Rees & Scott, 1969, 1971) predictions about lical conformation in water with the molecular parameters the conformation of glucan have been attested by experi- of 2170 nmÀ1 for molecular weight per contour length / / mental analysis, except for those of (1 3)-a,(1 2)-a, (ML), 150 nm for persistence length (q) and 0.30 nm for (1/2)-b,(1/6)-b- and (1/6)-a-glucan. This is mainly distance per turn of helix (h)(Kashiwagi et al., 1981). It due to the fact that the first three have poor packing ability was found that schizophyllan not only adopts a triple in organic cells and their native forms seldom occur in na- helical conformation in water, but also has a random coil ture; (1/6)-b- and (1/6)-a-glucan show typical flexible conformation in dimethylsulfoxide (DMSO) (Sato et al., chain characteristics which prevent further analysis. Burton 1983). When water was added to DMSO, the single chain & Brant (Burton & Brant, 1983) introduced an energy func- of schizophyllan associated because of the formation of tion for conformational analysis of glucans with various hydrogen bonds. McIntire and Brant (McIntire & Brant, linkages. They calculated molecular parameters including 1999), and Young and Dong (Young & Dong, 2000) have

Table 4. Quantitative comparison of extension and flexibility of several glucans

Polysaccharides CN q DSc Description of conformation

a-(1-3)-D-glucan 32 70 0.15 Extended conformation, no pseudohelicity b-(1-3)-D-glucan 3.1 15 1.1 Pseudohelical trajectory a-(1-4)-D-glucan 5.0 28 À1.1 Pseudohelical trajectory, moderately compact chain b-(1-4)-D-glucan 100 290 0.30 Extended conformation a-(1-6)-D-glucan 1.5 7 4.0 Great randomness and tortuosity, frequent and random change in direction b-(1-6)-D-glucan 1.7 6.9 3.4 Great randomness and tortuosity, frequent and random change in direction

Characteristic ratio (CN), persistance length (q) and configuration entropy (DSc, cal/Kmol residue). 12 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 independently shown that the triple helical structure could treatment of certain cancers such as human breast cancer be retrieved when the renaturation process was carried (MCF-7), human promyelocytic leukemia (HL-60), and out in considerably dilute solutions. Such dissociation of human liver cancer (HpG2). Elucidation of their conforma- triple helices and their reorganization has inspired re- tion and three dimensional structures will be crucial for searchers to study the interaction between a triple helical understanding their biological function. polysaccharide and DNA/RNA (Anada et al., 2004; Kou- moto, Mizu, Sakurai, Kunitake, & Shinkai, 2004; Yang Methods for conformation analysis et al., 2005; Mizu, Koumoto, Kimura, Sakurai, & Shinkai, High resolution NMR is the method of choice to study 2004). Sakurai dissociated schizophyllan into single chains the conformation and 3D structures of saccharides, with by adding DMSO into the aqueous solution to break down parameters represented by chemical shifts, coupling con- the inter-molecular hydrogen bonds (Bae et al., 2004). The stants, nuclear overhauser effects (NOEs), and also relaxa- single chains were then added to an aqueous solution con- tion time. Coupling constants can be used to evaluate the taining polynucleotides (poly(C)) for renaturation. It was magnitude of the torsion angles, and NOE measurements found that two single chains could include one poly(C) (Xu & Allen, 1996) can provide estimations of distances chain to form a new triple helical complex. The complexa- between protons located in rather close proximity. Relaxa- tion proceeds in a highly stoichiometric manner so that two tion time measurements give information on the mobility schizophyllan repeating units and three poly(C) units are and the behavior of molecules in solution. bound in the complex (Sakurai, Mizu, & Shinkai, 2001). The x-ray diffraction method is usually used to charac- Results from x-ray crystallographic studies indicated that terize the crystal structure of polysaccharides. Most poly- the newly formed triple helical complex was quite similar saccharides are amorphous and poorly crystalline. Only in molecular parameters to the original polysaccharide, a few helical polysaccharides with high rigidity showing but showed higher rigidity so that a good AFM spectrum ordered structure can form crystals for x-ray diffraction could be expected (Mizu, Koumoto, Kimura, Sakurai, & analysis. In most cases, x-ray diffraction measurements Shinkai, 2003). This interesting finding helps scientists