Antitumor activity of mushroom polysaccharides: a review

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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 fiber, minerals, and vitamins (in particularly,

vitamin D). Mushrooms are also recognized as functional foods for their bioactive compounds offer

huge beneficial 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

(polysaccharide–peptide complex) and PSK (polysaccharide–protein 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

affinities toward these receptors to trigger different host responses. Basically, their antitumor abilities

are influenced by the molecular mass, branching configuration, conformation, and chemical

modification 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 (408–438 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 mush-

rooms for over two thousand years due to their pleasant flavor

and texture. Traditional knowledge defines mushrooms as fleshy,

aerial umbrella-shaped, fruiting bodies of macrofungi.3 In the

literature, mushrooms are acceptably defined 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 confirmed by extensive studies. Zhang

et al.3 stated that in 1957, Lucas discovered the bioactivity of

School of Chemical Sciences, The University of Auckland, Auckland, NewZealand. E-mail: [email protected]

1118 | Food Funct., 2012, 3, 1118–1130

Basidiomycetes mushrooms for the first time by isolating a

substance from Boletus edulis which demonstrated a significant

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 polysaccharide–protein complexes, have been

proven to possess effective functions such as: immunomodula-

tory, anticancer,5 anti-inflammatory,6 and antioxidant7,8 effects,

along with lowering blood cholesterol levels effects.9 In partic-

ular, the commercialization of several polysaccharides and

polysaccharide conjugates has made patients benefit from such

anticancer therapies. They are schizophyllan, lentinan, grifolan,

PSP (polysaccharide–peptide complex) and krestin (poly-

saccharide–protein 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 under-

ground mycelia (hyphae) by the process of fructification. The

This journal is ª The Royal Society of Chemistry 2012

http://dx.doi.org/10.1039/c2fo10279j






http://pubs.rsc.org/en/journals/journal/FO

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

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bulk of fruiting bodies have a short lifetime of only about 10–14

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 puff-

ball) (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 benefits

from each other are called mutualistic symbiotic fungi.2 Mycelia

of the ectomycorrhizal species grows within the roots of plants,

Fig. 2 Photographs of mushrooms found in New Zealand.


such as trees. The terrestrial saprobic species snatch nutrients

mainly from organic compounds of the plant and animal

debris.10

3. Structural properties of polysaccharidesexhibiting 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-configuration

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 hetero-

linkages with respect to configuration 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

Table 1 Examples of homopolysaccharides (adapted from Izydorczyk11)

Polysaccharides Repeating unit: glycosidic linkage type/glycose unit

LinearAmylose a-(1/4)-GlcCellulose b-(1/4)-GlcXylan b-(1/4)-XylInulin b-(2/1)-FruLevan b-(2/6)-FruLaminaran b-(1/3)-GlcChitin b-(1/4)-GlcNAcb-Glucan b-(1/4, 1/3)-GlcBranchedAmylopectin a-(1/4, 1/6)-GlcDextran a-(1/2, 1/3, 1/4, 1/6)-GlcLevan b-(2/1, 2/6)-FruPullulan a-(1/6)-MaltotrioseScleroglucan b-(1/3, 1/6)-GlcGlycogen a-(1/4, 1/6)-Glc

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characterized by irregular sequences of monosaccharide units,

linkage positions, and configurations.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 chain’s 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 mono-

saccharide 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 modification can be performed via ester-

ification and/or etherification of chains. These modifications, 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 charac-

teristics of the polysaccharide are heterogeneous. For instance,

the most conservative is the configuration 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 cell–cell interac-

tions 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 filtrates of

basidiomycetes. These polysaccharides showing antitumor

activity have a great variety of chemical composition, structure

and antitumor activity.15 Since Lucas3 first reported the poly-

saccharides extracted from mushrooms indicating antitumor

activity in 1957, a great deal of polysaccharides that show anti-

tumor activity have been isolated from mushrooms and their

1120 | Food Funct., 2012, 3, 1118–1130

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)-b-

glucans and (1/3)-a-glucans. Also, there are some hetero-

glycans 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 demon-

strating 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. Anti-

tumor 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 mush-

room species, polysaccharides binding with proteins or peptides

as a polysaccharide–protein or polysaccharide–peptide 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 posi-

tions. 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 classified 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 poly-

saccharide nature, namely lentinan, schizophyllan and protein-

bound 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 poly-

saccharopeptide (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 five (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

400–800 � 103 Da.15,42

Schizophyllan is also a (1/3)-b-glucan containing a b-glu-

copyranosyl 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



Table 2 Reported polysaccharides showing antitumor activity from mushrooms

Mushroom species Polysaccharide source Antitumor type Reference

Cordyceps militaris Fruiting body Melanoma 16Lung cancer 17

Phellinus gilvus Fruiting body Lung cancer 18Phellinus linteus Mycelial culture Melanoma 19 and 20Pleurotus ostreatus Fruiting body Melanoma 21Ganoderma lucidum Fruiting body Breast cancer 22

