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REVIEW

Antitumor activity of mushroom polysaccharides: a review

Lu Ren, Conrad Perera and Yacine Hemar*
Received 16th December 2011, Accepted 1st July 2012 DOI: 10.1039/c2fo10279j Mushrooms were considered as a special delicacy by early civilizations and valued as a credible source of nutrients including considerable amounts of dietary ber, minerals, and vitamins (in particularly, vitamin D). Mushrooms are also recognized as functional foods for their bioactive compounds offer huge benecial impacts on human health. One of those potent bioactives is b-glucan, comprising a backbone of glucose residues linked by b-(1/3)-glycosidic bonds with attached b-(1/6) branch points, which exhibits antitumor and immunostimulating properties. The commercial pharmaceutical products from this polysaccharide source, such as schizophyllan, lentinan, grifolan, PSP (polysaccharidepeptide complex) and PSK (polysaccharideprotein complex), have shown evident clinical results. The immunomodulating action of mushroom polysaccharides is to stimulate natural killer cells, T-cells, B-cells, neutrophils, and macrophage dependent immune system responses via differing receptors involving dectin-1, the toll-like receptor-2 (a class of proteins that play a role in the immune system), scavengers and lactosylceramides. b-Glucans with various structures present distinct afnities toward these receptors to trigger different host responses. Basically, their antitumor abilities are inuenced by the molecular mass, branching conguration, conformation, and chemical modication of the polysaccharides. This review aims to integrate the information regarding nutritional, chemical and biological aspects of polysaccharides in mushrooms, which will possibly be employed to elucidate the correlation between their structural features and biological functions.

1. Introduction
The fossil record has proven the long existence of fungi as far back in time as the Paleozoic era (408438 million years ago) in the Silurian period.1 Mushrooms, as part of the fungal diversity for around 300 million years, might probably have been collected by prehistoric humans as food and possibly with medicinal aims.2 As the civilization of mankind progressed, mushrooms have been valued as edible and medicinal resources based on the long existing history in some Asian countries like China and Japan. Asian people have collected, cultivated and consumed mushrooms for over two thousand years due to their pleasant avor and texture. Traditional knowledge denes mushrooms as eshy, aerial umbrella-shaped, fruiting bodies of macrofungi.3 In the literature, mushrooms are acceptably dened as macrofungi comprising distinctive and visible fruiting bodies which can be hypogeous or epigeous.4 Mushrooms can be considered as a functional food for their great nutritional and medicinal values as dietary supplements, which has been sparked by the concerns about health and nutrition matters of consuming natural foods.2 The bioactivities of mushrooms have been conrmed by extensive studies. Zhang et al.3 stated that in 1957, Lucas discovered the bioactivity of

Basidiomycetes mushrooms for the rst time by isolating a substance from Boletus edulis which demonstrated a signicant inhibitory effect against Sarcoma S-180 tumor cells. Since then, numerous polysaccharides showing antitumor activity have been extracted from a variety of mushrooms. Recently, a huge amount of compounds isolated from mushrooms have been greatly highlighted for their sound pharmaceutical applications. These compounds, including lectins, polysaccharides, polysaccharide peptides, and polysaccharideprotein complexes, have been proven to possess effective functions such as: immunomodulatory, anticancer,5 anti-inammatory,6 and antioxidant7,8 effects, along with lowering blood cholesterol levels effects.9 In particular, the commercialization of several polysaccharides and polysaccharide conjugates has made patients benet from such anticancer therapies. They are schizophyllan, lentinan, grifolan, PSP (polysaccharidepeptide complex) and krestin (polysaccharideprotein complex).3

2. Mycological terms
The basic terminology used for the fruiting body of mushrooms is represented in Fig. 1. The gathered edible mushrooms are commonly described as higher fungi or macrofungi. The fruiting body (carpophore, mycocarp) in higher fungi is found mostly above ground. A fruiting body grows from spacious underground mycelia (hyphae) by the process of fructication. The
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School of Chemical Sciences, The University of Auckland, Auckland, New Zealand. E-mail: y.hemar@auckland.ac.nz

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such as trees. The terrestrial saprobic species snatch nutrients mainly from organic compounds of the plant and animal debris.10

3. Structural properties of polysaccharides exhibiting antitumor activity

3.1. Chemistry of polysaccharides Polysaccharides are condensation polymers, generally termed as glycans, in which large numbers of glycoses (monosaccharides) are mutually joined by O-glycosidic linkages. A glycosidic linkage is formed from the glycosyl moiety of hemiacetal (or hemiketal) and a hydroxyl group of another unit, acting as an acceptor molecule or aglycone.11 Glycosyl units indicate a monovalent character, while polycone shows the polyvalent nature. Branching is possible within the polysaccharide chains. It is impossible to have intramolecular cross linking by covalent bonds between adjacent chains through glycosidic linkages.11 Polysaccharides may be linear or branched. According to the number of different monomers present, polysaccharides are divided into two classes: homopolysaccharides comprise only one kind of monosaccharides, where as heteropolysaccharides comprise two of more kinds of monosaccharide units.12 As Table 1 demonstrates, homopolysaccharides can be subdivided by the type(s) of glycosidic linkages that link the monosaccharide units. The glycosidic linkage presents either an a- or b-conguration and at various positions, such as a-(1/2), a-(1/3), a-(1/4), b-(1/2), b-(1/3), b-(1/4). Both homopolysaccharides and heteropolysaccharides may possess homolinkages or heterolinkages with respect to conguration and/or linkage position. Furthermore, heteropolysaccharides have not only differing types and sequences of monosaccharide units, but also different types and sequences of glycosidic linkages. This leads to an almost limitless diversity in their structure.11 In addition, polysaccharides can be divided into three groups with respect to the type of sequence of sugar units. Periodic types are formed by a repeating patterns of sugar units. Interrupted types are formed by the chains that have repeating sequences that are separated by irregular sequences (kinds). Aperiodic types are

Fig. 1 Schematic image of a mushroom and basic mycological terms.

bulk of fruiting bodies have a short lifetime of only about 1014 days.10 Most types of mushrooms are commonly found in the shape of umbrella with pileus (cap) and stipe (stem). Nonetheless, some species additionally possess an annulus (ring), or a volva (cup), or have both. The forms of some unusual mushrooms look like pliable cups, golf balls, or small clubs.2 Our research team is presently dealing with some uncommon mushrooms found in New Zealand, e.g., Ileodictyon cibarium (resemble basket), Hericium clathroides (resembles coral), Auricularia cornea (resembles the human ear), Calvatia gigantea (resembles a puffball) (Fig. 2). Unlike green plants, mushrooms lack chlorophyll and so they cannot manufacture their own food from simple inorganic materials, such as water, carbon dioxide, and nitrates. They exploit foods from complex organic materials stored in dead or living tissues of plants and animals.2 Generally, they can be divided into three types of fungi according to their ecology. Those growing on dead organic material are termed saprophytic fungi. Those obtaining substances from living plants and animals and causing harm to the hosts are referred to as parasitic fungi. Those living with their hosts by symbiosis to gain vital benets from each other are called mutualistic symbiotic fungi.2 Mycelia of the ectomycorrhizal species grows within the roots of plants,