to are conducted using orientated fibers or films prepared connect the biological function of triple helical polysaccha- from concentrated polysaccharide solutions. During the rides to their interaction with DNA and RNA. Some Japa- fiber preparation, the molecules are forced to align approxi- nese researchers have found that the single chain of mately parallel along the helical axes. Although this schizophyllan forms a macromolecular complex with organization is artificial, the information obtained may poly(C), poly(A), poly(dA), or poly(dT), but doesn’t form help to understand the ordered structures of polysaccha- a complex with poly(G), poly(U), poly(I), poly(dG) and rides that occur in solution. A typical x-ray diffraction anal- poly(dC) (Saenger, 1984, chap. 6). This nucleotide specific- ysis may provide information about the helical structures ity presents evidence that the hydrogen bonds are essential such as repeat spacing of the helix, helical screw symmetry, to form the complex, because the former nucleotides have the unit cell dimensions and lattice type. However, the in- unoccupied hydrogen-bonding sites and the latter ones terpretation of x-ray data usually requires supplementation have used these hydrogen-bonding sites in the intramolec- with molecular modeling analysis using existing stereo- ular aggregation (Numata et al., 2003; Saenger, 1984, chemical information derived from crystal structures of chap. 6). These features imply that the single chain of related mono- or oligosaccharides (Rao, Qasba, Balaji, & schizophyllan can behave like a poly(G) in the double Chandrasekaran, 1998, chap. 2). Examples of x-ray diffrac- strand poly(C)/poly(G). This opens a door for new fields tion structures are given for curdlan (Deslandes & Marche- of application with polysaccharides in future gene technol- ssault, 1980) and (1/3)-a-D-glucan (Ogawa, Okamura, & ogy, such as antisense DNA drugs, nonvirus vectors and Sarko, 1981). affinity chromatography (Numata et al., 2003). X-ray dif- AFM has become an important tool during the past de- fraction analyses for another helical mushroom polysaccha- cades to examine the conformation of macromolecules ride, lentinan (Bluhm & Sarko, 1977), have predicted five (Ando et al., 2001). AFM employs particular atom probes models including one single helix, two double helices, to directly study the shape and conformation of biomacro- and two triple helices for the crystalline lentinan that is molecules such as protein, DNA and polysaccharides under (1/3)-b-D-glucan having two 1/6 branches for every circumstances similar to a physiological environment. five (1/3)-b-glucopyranoside. Xu et al. (Xu, Zhang, Much work in the microscopic observation of biomolecules Zhang, & Wu, 2004) indicated, by means of LLS and has been dedicated towards observation of the double helix AFM (atomic force microscopy), that lentinan predomi- of DNA (Hansma, Laney, Bezanilla, Sinsheimer, & nantly existed as triple helical chain in 0.5 M NaCl aqueous Hansma, 1995). Compared to protein and DNA, an AFM solution and as single flexible chain in DMSO. Zhang et al. spectrum of a polysaccharide is difficult to achieve due to (Zhang, Zhang, & Xu, 2005) also successfully observed the a more complicated structure, especially when branches dynamic transition of triple helicesesingle helix-random and non-carbohydrate substituted groups occur in polymer coil of this glucan. Polysaccharides that take on triple chains. The difficulty is also caused by the fact that the helical conformation have clinical applications for the radius of the tips of the probe is generally larger than the M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 13 pitch of the helix, and that the structure of the molecules is that is, straight synthesis of the polymer using deuterated generally fairly flexible, and probably distorted by the im- compounds, is often required. SANS is therefore best suited aging process. Successful observations are limited to a few where LLS fails, as for instance, with opaque systems such triple helical polysaccharides because they have a very as miceller suspensions in which multiple light scattering rigid structure that could be imaged by AFM (Camesano complicates the scattering pattern. As with LLS, SANS & Wilkinson, 2001; McIntire & Brant, 1998). McIntire provides information on the static structure of the system, (McIntire & Brant, 1998) and Stokke (Stokke, Elgsaeter, but molecular length is smaller. & Kitamura, 1994) and Zhang et al. (Zhang, Chen et