Lung cancer 23Prostate cancer 24Cervical cancer 25

Lentinula edodes Fruiting body Breast cancer 26Pleurotus geesteranus Fruiting body Breast cancer 27Pleurotus tuber-regium Sclerotia Breast cancer 28Clitocybe alexandri Fruiting body Lung cancer 29Lepista inversa Fruiting body Lung cancer 29Sparassis crispa Fruiting body Lung cancer 30Agaricus blazei Fruiting body Prostate cancer 31Grifola frondosa Fruiting body Prostate cancer 32Trametes versicolor Fruiting body Prostate cancer 33Angelica sinensis Mycelia Cervical cancer 34

Table 3 Antitumor activities of extracts of mushroom species (againstsarcoma-180 in mice)

Mushroom speciesTumor inhibition(%) Reference

AgaricaceaeAgaricus bisporus 2 35AuriculariaceaeAuricularia auricula-judae 42 35CorticiaceaeLaetisaria arvalis 95 36PleurotaceaePleurotus tuber-regium: carboxymethylated hot alkali extracts (CMHZE)CMHZE-1 64 37CMHZE-2 48 37CMHZE-3 75 37CMHZE-4 53 37CMHZE-5 46 37CMHZE-6 43 37PolyporaceaeGanoderma tsugae 77 35Coriolus versicolor 77 35Trametes gibbosa 49 35Fomes fomentarios 5 35RussulaceaeRussula lepida 67.6 38StrophariaceaePholiota nameko 86 35TricholomataceaeLentinus edodes 80 35Flammulina velutipes 81 35Pleurotus ostreatus 75 35Tricholoma matsutake 91 35

Fig. 3 Structure units of polysaccharides showing antitumor activity:

lentinan (A), schizophyllan (B).

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PSK (Krestin) is a b-glucan–protein 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 galac-

tose. PSK has a (1/4)-b-glucan with (1/6)-b-glucopyranosidic


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 versi-

color in China has a similar glucan structure to PSK in Japan.44

A polysaccharide–protein complex (PSPC), that was extracted

from the culture filtrates of Tricholoma lobayense, consists of

54.3% polysaccharides containing galactose, glucose, arabinose,

xylose, rhamnose, fucose and mannose, and 35.9% protein con-

taining majorly aspartic acid, glutamic acid, serine, glycine,

lysine and threonine. PSPC shows the characteristics of a poly-

saccharide 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 classified as a Ganoderma species which are the most

Food Funct., 2012, 3, 1118–1130 | 1121


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well known medicinal fungi in the Orient. It is an immuno-

modulatory 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 Gano-

derma applanatum comprise b-glucan, heteroglycans and glycan-

protein 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-gluco-

pyranan with 1–15 (1/6)-b-monoglucosyl side chains.47 Seven

potent antitumor polysaccharide–protein complexes have been

extracted from Ganoderma tsugae. Two of them were protein-

containing glucogalactans related to mannose and fucose. And

five are protein-containing (1/3)-b-glucans.15

4. Connection between structure and antitumoractivities of mushroom polysaccharides

Polysaccharides possess a huge variety of chemical compositions

and configurations and physical properties. The antitumor

activity of the polysaccharides can be influenced 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 anti-

tumor activity. In the study based on seven potent antitumor

polysaccharide–protein complexes from Ganoderma tsugae, it

was found that polysaccharides showing antitumor activity with

high activity derived from fruiting bodies were mainly hetero-

polysaccharides 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 poly-

saccharides. Polysaccharides that show antitumor activity may

contain other chemical structures, such as hetero-b-glucans,40

heteroglycan,50 b-glucan–protein,51 a-manno-b-glucan,40

a-glucan–protein40 and heteroglycan–protein complexes.52 For

instance, PSK and PSP have a b-glucan–protein, while PSPC

isolated from the Tricholoma species are a heteroglycan–protein

complex.