Table 1 Examples of homopolysaccharides (adapted from Izydorczyk11) Polysaccharides Linear Amylose Cellulose Xylan Inulin Levan Laminaran Chitin b-Glucan Branched Amylopectin Dextran Levan Pullulan Scleroglucan Glycogen Fig. 2 Photographs of mushrooms found in New Zealand. Repeating unit: glycosidic linkage type/glycose unit a-(1/4)-Glc b-(1/4)-Glc b-(1/4)-Xyl b-(2/1)-Fru b-(2/6)-Fru b-(1/3)-Glc b-(1/4)-GlcNAc b-(1/4, 1/3)-Glc a-(1/4, 1/6)-Glc a-(1/2, 1/3, 1/4, 1/6)-Glc b-(2/1, 2/6)-Fru a-(1/6)-Maltotriose b-(1/3, 1/6)-Glc a-(1/4, 1/6)-Glc

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characterized by irregular sequences of monosaccharide units, linkage positions, and congurations.11 Polysaccharides have various degrees of polymerization (DP) which is determined by the number of monosaccharide units in a chain. Only a few polysaccharides are found having DPs below 100. Polysaccharides are secondary gene products, which is different with proteins. Therefore, the products from various biosynthetic enzymes (glycosyl transferases) in nature are not under strict and direct genetic control in their synthesis. The mechanisms controlling certain biosynthetic events, such as the density and distribution of branches along the polysaccharide chain or the chains length, are not fully elucidated, although the general biosynthetic pathways of many polysaccharides have been well studied.11 There is a general agreement that different transferases are demanded for the addition of each monosaccharide unit to the growing chain. The backbone growth most likely happens by adding new sugar residues to the nonreducing end (tailward growth). Whereas, the addition of new residues happens at the reducing end (headward growth) when involving lipid intermediates. Most polysaccharides are considered to undergo precise synthesis. Its backbone growth is concurrent with the addition of side chains. The synthesis and transport of the plant cell wall polysaccharides are conducted by the membrane systems of the endoplasmic reticulum, Golgi bodies, and plasma lemma. The rate of transfer and deposition of the newly synthesized polysaccharide in the target tissue are considered to play some role in determining its chain length.11 Post-polymerization modication can be performed via esterication and/or etherication of chains. These modications, in some cases, may take place simultaneously with polymerization of the backbone chains. Lacking strict genetic control during the synthesis of each polysaccharide chain results in a great degree of heterogeneity of those polymers based on their molecular mass and DP, and certain aspects of molecular structure, such as ratio of different monosaccharides, linkage distribution, degree and distribution of branches. Hence, polysaccharides are regarded as polydispersed polymers. Nonetheless, not all structural characteristics of the polysaccharide are heterogeneous. For instance, the most conservative is the conguration of glycosidic bonds in polysaccharides, while the most variable characteristic is the molecular weight.11 In other words, polysaccharides belong to a structurally diverse class of macromolecules. The monosaccharide units in polysaccharides can interconnect at several points to produce various branched or linear structures.13 This vast potential variability in polysaccharide structure could offer possibilities to the precise regulatory mechanisms of various cellcell interactions in higher organisms.14 3.2. Mushroom polysaccharides showing antitumor activity

Polysaccharides showing antitumor activity have been isolated from the fruiting bodies, cultured mycelia and culture ltrates of basidiomycetes. These polysaccharides showing antitumor activity have a great variety of chemical composition, structure and antitumor activity.15 Since Lucas3 rst reported the polysaccharides extracted from mushrooms indicating antitumor activity in 1957, a great deal of polysaccharides that show antitumor activity have been isolated from mushrooms and their
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antitumor activities have been extensively studied. Some studies on those polysaccharides are shown in Table 2. Furthermore, although mushroom polysaccharides exhibit remarkable antitumor activity, their tumor inhibition ability varies greatly. For example, some reported polysaccharides present different antitumor activities in vivo through screening studies using sarcoma-180 in mice (Table 3). The main polysaccharides in mushrooms are glucans with different types of glycosidic linkages, such as (1/3)-, (1/6)-bglucans and (1/3)-a-glucans. Also, there are some heteroglycans present in mushrooms. The others are PSP complexes which are mostly bound to protein residues.39 Although, the fungal cell wall is the main source of polysaccharides demonstrating antitumor activity, chitin and chitosan (fungal chitin) show no antitumor activity.40 Particularly, polysaccharides that exhibit strong antitumor action are greatly different in their chemical structures. Antitumor activity is demonstrated by a wide range of glycans which extend from homopolymers to highly complex heteropolymers.41 Some monosaccharide types of the polysaccharides showing antitumor activity consist of glucose, galactose, mannose, xylose, arabinose, fucose, ribose and glucuronic acid. In some mushroom species, polysaccharides binding with proteins or peptides as a polysaccharideprotein or polysaccharidepeptide complex indicate higher potent antitumor activity.3 Apart from the well-known antitumor (1/3)-b-glucans, those biologically active glucans are linear or branched molecules which contain a backbone composed of a- or b-linked glucose units; some of them have side chains attached at different positions. Heteroglucan side chains hold glucuronic acid, xylose, galactose, mannose, arabinose, or ribose, which may be in different combinations. Heteroglycans are another large group of bioactive polysaccharides that are classied as galactans, fucans, xylans, and mannans by individual sugar components in the backbone. Likewise, heteroglycan side chains may hold arabinose, mannose, fucose, galactose, xylose, glucuronic acid, and a glucose moiety as a main component.3 In the seventies and eighties, three antitumor agents of polysaccharide nature, namely lentinan, schizophyllan and proteinbound polysaccharide (PSK, Krestin), were isolated from Lentinus edodes, Schizophyllum commune and Coriolus versicolor, respectively. They have since become large market items in Japan.40 Lentinan and schizophyllan belong to pure b-glucans, while PSK is a protein-bound b-glucan. In China, a polysaccharopeptide (PSP) has been isolated and employed as an anti-cancer and immunomodulatory agent in clinical treatments.15 Lentinan is a representative mushroom b-glucan, which shows effective antitumor and immunopotentiating activity. Its primary structure is a (1/3)-b-glucan containing ve (1/3)-b-glucose residues in a linear linkage and two (1/6)-b-glucopyranoside branches in side chains (Fig. 3A). This leads to a right-handed triple helical structure. The molecular weight of lentinan is about 400800 103 Da.15,42 Schizophyllan is also a (1/3)-b-glucan containing a b-glucopyranosyl group joined by a b-(1/6) linkage to every third or fourth residue of the main chain (Fig. 3B). It has a similar triple helix structure and biological activity to lentinan, and possesses a molecular weight of about 450 103 Da.15
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Table 2 Reported polysaccharides showing antitumor activity from mushrooms Mushroom species Cordyceps militaris Phellinus gilvus Phellinus linteus Pleurotus ostreatus Ganoderma lucidum Polysaccharide source Fruiting body Fruiting body Mycelial culture Fruiting body Fruiting body Antitumor type Melanoma Lung cancer Lung cancer Melanoma Melanoma Breast cancer Lung cancer Prostate cancer Cervical cancer Breast cancer Breast cancer Breast cancer Lung cancer Lung cancer Lung cancer Prostate cancer Prostate cancer Prostate cancer Cervical cancer Reference 16 17 18 19 and 20 21 22 23 24 25 26 27 28 29 29 30 31 32 33 34