al., Computer programs have been developed to generate 3D 2005) have independently observed three triple helical structures of polysaccharides from their primary sequence (1/3)-b-D-glucans (scleroglucan, schizophyllan, and len- (Tvaroska, 1989). Computer modeling has been used to tinan, respectively) with AFM, and they have found that analyze the conformational effects of steric interactions the natural glucans exhibited a rod-like architecture, which between atoms of the polymer skeletons for related poly- is expected from the studies on the dilute solution proper- saccharides. Over 99% of the conformations are thus ties of those polymers. However, the resolution was not excluded and virtually all of the remainders for each poly- good enough to observe the helicity of these polysaccha- saccharide lie close together. In brief, the simulation in- rides. Kunitake & Ohira (Kunitake & Ohira, 2002) also cludes four steps (Perez, Kouwijzer, Mazeau, & Engelsen, reported helical pattern from AFM observation on the 1996). The first step starts with a description of the confor- schizophyllan molecule. Nevertheless, the observed helicity mations of the monosaccharides and a thorough description was not consistent with the crystallographic data. As men- of the conformation space that is available for a repeating tioned previously, a single chain of schizophyllan forms unit. The results were used in the second step, aimed at a high rigid complex with DNA/RNA so that a good generating a disordered polymer chain based on the as- AFM spectrum could be expected. This finding provides sumption that due to the size and relative rigidity of the in- a good method to directly observe the helical structure of tervening monosaccharide units, the rotations at a particular polysaccharides using AFM. McIntire (McIntire & Brant, linkage can be, under certain conditions, independent of the 1998) successfully observed the triple helical conformation nearest neighboring interactions. In the third step, the ap- of scleroglucan that possesses the primary structure of 1/6 propriate modeling techniques will be used to construct branched (1/3)-b-D-glucan. They also observed triple the ordered state of polysaccharide strands. The generation helicesesingle helix-random coil transition when a treat- of multiple helices can be then attempted in order to ex- ment of heat was applied to this polysaccharide. Moreover, plore the occurrence of such multi-strand arrangements recent experiments (Piotr, Hongbin, Andres, Oberhauser, & that may be energetically stable. The final step in the deter- Fernandez, 2002) have demonstrated that a force field can mination of the 3D structure of polysaccharides in the or- trigger conformational transitions in these molecules which dered states, is the investigation of the possible cannot be observed by traditional NMR or x-ray crystallo- interactions among helices and interactions between helices graphic techniques. Therefore, AFM should be increasingly and target molecules. Molecular simulation allows auto- applied in analyzing the branching and the length of the matic searches for meaningful correlations between struc- branches in the polysaccharide, cross-linked network, as tures and functions, through exploratory data analysis. well as the dynamic procedure. Structure-function or structure-property correlation could LLS and SAXS (small-angle x-ray scattering) have been then be used to model and predict changes arising from widely used to characterize polymer chains in solution. The structural alterations. weight average molecular weight (Mw), the radius of gyra- 2 1/2 tion ( ) and the second virial coefficient (A2) can be Antitumor activity and cellular mechanism determined (Johnson & Gabriel, 1994). The shape of the The involvement and importance of polysaccharides in polymer molecule, whether it is spherical, random coiled, tumor and cancer treatment were first recognized more or rod like, can be analyzed from data of 1/2 or [h] than 100 years ago when it was found that certain polysac- as a function of molecular weight. These data can be ana- charides could induce complete remission in patients with lyzed in terms of Yamakawa’s theories for unperturbed cancer (Nauts, Swift, & Coley, 1946). Ever since antitumor wormlike chains, in order to provide molecular parameters activity of macrofungal polysaccharides was first published such as molecular weight per contour length (ML), persis- by Chihara in the 1960s (Chihara, 1969), researchers have tence length (q) and