4.1. The influence of molecular mass

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

1122 | Food Funct., 2012, 3, 1118–1130

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 schizo-

phyllan, present the same antitumor activity against Sarcoma

180 as those with higher molecular weights. The divergent results

remain to be clarified.15

4.2. The effect of the branching configuration

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 five 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

five 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 kg�1 for five days in mice.15

4.3. The impact of conformation

The conformations of polysaccharides demonstrating antitumor

activity include single helix, triple helix and random coil. Len-

tinan, schizophyllan and PSK all consist of a triple helix struc-

ture.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 dena-

tured 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-1,3-

D-glucan containing a single helix conformer, which is not bio-

logically active against tumor growth. However, when it becomes

pachymaran by periodate oxidation and mild hydrolysis, the

newly formed conformer shows pronounced antitumor activity.55

Schizophyllan–OH 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 immuno-

pharmacological activities demonstrated by mushroom

b-(1/3)-glucan, such as macrophage nitrogen oxide synthesis

and limulus factor G activation, are determined by the triple-

helix conformation, whereas others are not dependent on this

conformation, e.g., synthesis of interferon-g and colony stimu-

lating 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 polysaccharide–protein complexes

implies that the existence of biological systems within the

host body recognize the configurational structure of

polysaccharide.15

4.4. The effect of solubility

The solubility of b-glucans is affected by their degree of poly-

merization and thus their physical organization.57 When the

alkali-insoluble, branched (1/3)-b-D-glucan, isolated from

Auricularia auricular-judae, was modified by controlled periodate

oxidation, borohydride reduction, and mild acid hydrolysis, its

water solubility was increased by having covalently linked

D-glucosyl residues, which demonstrated significantly potent

antitumor activity, whereas in the native state it had no such

activity.58

4.5. Enhancement of antitumor activity by chemical

modification

The improvement of the biological activity of polysaccharides

that show antitumor activity can be achieved by chemical

modification. Various carboxymethylated (CM), hydroxylated,

formylmethylated, aminethylated and sulfated products have

been designed.

The successful schemes for chemical improvement of mush-

room polysaccharides have been designed for Ganoderma luci-

dum, Grifola frondosa and Leucopaxillus giganteus. Two main

procedures are involved in these schemes: modification of

mushroom polysaccharides by Smith degradation (oxydo-

reducto-hydrolysis) and activation by the method of for-

molysis.49 During the Smith degradation modification, original

polysaccharide solutions are first oxidized to polyaldehydes by

0.1 M NaIO4 in darkness. They are then are reduced into poly-

alcohols by NaBH4 in an alkaline medium adjusted to pH 8 with

2 M NaOH, and hydrolyzed by 1 M H2SO4 at room tempera-

ture.59 Chemical activation of the mushroom polysaccharides by

means of formolysis consists of degradation of the poly-

saccharides 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 significant activity. Furthermore, polyaldehyde, and

polyol–polysaccharides 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 MNaOH

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 0–6 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 modification, the carboxymethylated


linear (1/3)-a-glucans indicate strong antitumor activity

against Sarcoma 180 and immunomodulating activity in mice.15

Chemical modification of branched mushroom poly-

saccharides 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-1,3-

D-glucan showing more effective antitumor activity.15 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 immuno-

modulatory and anticancer properties.62

The antitumor activity of the formylmethylated and amino-

ethylated derivatives of schizophyllan against Sarcoma 180 solid

tumor in mice is largely augmented compared with the native

schizophyllan.63 The sulfated lentinan and schizophyllan prod-

ucts show potent anti-human immunodeficiency 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

modifications.15

5. Immunomodulating activities and mechanisms ofantitumor activity by mushroom polysaccharides

5.1. Cancer

A neoplasm is defined 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 trans-

formation 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-sufficiency in growth signals, insensitivity to growth-

inhibitory 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

inflammation 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 sufficient 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 metasta-

size. 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

modifiers.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 nonspecific action on the body,

supporting some or all of the major systems, such as nervous,

hormonal, and immune systems, as well as regulatory func-

tions.67 Substances that are capable of interacting with the

immune system can either upregulate or downregulate specific

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 adminis-

tration 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, nonspecifically

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 recog-

nized 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 orches-

trated interplay of innate and acquired immune responses

(Fig. 4). Innate immunity, containing macrophages, neutrophils,

natural killer cells (NKs) and dendritic cells (DCs) as gate-

keepers, is regulated by chemical-messengers or cytokines and by

activating inflammatory and acute phase responses.42 The

mononuclear phagocyte system (e.g., macrophages and mono-

cytes), DCs and certain lymphocytes (e.g., NK cells) exact

numerous important functions involving the recognition and

destruction of abnormal cells. Specific immunity to abnormal

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

1124 | Food Funct., 2012, 3, 1118–1130

cells or tissues contains humoral (e.g., generates antibodies) and

cell-mediated immunity (also enhances inflammatory responses

and ultimately kills infected or abnormal cells). Therefore, an

adequately functional immune response is critical to the recog-

nition 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 virus-

infected 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 identified in

humans, such as dectin-1, complement receptor 3 (CR3), scav-

enger 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 inflamma-

tory cytokines, have been identified. One pathway is that dectin-1

works synergistically with TLR to generate strong inflammatory

responses by stimulating cytokines such as tumor necrosis factor-

alpha (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 inflammatory 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 cyto-