Lentinula edodes Pleurotus geesteranus Pleurotus tuber-regium Clitocybe alexandri Lepista inversa Sparassis crispa Agaricus blazei Grifola frondosa Trametes versicolor Angelica sinensis

Fruiting body Fruiting body Sclerotia Fruiting body Fruiting body Fruiting body Fruiting body Fruiting body Fruiting body Mycelia

Table 3 Antitumor activities of extracts of mushroom species (against sarcoma-180 in mice) Tumor inhibition (%)

Mushroom species

Reference

Agaricaceae Agaricus bisporus 2 35 Auriculariaceae Auricularia auricula-judae 42 35 Corticiaceae Laetisaria arvalis 95 36 Pleurotaceae Pleurotus tuber-regium: carboxymethylated hot alkali extracts (CMHZE) CMHZE-1 64 37 CMHZE-2 48 37 CMHZE-3 75 37 CMHZE-4 53 37 CMHZE-5 46 37 CMHZE-6 43 37 Polyporaceae Ganoderma tsugae 77 35 Coriolus versicolor 77 35 Trametes gibbosa 49 35 Fomes fomentarios 5 35 Russulaceae Russula lepida 67.6 38 Strophariaceae Pholiota nameko 86 35 Tricholomataceae Lentinus edodes 80 35 Flammulina velutipes 81 35 Pleurotus ostreatus 75 35 Tricholoma matsutake 91 35

Fig. 3 Structure units of polysaccharides showing antitumor activity: lentinan (A), schizophyllan (B).

PSK (Krestin) is a b-glucanprotein complex consisting of 25 38% protein residues. Its average molecular weight, measured by ultracentrifuge analysis, is about 94 103 Da. It mainly contains acidic amino acids, such as aspartic acid and glutamic acid, and neutral amino acids, such as valine and leucine, and small amounts of basic amino acid, such as lysine and arginine. The main constituent monosaccharide is glucose with small amounts of other sugar residues like mannose, fucose, xylose and galactose. PSK has a (1/4)-b-glucan with (1/6)-b-glucopyranosidic
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side chains for every fourth glucose unit. It possesses branches at the 3- and 6-positions in a proportion of one every several residual groups of (1/4) bonds.43 A polysaccharopeptide (MW 100 103 Da) that was isolated from a strain of Coriolus versicolor in China has a similar glucan structure to PSK in Japan.44 A polysaccharideprotein complex (PSPC), that was extracted from the culture ltrates of Tricholoma lobayense, consists of 54.3% polysaccharides containing galactose, glucose, arabinose, xylose, rhamnose, fucose and mannose, and 35.9% protein containing majorly aspartic acid, glutamic acid, serine, glycine, lysine and threonine. PSPC shows the characteristics of a polysaccharide and intermolecular hydrogen bonds by inspection of the infrared spectra. The polysaccharide moiety of PSPC belongs to a unique heteroglycan with a molecular weight of about 154 103 Da.45 Ganoderan (MW 20 kDa) was isolated from Ganoderma lucidum and classied as a Ganoderma species which are the most
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well known medicinal fungi in the Orient. It is an immunomodulatory b-glucan, which induces potent antitumor immunity in tumour-bearing mice. It mostly contains glucose and 4% protein.46 Moreover, the fruiting bodies and mycelia of Ganoderma applanatum comprise b-glucan, heteroglycans and glycanprotein complexes. These polysaccharides indicating antitumor activity have the molecular weights ranging from 30 103 to 10 105 Da. Their basic chemical structure is (1/3)-b-glucopyranan with 115 (1/6)-b-monoglucosyl side chains.47 Seven potent antitumor polysaccharideprotein complexes have been extracted from Ganoderma tsugae. Two of them were proteincontaining glucogalactans related to mannose and fucose. And ve are protein-containing (1/3)-b-glucans.15

possibility that polysaccharides showing antitumor activity may not always be multiple enhancers of the host defense system, and that a high molecular mass is needed for extensive enhancement of immunological and antitumor activities. However, some low molecular weight polysaccharides, such as lentinan and schizophyllan, present the same antitumor activity against Sarcoma 180 as those with higher molecular weights. The divergent results remain to be claried.15 4.2. The effect of the branching conguration

4. Connection between structure and antitumor activities of mushroom polysaccharides

Polysaccharides possess a huge variety of chemical compositions and congurations and physical properties. The antitumor activity of the polysaccharides can be inuenced by the size of the molecules, degree of branching, form, and solubility in water.14 Generally, the greater the molecular weight and the higher the water solubility of these polysaccharides, the higher the antitumor activity. In the study based on seven potent antitumor polysaccharideprotein complexes from Ganoderma tsugae, it was found that polysaccharides showing antitumor activity with high activity derived from fruiting bodies were mainly heteropolysaccharides that had molecular weights of about 10 103 Da, containing galactose, glucose, mannose and fucose. However, highly active polysaccharides isolated form mycelia were mostly protein-containing glucans with molecular weights of around 10 103 Da.40,48 Most polysaccharides exhibiting antitumor activity have been reported to have the same basic b-glucan structure with different types of glycosidic linkages. Hence, the antitumor action requires structural features such as b-(1/3) linkages in the main chain of the glucan and additional b-(1/6) branch points. b-Glucan, comprising mainly (1/6) linkages possesses less activity.49 Nonetheless, there are other obvious variations in those polysaccharides. Polysaccharides that show antitumor activity may contain other chemical structures, such as hetero-b-glucans,40 heteroglycan,50 b-glucanprotein,51 a-manno-b-glucan,40 40 a-glucanprotein and heteroglycanprotein complexes.52 For instance, PSK and PSP have a b-glucanprotein, while PSPC isolated from the Tricholoma species are a heteroglycanprotein complex. 4.1. The inuence of molecular mass