distance per turn of helix (h). isolated structural diversified polysaccharides with strong Small-angle neutron scattering (SANS) has become antitumor activity. Unlike traditional antitumor drugs, these a preferred tool to analyze a variety of polymer systems, substances produce an antitumor effect by activating vari- including pure and blend bulk polymers, phase-separated ous immune responses in the host and cause no harm to systems, miceller suspensions, and solutions, especially the body (Wasser & Weis, 1999). concentrated ones (Higgins & Benoit, 1997). Unlike light Mushroom polysaccharides have shown widely inhibi- scattering, it is available only in a limited number of facil- tory effects towards many kinds of tumors including Sar- ities around the globe. Labeling of polymers by deuterium, coma 180 solid cancers, Ehrlich solid cancer, Sarcoma 14 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19

37, Yoshida sarcoma and Lewis lung carcinoma (Wasser & the prolonged survival time of the tumor bearing mice, Weis, 1999). The proposed mechanism by which mush- were evaluated as the inhibitory effect, indicating the im- room polysaccharides exert antitumor effect include: 1) muno-enhancing activity of the mushroom polysaccharides. the prevention of the oncogenesis by oral administration The host-mediated immunomodulating activity has been of polysaccharides isolated from medicinal mushrooms demonstrated by a number of studies. The antitumor effect (cancer-preventing activity); 2) enhancement of immunity of polysaccharides was lost in neonatal thymectomized against the bearing tumors (Immuno-enhancing activity); mice (mice that have no thymus-dependent immune sys- and 3) direct antitumor activity to induce the apoptosis of tem), and decreased significantly by administration of tumor cells (Direct tumor inhibition activity). anti-lymphocyte serum (Maeda & Chihara, 1971). The re- The cancer-preventing activity of medicinal mush- sults suggest that the antitumor action of polysaccharides room polysaccharides has been found with Hypsizygus requires an intact T-cell component and that the activity marmoreus and its mechanism was due to the immuno- is mediated through a thymus-dependent immune mecha- potentiation of polysaccharides (Ikekawa, 2001). The nism. The possible pathways of host-mediated actions cancer-preventive activity of medicinal mushroom polysac- from lentinan have been suggested (Chihara, 1992) and charides has been observed in farmers whose main occupa- later modified by S. P. Wasser (Wasser & Weis, 1999). tion was producing medicinal mushrooms such as The pathway explained the possible immune mechanism Flammulina velutipes in Japan and Agaricus blazei in Brazil of lentinan, showing that the administration of lentinan (Ikekawa, 2001). These farmers’ cancer death rate was re- can promote potentiation of response of precursor T cells markably lower than that of the general population by 40%. and macrophages to cytokines produced by certain groups Consequently, an animal study was conducted by feeding of lymphocytes after specific recognition of tumor cells the control mice with an ordinary diet and the treated (Chihara, 1992). The induction of a marked increased in mice with medicinal mushroom polysaccharides from F. the amounts of TNF-a (tumor necrosis factor), IL-1 (inter- velutipes and A. blazei in a diet, before they were inoculated leukin-1), IL-3 (interleukin-3) and IFN (interferon) by len- with tumors. At the end of the experiment, the number of tinan results in maturation, differentiation, and proliferation mice that developed tumors on the test diet was compared of immunocompetent cells for host defense mechanisms with that in the control group. This decreased number of tu- (Chihara, 1992). In addition, lentinan is able to restore mor bearing mice indicates the cancer-preventive activity the suppressed activity of helper T cells in the tumor bear- of the polysaccharides (Ikekawa, 2001). ing host to their normal state, leading to the complete res- Mushroom polysaccharides exert their antitumor action toration of humoral immune responses (Maeda, Watanabe, mainly via activation of the immune response of the host Chihara, & Rokutanda, 1988). Moreover, it has been organism (Immuno-enhancing activity). In other words, reported that the delayed-type hypersensitivity response mushroom polysaccharides do not directly kill tumor cells. induced at tumor sites by lentinan, and the subsequent infil- They help the host to adapt to various biological