toxicity is blocked by an anti-CR3 antibody.83



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Scavenger receptors that are located in myeloid and endothe-

lial 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, low-

density 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 sufficient

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 confirmed this. For example, the anti-

tumor effect of polysaccharides is lost in neonatal thymectom-

ized mice, or is decreased significantly 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 specific 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, differenti-

ation, and proliferation of immunocompetent cells for host

defence mechanisms.42 Lentinan is also able to restore the sup-

pressed 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 infiltration 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 anti-

proliferation 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


elucidates the in vitro anti-proliferative effect of poly-

saccharides.91 A polysaccharide–peptide complex (PSP) isolated

from Trametes versicolar was reported to significantly decrease

proliferation of MAD-MB-231 breast cancer cells.92 These

results suggest that mushroom polysaccharides not only stimu-

late 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 stim-

ulation 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

ethanol–water 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 pro-

apoptotic 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 nuclear-

encoded 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 mitochon-

drial membranes, either by forming channels,101 by interacting

with components of the permeability transition pore,101 or by

altering fission 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 trans-

membrane 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, con-

sisting of phagocytosis of pathogens and apoptotic cells,

production of cytokines, and proteolytic processing and presen-

tation 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 poly-

saccharides purified 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 patho-

physiological 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 iNOSgene.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 ofNOand its derivatives on target cells are based

on several mechanisms, which include (i) inactivating iron–sulfur

cluster-containing enzymes through loss of iron from cells; (ii)

inhibiting DNA-binding activity of zinc finger-type transcrip-

tional factors by the induction of the release of zinc from zinc-

containing 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 Like-

wise, carboxymethylated polysaccharides are proposed to prob-

ably bind non-specifically to DNA-interacting enzymes. Despite

that non-specific binding by individual carboxymethylated

groups might be weaker than the specific binding by DNA. The

large polysaccharidic molecules might be able to cover the locus

1126 | Food Funct., 2012, 3, 1118–1130

of the enzymes due to the multivalent nature of carboxymethy-

lated polysaccharides, blocking their reaction with the DNA

molecules.28

In addition, recent studies have reported that anti-angiogen-

esis might be one of the important mechanisms of antitumor

activity. Angiogenesis is based on several aspects that the endo-

thelial 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 endoge-

nous inducers and inhibitors of angiogenesis. Primary tumors

cannot grow greater than 2–3 mm without eliciting neo-

vascularisation. 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 neo-

vascularisation and growth of the tumor.113 Several endogenous

inhibitors of angiogenesis, such as angiostatin, endostatin,

interferons, thrombospondin-1 (TSP-1), tissue inhibitor of met-

alloproteinases (TIMP), and tumstatin, have been identified. 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 poly-

saccharides 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 simul-

taneously 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 poly-

saccharides, 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

specific 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 quantification approaches

including spectrophotometric and fluorimetric techniques to

numerate appropriately labelled cells.115 Technically, cancer cells

are cultured in flasks 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 anti-

tumor 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 poly-

saccharides or anticancer medicines and further cultured for 2–5

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 tetra-

zolium 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-carboxy-

methoxyphenyl)-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-6-

diethylazaniumylidene-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 quantifi-

cation 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 ð%Þ ¼ 1� absorbance of sample

absorbance of control� 100 (1)

Using similar cell culture procedures as stated above, many

standard assays have been developed to measure the cell prolif-

eration 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 andWST-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


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

fluorometric 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 mitochon-

drial electron transport chain.121 The system incorporates

an oxidation–reduction (REDOX) indicator that leads to a

corresponding shift in its absorbance and fluorescence.122 The

alamarBlue assay has been considered superior to classical tests,

such as theMTT test, due to its advantages of high stability, non-

toxicity 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 appli-

cation of WST-1 and alamarBlue assays to investigate the cyto-

toxic 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:

Food Funct., 2012, 3, 1118–1130 | 1127


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inhibition ratio ð%Þ ¼�A� B

A

�� 100 (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 ¼ LþW

2� �

L�W�� 0:5236 (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 anti-

tumor 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 scientific 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 vari-

ability makes the precise regulatory mechanisms of cell–cell

interactions flexible 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 recep-

tors, 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 anti-

angiogenesis, more scientific insight is needed to build upon the

theories. In particularly, the structure and function relationship

is not fully understood. The biochemical affinities and passages

behind those reactions and functions are still unclear.

Therefore, further scientific studies are required to clarify the

mechanisms andcharacterize the responsible structural parameters

by utilizing chemical routes and biological molecular techniques.

After the map of structural features corresponding to specific

bioactive functions is elucidated by purification and screening of

polysaccharides, the predicted properties can be designed for the

synthesis of significantly pharmaceutical polysaccharides.

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Antitumor activity of mushroom polysaccharides: a review