If b-glucans are mostly linear, containing branches that are not excessively long, they will exhibit antitumor activity. For instance, pachyman, which is separated from Poria cocos, is inactive although it is a branched b-glucan. Nonetheless, pachymaran, that is formed by debranching pachyman using periodate oxidatiton and mild hydrolysis shows pronounced antitumor activity. Miyazaki et al.53 suggested that the optimal branching frequency is from 0.2 (1 in 5 backbone residues) to 0.33 (1 in 3 backbone residues). Lentinan (2/5) is a b-1,3-D-glucan possessing two branches for every ve D-glucopyranosyl residues. Schizophyllan (1/3) is also a b-1,3-D-glucan having one branch for every three D-glucopyranosyl residues. The polysaccharide of PSK (1/5) is a (1/3)-b,(1/4)-D-glucan of one branch for every ve D-glucopyranosyl residues. Their antitumor activities are not apparently different even though the degree of their branches differs. However, the debranched lentinan indicates a more effective antitumor activity than the native lentinan at a dose of 2.0 mg kg1 for ve days in mice.15 4.3. The impact of conformation

A high molecular mass is necessary for extensively enhancing immunological and antitumor activities. Four fractions of PSK have been successively separated. The highest molecular mass fraction shows the strongest immunomodulatory activity. A (1/3)-b-glucan, isolated from the cultured mycelium of Grifola frondosa, indicates changes in biological activities with various molecular masses, which was obtained by heat treatment for different lengths of time at 150  C. The fraction with the highest molecular mass (800 103 Da) shows the most potent antitumor and immunomodulatory activities. These studies highlight the
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The conformations of polysaccharides demonstrating antitumor activity include single helix, triple helix and random coil. Lentinan, schizophyllan and PSK all consist of a triple helix structure.15 It is known that a triple-helical tertiary conformation of medicinal mushroom (1/3)-b-glucans is crucial for their immune-stimulating activity. The tertiary structure of lentinan is lost while its primary structure is not affected when it is denatured with dimethyl sulfoxide, urea, or sodium hydroxide. Its tumor inhibition properties are reduced during progressive denaturation.54 Pachyman isolated from Poria cocos is a b-1,3D-glucan containing a single helix conformer, which is not biologically active against tumor growth. However, when it becomes pachymaran by periodate oxidation and mild hydrolysis, the newly formed conformer shows pronounced antitumor activity.55 SchizophyllanOH with a single helix structure, that is obtained from the alkaline-treated schizophyllan, exhibits a reduced ability to inhibit tumor growth as compared with the native schizophyllan.42 Yadomae56 explained that many biological and immunopharmacological activities demonstrated by mushroom b-(1/3)-glucan, such as macrophage nitrogen oxide synthesis and limulus factor G activation, are determined by the triplehelix conformation, whereas others are not dependent on this conformation, e.g., synthesis of interferon-g and colony stimulating factor. Hence, it can be seen that the antitumor activity of polysaccharide is dependent on the helical conformation. The correlation between conformation and antitumor activity of
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the polysaccharides or polysaccharideprotein complexes implies that the existence of biological systems within the host body recognize the congurational structure of polysaccharide.15 4.4. The effect of solubility

The solubility of b-glucans is affected by their degree of polymerization and thus their physical organization.57 When the alkali-insoluble, branched (1/3)-b-D-glucan, isolated from Auricularia auricular-judae, was modied by controlled periodate oxidation, borohydride reduction, and mild acid hydrolysis, its water solubility was increased by having covalently linked D-glucosyl residues, which demonstrated signicantly potent antitumor activity, whereas in the native state it had no such activity.58 4.5. Enhancement of antitumor activity by chemical modication The improvement of the biological activity of polysaccharides that show antitumor activity can be achieved by chemical modication. Various carboxymethylated (CM), hydroxylated, formylmethylated, aminethylated and sulfated products have been designed. The successful schemes for chemical improvement of mushroom polysaccharides have been designed for Ganoderma lucidum, Grifola frondosa and Leucopaxillus giganteus. Two main procedures are involved in these schemes: modication of mushroom polysaccharides by Smith degradation (oxydoreducto-hydrolysis) and activation by the method of formolysis.49 During the Smith degradation modication, original polysaccharide solutions are rst oxidized to polyaldehydes by 0.1 M NaIO4 in darkness. They are then are reduced into polyalcohols by NaBH4 in an alkaline medium adjusted to pH 8 with 2 M NaOH, and hydrolyzed by 1 M H2SO4 at room temperature.59 Chemical activation of the mushroom polysaccharides by means of formolysis consists of degradation of the polysaccharides by formic acid in 99% HCOOH solution. Fractions are obtained by alcohol (99% EtOH) precipitation.59 The two original polysaccharides do not possess activity, however, their polyaldehyde polyol, formylated, and formolysis derivatives exhibit signicant activity. Furthermore, polyaldehyde, and polyolpolysaccharides obtained from a polysaccharide that has low antitumor activity indicates activity that is stronger than the original polysaccharide.59 The method of carboxymethylation can be employed to transform b-glucans into a water-soluble form. For instance, the fruit bodies of Pleurotus ostreatus are treated with 0.15 M NaOH solution at 95  C for 2 h. The residue is collected and washed with water until neutral. It is then suspended in 0.06% NaCl solution, adjusted to pH 4.5 with acetic acid, and stirred for 6 h at 50  C. The polysaccharide thus obtained is a b-(1/3)-linked glucan with every fourth glucopyranosyl residue substituted at 06 with single D-glucopyranosyl groups. Carboxymethylated glucan P. ostreatus shows immunomodulatory effects, and elevated phagocytic activity.60,61 The linear (1/3)-a-glucans derived from Amanitamuscaria and Agrocybe cylindracea show little antitumor activity. After modication, the carboxymethylated
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linear (1/3)-a-glucans indicate strong antitumor activity against Sarcoma 180 and immunomodulating activity in mice.15 Chemical modication of branched mushroom polysaccharides resulting in side-chain reduction can be performed not only by Smith degradation but also by enzymatic reactions. Debranched pachymaran and CM-pachynmaran is a b-1,315 D-glucan showing more effective antitumor activity. Linear low molecular weight a-(1/4)-glucans that are prepared after enzymatic reduction of the side chains and protein component (active hexose correlated compounds AHCC) show immunomodulatory and anticancer properties.62 The antitumor activity of the formylmethylated and aminoethylated derivatives of schizophyllan against Sarcoma 180 solid tumor in mice is largely augmented compared with the native schizophyllan.63 The sulfated lentinan and schizophyllan products show potent anti-human immunodeciency virus activity though with reduced antitumor effect. These investigations give the direction that the improvement of the biological activities of polysaccharides may be effectively approached by chemical modications.15