stresses tration of immune effector cells, such as natural killer cells and exert a nonspecific action on the host, supporting and cytotoxic T lymphocytes, is an important mechanism of some or all of the major systems. Mushroom polysaccha- antitumor action for lentinan (Suzuki, Iwashiro, Takatsuki, rides cause no harm and place no additional stress on the Kuribayashi, & Hamuro, 1994). In recent years, it has been body, therefore, they are regarded as biological response proposed that lentinan inhibits hepatic metastasis in adeno- modifiers. Immuno-enhancing activity has been found in carcinoma-26 bearing mice by activating Kupffer cells many mushroom polysaccharides. Mushroom polysaccha- (Taki et al., 1995). Therefore, it remains to be clarified rides have been shown to produce over 50% reduction in which immunomodulatory effects induced by lentinan are tumor size and prolong the survival time of tumor bearing critical for tumor rejection. Schizophyllan is similar to len- mice (Wasser, 2002). Lentinan produced over 90% reduc- tinan in composition and antitumor activity, as well as in tion in tumor size, or complete regression in most of the their mechanism for antitumor action (Jong et al., 1991). tested animals. In addition, it showed prominent antitumor However, the kinetics of gene expression of cytokines in activity not only against allogenic tumors, but also against schizophyllan is different in peritoneal exudate cells, sple- various synergic and autochthonous tumors (Wasser, 2002). nocytes, and liver cells (Nemoto, Ohno, Saito, Adachi, & Schizophyllan displayed antitumor activity against both the Yasomae, 1993; Okazaki, Adachi, Ohno, & Yadomae, solid and ascite forms of Sarcoma 180, as well as against 1995). Grifolan, which is isolated from Grifola frondosa the solid form of Sarcoma 37, Erlich sarcoma, Yoshida sar- and similar to schizophyllan in primary structure, is a novel coma and Lewis lung carcinoma (Wasser, 2002). Most of macrophage activator that enhances mRNA levels of IL-6, the experimental immuno-enhancing activity testing was IL-1, and TNF-a macrophages (Adachi, Okazaki, Ohno, & performed by inoculating the mice with tumor cells (Ooi Yadomae, 1994). & Liu, 1999). The polysaccharides were administered intra- Direct tumor inhibition activity has been documented peritoneally to the mice for a short period of time, after in many mushroom polysaccharides (Wang et al., 2002). tumors had developed. The reduction of the tumor size or Although the anti-proliferative effect of polysaccharides the number of the complete regressive test animals, and towards tumor lines in vitro remains unclear, some studies M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19 15 indicate that incubation of polysaccharides together with points, have been indicated as important factors in antitu- tumor cells could change the expression of signals within mor action. b-glucans containing mainly 1/6 linkages tumor cells. That could arrest the cell cycle and generate exhibit less activity, possibly due to their inherent flexibility apoptosis, which explains the in vitro anti-proliferative of having too many possible conformations. However, effect of polysaccharides (Chen & Chang, 2004; Li, Kim, antitumor polysaccharides may have other chemical struc- Kim, & Park, 2004; Lin et al., 2003). It has been reported tures, such as hetero-b-glucans (Mizuno, Saito, Nishitoba, that a polysaccharideepeptide complex (PSP) extracted & Kawagashi, 1995), heteroglycan (Gao, Seljelid, Chen, & from Trametes versicolor significantly reduced prolifera- Jiang, 1996), b-glucaneprotein (Kawagishi, Kanao et al., tion of MAD-MB-231 breast cancer cells (Chow, Lo, 1990), a-mannoeb-glucan (Mizuno et al., 1995), a- Loo, Hu, & Sham, 2003), as compared with the control. glucaneprotein (Mizuno et al., 1995) and heteroglycane It was also found that a protein bound polysaccharide protein complexes (Mizuno et al., 1996; Zhuang et al., (PBP) isolated from Phellinus linteus had an anti-prolifera- 1993). It has been postulated that mushroom polysaccharides tive effect for SW480 human colon cancer cells (Li et al., containing glucose and mannose may have some antitumor 2004). In addition, polysaccharides isolated from P. cocos, action because a polysaccharide receptor has been found Lycium barbarum and Cladonia furcata have been demon- on human macrophages, which has demonstrated high spec- strated to have novel anti-proliferative activities (Chen & ificity for glucose and mannose (Lombard, 1994). Chang, 2004; Li et al., 2004; Lin et al., 2003; Zhang, Triple helical conformation of (1/3)-b-glucans is Chen et al., 2005). These results suggest that mushroom regarded as an important structural feature for their polysaccharides not only stimulate the proliferation of T immuno-stimulating activity. (1/3)-b-glucans exhibit a lymphocytes and the immune function through the immu- variety of biological and immuno-pharmacological activi- nopotentiation, which has been revealed in the last three de- ties related to their triple helical conformation. For exam- cades (Zaidman, Yassin, Mahajna, & Wasser, 2005), but ple, when lentinan was denatured with DMSO, urea, or also have a direct action on the tumor cells. Nevertheless, sodium hydroxide, its tertiary structure was lost while its little is known about the direct effect of polysaccharides primary structure was maintained; but its tumor inhibitory on cancer cells. In recent years, some immunostaining tech- effect was lowered with progressive denaturation (Maeda niques have shown that PSP increased the p21 expression et al., 1988). The same results were obtained when a corre- and decreased the cyclin D1 expression (Chow et al., lation between antitumor activity and triple helical structure 2003). Molecular techniques have also been used to study was investigated in schizophyllan (Yanaki, Ito, & Tabata, the effect of PBP from P. linteus. It was found that the di- 1986). However, exactly how the triple helical conforma- rect cytotoxicity of PBP was mediated by induction of ap- tion of (1/3)-b-glucan affects their antitumor action still optosis, by G2/M cell cycle arrest associated with remains unclear. Many of the biological and immuno-phar- a decrease in Bcl-2, by an increase in the release of cyto- macological activities such as macrophage nitrogen oxide chrome c, and by reduced expression of cyclin B1 (Li synthesis and limulus factor G activation, are dependent et al., 2004). Lycium Barbarum polysaccharide (LBP) treat- on the triple helical conformation; while other activities, ment caused inhibition of human hepatoma QGY7703 cell such as synthesis of interferon-g and colony stimulating growth with cell cycle arrest in S phase and apoptosis induc- factor (Yadomae & Ohno, 2000), are independent from tion (Zhang, Chen et al., 2005). In our previous study of car- the triple helical conformation. It has been found that the boxymethylated polysaccharides (CMPTR) from Pleurotus (1/3)-b-glucan backbone structure is of more tuber-regium it was found that CMPTR-treated MCF-7 can- importance than the tertiary structure of the molecule, cer cells were associated with reduced expression of the cell and that helps to explain why (1/3)-a-mannan, having cycle related proteins, cyclin D1 and cyclin E, and with similar backbone conformation with (1/3)-b-glucan, has decreased expression of Bcl-2 and increased expression of shown comparable antitumor action to (1/3)-b-glucan. Bax. This suggests that CMPTR can directly inhibit the pro- Mizuno indicated that high molecular weight glucans ap- liferation of MCF-7 by blocking the cell cycle and generating pear to be more effective than those of low molecular apoptosis. Recent studies on direct cytotoxicity of poly- weight. However, unlike (1/3)-b-glucans with medicinal saccharides have also indicated that polysaccharides can properties that are strongly dependent on high molecular directly inhibit the cancer cell proliferation in a dose- and weight, ranging from 500 to 2000 kDa (Mizuno et al., time-dependent manner, which could be mediated through 1996), medical properties of some mushroom poly- up-regulation of p21 and down-regulation of cyclin D1. saccharides like (1/3)-a-glucuronoxylomannans, are not They could also directly induce apoptosis in cancer cells strongly dependent on molecular weights. Their hydrolysate which might be mediated by up-regulation of a pro-apoptosis fractions containing glucuronoxylomannans with molelcu- Bax protein (Zaidman et al., 2005). lar weights from 53 to 1000 Da are as effective as those frac- tions having higher molecular weights (Gao et al., 1996). It Structure-antitumor activity relationship was also reported by Gao that differences in molecular Structural features such as (1/3)-b- linkages in the weight had no obvious influence on the activity of the hetero- main chain of the glucan and additional (1/6)-b- branch glycans (Gao et al., 1996). 16 M. Zhang et al. / Trends in Food Science & Technology 18 (2007) 4e19

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