5. Immunomodulating activities and mechanisms of antitumor activity by mushroom polysaccharides

5.1. Cancer A neoplasm is dened as an abnormal mass or colony of cells formed by a relatively autonomous new growth of tissue.64 Most of the neoplasms originate from the clonal expansion of a single cell which has undergone neoplastic transformation. The transformation of a normal cell to a neoplastic cell can be triggered by chemical, physical, or biological agent (or event), which directly and irreversibly changes the cell genome. Neoplastic cells have the characteristics exhibited by the loss of some specialized functions and the acquisition of new biological properties, such as self-sufciency in growth signals, insensitivity to growthinhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Neoplastic cells deliver their heritable biological characteristics to progeny cells.64 Cancer is a generic term used for malignant neoplasms.64 A characteristic property of cancer cells is anaplasia, which denotes a lack of normal structural and functional characteristics. Literally, a tumor describes a swelling of any type, such as an inammation of other swelling. Generally, the development of a cancer includes three stages. In the initial stage, a mutagen binds to the cell DNA and results in damage. Usually, the initiation is not sufcient to trigger tumor production by itself. The second stage is called activation, in which a tumor promoter is activated to cause the formation of small benign tumors. In the third stage of progression, the loss of the normal tight control over the cell cycle leads to uncontrolled cell proliferation.65 Furthermore, the biological behavior or clinical course of neoplasm can be divided into benign or malignant. A malignant neoplasm, that presents a greater degree of autonomy, is capable of invasion and metastatic activity, which may be resistant to treatment and leads to death. A benign neoplasm, with a lesser degree of autonomy, is usually not invasive, does not metastasize. It usually generates no great harm if treated adequately.64
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5.2.

Antitumor activity by mushroom polysaccharides

Mushroom polysaccharides perform their antitumor action mainly via activation of the immune response of the host organism. These substances are considered as biological response modiers.66 Basically, this suggests that: (1) they cause no harm and exert no additional stress on the body; (2) they assist the body to adapt to a variety of environmental and biological stresses; and (3) they place a nonspecic action on the body, supporting some or all of the major systems, such as nervous, hormonal, and immune systems, as well as regulatory functions.67 Substances that are capable of interacting with the immune system can either upregulate or downregulate specic aspects of the host response. Whether certain substances enhance or suppress immune responses are dependent on many factors including dose, route of administration, and timing of administration of the substances in question. The type of activity can also be determined by their mechanism of action or the site of activity.68 Mushroom polysaccharides have been proven in a wide range of antitumor activities. Numerous studies have reported that the compounds, particularly b-D-glucan derivatives, nonspecically activate cellular and humoral components of the host immune system so that they raise functional activity of macrophages, monomuclear cells, and neutrophils.69 b-D-Glucans are recognized by the human immune systems as foreign molecules since they are not synthesized by humans. These compounds can cause both innate and adaptive immune responses.70 The defence of the body against microbial attack and against spontaneously occurring malignant tumor cells consists of a dynamic orchestrated interplay of innate and acquired immune responses (Fig. 4). Innate immunity, containing macrophages, neutrophils, natural killer cells (NKs) and dendritic cells (DCs) as gatekeepers, is regulated by chemical-messengers or cytokines and by activating inammatory and acute phase responses.42 The mononuclear phagocyte system (e.g., macrophages and monocytes), DCs and certain lymphocytes (e.g., NK cells) exact numerous important functions involving the recognition and destruction of abnormal cells. Specic immunity to abnormal

Fig. 4 Immune responses stimulated by fungal b-glucans.

cells or tissues contains humoral (e.g., generates antibodies) and cell-mediated immunity (also enhances inammatory responses and ultimately kills infected or abnormal cells). Therefore, an adequately functional immune response is critical to the recognition and removal of tumor cells.71 For instance, the activated phagocytes can eliminate pathogens by phagocytosis.72 The macrophages target and remove dead cells and intracellular pathogens.73 NKs circulating in blood lyse cancer and virusinfected cells. Neutrophils attack pyogenic bacteria.74 The adaptive immune system works on the despondence to the introduction of foreign antigens, involving both B and T cells. B cells generate antibodies to mediate humoral immunity, while T cells trigger cell-mediated immunity.73 Cytokines potentiate T cell differentiation to helper T cells 1 (Th1) and 2 (Th2), which mediate cell and humoral immunities, respectively.75 DCs, derived from monocytes, are involved in the adaptive immune response, presenting antigens to T cells to activate immune responses.73 Multicellular organisms contain receptors which are called pattern recognition receptors (PRRs), detecting innately foreign structures like pathogen-associated molecular patterns (PAMPs). Fungal b-glucans are considered as PAMPs and are recognized by appropriate cell surface receptors to initiate immune responses. Some receptors have been identied in humans, such as dectin-1, complement receptor 3 (CR3), scavenger receptors, lactosylceramide (LacCer), and the toll-like receptor (TLR).70 Dectin-1 is a lectin which contains four components, namely an extracellular carbohydrate-recognition domain (CRD), a stalk, a transmembrane region, and an intracellular cytoplasmic tail.76 Dectin-1 is evidenced to be of the most importance in activating innate immune responses in macrophages,77 since the abolition of all macrophage-mediated responses can be caused when blocked with an anti-dectin-1 antibody and knockout of the detin-1 gene.78 Several signaling pathways contributed to by the dectin-1 binding with the ligand, promoting innate immune responses through the activation of phagocytosis, reactive oxygen species (ROS) production, and induction of inammatory cytokines, have been identied. One pathway is that dectin-1 works synergistically with TLR to generate strong inammatory responses by stimulating cytokines such as tumor necrosis factoralpha (TNF-a), interleukine-2 (IL-2) and IL-12.79 The second pathway is independent of TLR, which is mediated via spleen tyrosine kinase (Syk) to yield other cytokines, such as the macrophage inammatory protein-2 (MIP2, CXC2) and IL-2 and IL-10 in mice DC cells.80 In addition, another independent signaling pathway that is activated by the dectin-1 receptor is phagocytosis in macrophages.79 The CR3 receptor, that comprises CD11b and CD18 domains, recognizes a large range of microbial cells and acts as an adhesion molecule. It is presented primarily on neutrophils, monocytes and NK cells, but not macrophages.81 CD11b contains two binding sites. The one located within the C terminus is for b-glucans, whereas the other which is located within the N-terminus, is for iC3b (cleaved component 3 fragment of serum complement system).82 When b-glucans are bound to CR3, the adhesion to microbial cells is increased and the iC3b pathway is activated to result in tumor cytotoxicity.82 It is essential that both binding sites are occupied to trigger this activation, since cytotoxicity is blocked by an anti-CR3 antibody.83
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Scavenger receptors that are located in myeloid and endothelial cells consist of a heterogeneous group of proteins with two transmembrane domains, two intracellular domains and one extracellular domain, recognizing a range of foreign cells, lowdensity lipoprotein (LDL), high-density lipoprotein (HDL) and selected polyanionic ligands.84 The Src receptor activates multiple signaling pathways involving Src family kinase(s), phosphatidylinositol-3 kinase (P13K), Akt kinase, and p38 mitogen-activated protein kinase (MAPK), and an endothelial nitric oxide synthase (eNOS).84 However, Chen and Seviour74 think that they are not important due to the lack of sufcient evidence to understand the biological effects mediated by fungal b-glucans. LacCer, located in neutrophil and endothelial cells, which is a glycolipid, possessing a hydrophobic ceramid lipid and hydrophilic sugar moiety, recognizes both microbial cells and b-(1/3)-glucans.85 TLRs are transmembrane receptors of a novel protein family, responding to the presence of a diverse group of microbes, such as fungi, bacteria, viruses and protozoa.86 Nonetheless, further investigation is required to clarify the pathway that the immune responses are activated by b-(1/3)-glucans via these receptors. Polysaccharides derived from mushrooms do not attack cancer cells directly, while they generate their antitumor effects via the activation of different immune responses in the host. Many experiments have conrmed this. For example, the antitumor effect of polysaccharides is lost in neonatal thymectomized mice, or is decreased signicantly after administration of anti-lymphocyte serum.87 The results imply that the antitumor action of polysaccharides needs an intact T cell component and that the activity is mediated through a thymus-dependent immune mechanism.42 The pathway of the possible immune mechanism shows that the administration of lentinan can promote potentiation of the responses of precursor T cells and macrophages to cytokines that are produced by certain groups of lymphocytes after specic recognition of tumor cells.42 The induction of the marked rise in the amount of TNF-a, IL-1, IL-3 and interferon (IFN) by lentinan causes maturation, differentiation, and proliferation of immunocompetent cells for host defence mechanisms.42 Lentinan is also able to restore the suppressed activity of helper T cells in the tumor bearing host to their normal state, resulting in the complete restoration of humoral immune responses.54 Furthermore, lentinan-induced delayed-type hypersensitivity response at tumor sites plays a role in eradicating tumors by regulating inltration of activated immune effector cells, such as natural killer cells and cytotoxic T lymphocytes.88 Compared with lentinan, schizophyllan has a similar composition, antitumor activity, as well as a mechanism for antitumor action. Grifolan derived from Grifola frondosa is similar to schizophyllan in primary structure. It is a novel macrophage activator enhancing mPNA levels of IL-6, IL-1, and TNF-a macrophages.89,90 Direct tumor inhibition activity of mushroom polysaccharides has also been documented. Despite the mechanism of antiproliferation of polysaccharides towards tumor lines in vitro being unclear, some researchers have demonstrated that the expression of signals within tumor cells could be changed by the incubation of polysaccharides together with tumor cells. This could arrest the cell cycle and produce apoptosis,which
This journal is The Royal Society of Chemistry 2012

elucidates the in vitro anti-proliferative effect of polysaccharides.91 A polysaccharidepeptide complex (PSP) isolated from Trametes versicolar was reported to signicantly decrease proliferation of MAD-MB-231 breast cancer cells.92 These results suggest that mushroom polysaccharides not only stimulate the proliferation of T lymphocytes and the immune function through the immunopotentiation, but also exact a direct action on the tumor cells. However, little is known regarding the direct effect of polysaccharides on cancer cells.3 5.3. Mechanisms of antitumor by mushroom polysaccharides

The proliferation of tumor cells can be prevented through diverse mechanisms, including cell cycle arrest, induction of tumor cell death by apoptosis and secondary necrosis, together with stimulation of the antitumor activity of macropahges.93 In the eukaryotic cell cycle, cyclins and cyclin-dependent kinases (Cdks) are critical regulators. Cell cycle progression is regulated at several irreversible transition points. The passage is controlled by the activity of Cdks.94 At least three differing mechanisms, namely binding of cyclin proteins, phosphorylation, and binding of the cyclin-dependent kinase inhibitors (CKIs), have been discovered in the activity of Cdks. The accumulation of cyclins D, E, and A, which bind to and activate different Cdk catalytic subunits, is able to promote the progression from G1 to S phase in mammalian cells.95 The research group of Hsieh96 found that ethanolwater C. versicolor extracts could induce G0/G1 phase arrest in tumor cells. The death of tumor cells undergoing antitumor therapy can be caused by apoptosis and/or necrosis. Apoptosis is a form of cell death in which a programmed sequence of events results in the ingestion of cell remains by surrounding cells without releasing harmful substances.93 Apoptosis is tightly controlled by a number of gene products that either promote or block cell death at different stages of the cell cycle.28 One of the major gene groups that regulate apoptosis is the Bcl-2 family, which is composed of a large number of proteins. They all belong to three sub-families based on the number of Bcl-2-homology (BH) domains present in these proteins; (i) a subfamily consisting of Bcl-2, Bcl-xL and Bcl-w performs anti-apoptotic activities and shares sequence homology, especially in four regions, BH1 through BH4; (ii) a subfamily including Bax, Bad and Bak shares sequence homology at BH1, BH2 and BH3, exerting proapoptotic activity; (iii) a subfamily containing Bik and Bid shares sequence homology within the BH3 domain only, which shows pro-apoptotic activity.97 Bax is a 21 kD protein of 192 amino acids98 that shares homology with Bcl-2 in conserved regions, including BH1 and BH2. Hence, Bax may heterodimerize with Bcl-2 or other proteins and/or hodimerize.99 Bax is a nuclearencoded protein present in higher eukaryotes which can pierce the mitochondrial outer membrane to mediate cell death by apoptosis.100 During apoptosis, Bax may form oligodimers that are considered to cause the permeabilization of the mitochondrial membranes, either by forming channels,101 by interacting with components of the permeability transition pore,101 or by altering ssion and fusion processes.102 For instance, the water extract of Cordyceps militaris (WECM) induced apoptosis in human lung carcinoma A549 cells. The data revealed that there was a concentration-dependent increase of Bax expression in
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WECM treated A549 cells, but a decrease of Bcl-2.17 In another study, the treatment with a novel polysaccharide isolated from Angelica sinensis can reduce Bcl-2 and Bcl-X1 expression, and raise Bax and Bak expression in HeLa cells, triggering apoptosis.34 On the other hand, polysaccharides are known to induce necrosis.20,103 Necrosis is accidental cell death without a precise mechanism, leading to the break-down of the cell membrane and release of intracellular compounds into surrounding tissue.104 TNF is a multifunctional cytokine which is a protein produced by many cell types such as monocytes, macrophages, T cells, and B cells with appropriate stimulation. The human TNF protein is expressed as a 26 kDa (233 amino acid long) integral transmembrane precursor protein. A 17 kDa (157 amino acids) mature TNF protein can be released from the precursor protein into the medium by proteolytic cleavage, possibly involving a serine protease.105 TNF is not only cytotoxic or cytostatic to some tumor cell lines in vitro106 but also destroys actively proliferating endothelial cells in primary culture.107 The hot water extract from Polyporus rhinocerus has been proved to increase TNF-a production.103 Macrophages defend the host by playing critical roles, consisting of phagocytosis of pathogens and apoptotic cells, production of cytokines, and proteolytic processing and presentation of foreign antigens. Macrophages can be stimulated by polysaccharides to release a broad spectrum of cytokines like interleukins, TNF-a, and nitric oxide (NO), which are referred to as the inhibitory factors of cancer.22 Two fractions of polysaccharides puried from Ganoderma lucidum were reported to have a proliferative effect on macrophages up to 160% of the control cells.22 NO has been studied in the last few years and is recognized as a crucial messenger that indicates diverse pathophysiological functions, such as neuronal transmission, vascular relaxation, immune modulation, and cytotoxicity against tumor cells.108 NO has been proved to be a main effector molecule destructing tumor cells by activated macrophages.109 Basically, macrophages that are stimulated by TNF-a to generate NO through the expression of the iNOS gene. Moreover, the induction of NO and TNF-a production and gene expression by activated macrophages can have a cytotoxic impact on malignant cells.110 The toxic effects of NO and its derivatives on target cells are based on several mechanisms, which include (i) inactivating ironsulfur cluster-containing enzymes through loss of iron from cells; (ii) inhibiting DNA-binding activity of zinc nger-type transcriptional factors by the induction of the release of zinc from zinccontaining proteins; and (iii) destructing the mitochondrial membrane potential by affecting the activity of ion channels.111 It was found that NO and TNF-a elicited by acidic polysaccharides isolated from Phellinus linteus may contribute in vivo to its immunomodulatory and anti-tumoricidal activities.20 The antitumor activity of an anionic sulfated polysaccharide was performed by binding to positively charged DNA-binding locus of enzymes via electrostatic interaction on the cell surface of human cancer cells, inhibiting its binding to DNA.112 Likewise, carboxymethylated polysaccharides are proposed to probably bind non-specically to DNA-interacting enzymes. Despite that non-specic binding by individual carboxymethylated groups might be weaker than the specic binding by DNA. The large polysaccharidic molecules might be able to cover the locus
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of the enzymes due to the multivalent nature of carboxymethylated polysaccharides, blocking their reaction with the DNA molecules.28 In addition, recent studies have reported that anti-angiogenesis might be one of the important mechanisms of antitumor activity. Angiogenesis is based on several aspects that the endothelial cells must proliferate to offer the necessary number of cells for the growing vessels, and the cells are able to migrate.23 Angiogenesis can be tightly controlled by a balance of endogenous inducers and inhibitors of angiogenesis. Primary tumors cannot grow greater than 23 mm without eliciting neovascularisation. In the initial stage of tumor progression, an imbalance of angiogenesis regulators takes place, which favours an angiogenic environment. This angiogenic change leads to the oversecretion of angiogenesis inducers, such as vascular endothelial growth factor (VEGF), and the subsequent neovascularisation and growth of the tumor.113 Several endogenous inhibitors of angiogenesis, such as angiostatin, endostatin, interferons, thrombospondin-1 (TSP-1), tissue inhibitor of metalloproteinases (TIMP), and tumstatin, have been identied. The understanding of the basic science of angiogenesis involving those inducers and inhibitors has resulted in the development of anti-angiogenic therapies.113 Ganoderma lucidum has been found to suppress capillary morphogenesis of aortic endothelial cells. This is affected by the mediation through the inhibition of secretion of angiogenic factors like VEGF and transforming growth factor-b 1 (TGF-b 1) from prostate cancer cells PC-3. G. lucidum inhibits functions of kinases Erk1/2 and Akt resulting in the inhibiton of AP-1, which causes the down-regulation of expression of VEGF and TGF-b 1.24 Similarly, Cao and Lin23 reported that G. lucidum polysaccharide peptides exerted the inhibitory actions not only on vascular cell proliferation, but also on the secretion of VEGF by human lung carcinoma cells in hypoxia. Most of the reported antitumor mechanisms of polysaccharides were raised from the cytological studies, such as cell cycle arrest, apoptosis, necrosis, and stimulation of immune responses. However, each mechanism does not occur solely in a one-way lane, which can connect with other mechanisms simultaneously in a complicated matrix. For example, in the theory of necrosis, the functioning cytokine TNF can be produced by numerous cell types like monocytes, macrophages, T cells, and B cells under appropriate stimulation. This suggests that the death of tumor cells during necrosis can be triggered by different passages together at the same time, if the polysaccharides are able to stimulate a number of those types of cells simultaneously. Polysaccharides derived from mushrooms can perform their antitumor activities under different mechanisms. A good example is the extracts from Ganoderma lucidum, which have been reported to demonstrate antitumor abilities by stimulation of macrophages22 or by anti-angiogenesis.23 Note however, that as these two studies were carried out separately, it is not known if the effective compounds are identical or not. Anti-angiogenesis can be regarded as a different approach, even though it can still be placed in the biological scope. However, except in the case of nitric oxide, which is produced through the stimulation of the cell by mushroom polysaccharides, the exact mechanisms related to structural chemistry and chemical interactions have not been fully researched. More
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studies are needed to develop theories clarifying why and how the specic conformations of polysaccharides are related to their antitumor properties. 5.4. Methods used to quantify antitumor activity of mushroom polysaccharides Quantitative assessments of antitumor activity that relies on analyzing the changes of entities of cells cultured in different conditions can be divided into two groups, as in vitro and in vivo analyses. The in vitro analysis is mainly performed by modern colorimetric cell-based proliferation or toxicity assays using compounds that stain the cells directly or that are metabolized into coloured products,114 followed by quantication approaches including spectrophotometric and uorimetric techniques to numerate appropriately labelled cells.115 Technically, cancer cells are cultured in asks with medium and antibiotics. Once their growth reaches the desired density they are harvested. To determine the total amount of the cells, an aliquot is taken and stained, and then counted by a hemocytometer. For the antitumor activity test, the cells at a certain concentration are seeded in multiwall plates and cultured overnight. The following day, the cells are drugged with cytotoxic compounds such as polysaccharides or anticancer medicines and further cultured for 25 culture doubling times. At the end of incubation, the cells are treated with color reagents which are indicator dyes. These color reagents include 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium 30 -[1-(phenylaminocarbonyl)3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 4-[3(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), 7-hydroxy-3H-phenoxazin-3-one 10-oxide (alamarBlue), and sulforhodamine B [2-(3-diethylamino-6diethylazaniumylidene-xanthen-9-yl)-5-sulfo-benzenesulfonate] (SRB). Examples of the application of these indicator dyes are given below. The last step of the in vitro method is the quantication of cells in the control and drugged cells using colorimetric devices such as plate readers. The inhibition rate is calculated according to the formula116 below: inhibition rate % 1 absorbance of sample 100 (1) absorbance of control

Using similar cell culture procedures as stated above, many standard assays have been developed to measure the cell proliferation or the cell viability, which are normally termed according to the names of the indicator dye used in the detection stage, e.g., MTT assay, XTT assay, and so on. Most of the indicator dyes work on the principle that they can be metabolized by living cells. Mitochondrial reduction of dyes has been developed as an assessment of lymphocyte growth. Metabolism of tetrazolium salts, such as MTT, XTT, MTS and WST-1, produce the basis of colorimetric assays.117 MTT has been most widely used, as it can be cleaved by functional mitochondria to produce formazan in viable cells,118 resulting in measurable colour changes in the culture. Nonetheless, it is impossible to follow-up cell cultures as the MTT determination necessitates destruction of the cells.115 WST-1 has a similar working principle to MTT, through the reaction with the mitochondrial succinate-tetrazolium reductase
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to generate the formazan dye. The WST-1 reagent forms a water-soluble formazan rather than the water-insoluble product formed by MTT.119 This makes the WST-1 assay a convenient and common tetrazolium salt technique in microplate format. In contrast, the alamarBlue assay involves a colorimetric and uorometric growth indicator that can be used to detect the metabolic activity of cells.120 Basically, the native, oxidized form of resazurin can be taken up readily by viable cells, which is reduced intracellularly by oxidoreductases and the mitochondrial electron transport chain.121 The system incorporates an oxidationreduction (REDOX) indicator that leads to a corresponding shift in its absorbance and uorescence.122 The alamarBlue assay has been considered superior to classical tests, such as the MTT test, due to its advantages of high stability, nontoxicity to the cells, and the possibility of continuous monitoring of cultures over time.123,124 Another major technique for measuring cytotoxicity is the SRB protein staining assay determining the cellular protein content of adherent and suspension cultures, which is adopted for routine use in the U.S. National Cancer Institute in vitro anticancer screen.125 This assay depends on binding of the dye to basic amino acids of cellular proteins. Its colorimetric evaluation offers an estimate of total protein mass, which is directly proportional to the cell mass.126 SRB staining is independent of cell metabolic activity, thus cannot distinguish between viable and dead cells.127 MTT and SRB assays have been successfully used to test the antitumor activity of polysaccharides derived from mushrooms. For instance, MTT assays were performed to determine the cytotoxicity of the polysaccharides isolated from Cordyceps militaris on B16-F10 melanoma cells.16 Similarly, the anticancer ability of polysaccharides from Phellinus linteus against B16-F10 cells was evaluated utilizing a SRB assay.19 However, the application of WST-1 and alamarBlue assays to investigate the cytotoxic effect of mushroom polysaccharides on cancer cells has rarely been reported. Despite the MTT assay being dominantly used on this kind of study in the past, more recent techniques, namely WST-1 and alamarBlue have a potential to replace the MTT assay. Three cytotoxicity testing methods, including SRB, WST-1 and alamarBlue assays, have been employed in our research for screening mushroom polysaccharides that can present antitumor activities. Overall, the SRB assay does not demand time-sensitive measurements and possesses a practical advantage for large-scale screening compared to WST-1 and alamarBlue assays. On the other hand, both WST-1 and alamarBlue assays are faster, easier, and are less technique-sensitive than the SRB assay, which involves multiple manual washing and drying steps. More importantly, plates in WST-1 and alamarBlue tests can be read and returned several times to the incubator for further color development. The in vivo methods used to study the antitumor activity are conducted on animals, such as mice,128 dogs,129 and pigs.130 The commonly used procedures involve implanting tumor cells into animals, then administrating animals with anticancer compounds such as polysaccharides for a period of time, and detecting tumor changes compared to the control animals. The inhibition ratio is calculated by the formula128 below:
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inhibition ratio %

  AB 100 A

(2)

where A and B were the mean tumor weights of the negative control and treated groups respectively. In addition, the antitumor activity can be estimated by calculating tumor volume (TV), which is based on the tumor size measured with a calliper, using:34 TV LW L W 0:5236 2 (3)

where L and W are the maximum diameter and the minimum diameter of the tumor, respectively. The in vivo analysis has been applied to determine the antitumor activity of polysaccharides derived from mushrooms. For example, Ding et al.128 successfully studied the antitumor activity of a novel polysaccharide isolated from the Lactarius deliciosus mushroom against S180 tumors in mice using the in vitro procedures described above.

6. Conclusions
Mushrooms have been considered and consumed as a delicacy for millenniums. Historic practices and scientic studies have also highlighted that mushrooms are a bunch of highly recommended dietary supplements due to their evidently nutritional values. Polysaccharides found in mushrooms demonstrate a limitless structural diversity that provides the largest capacity and potential for creating biological functions. The structural variability makes the precise regulatory mechanisms of cellcell interactions exible in higher organisms. These features have been successfully exhibited in an excellent example of b-glucans which act to recover the impaired immune systems of humans and particularly against cancer and infectious diseases. The antitumor abilities of polysaccharides from mushrooms have been proven to work by activating different immune responses in the host. The research data shows that the antitumor action of polysaccharides is dependent on their capabilities to bind to cell receptors such as dectin-1, CR3, LacCer, and scavenger receptors, resulting in boosting of immune responses in affected cells by activating multiple signal pathways. Although several preliminary antitumor mechanisms have been reported, such as cell cycle arrest, induction of tumor cell death by apoptosis and secondary necrosis, stimulation of macrophages, and antiangiogenesis, more scientic insight is needed to build upon the theories. In particularly, the structure and function relationship is not fully understood. The biochemical afnities and passages behind those reactions and functions are still unclear. Therefore, further scientic studies are required to clarify the mechanisms and characterize the responsible structural parameters by utilizing chemical routes and biological molecular techniques. After the map of structural features corresponding to specic bioactive functions is elucidated by purication and screening of polysaccharides, the predicted properties can be designed for the synthesis of signicantly pharmaceutical polysaccharides.

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