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Critical Reviews in Biotechnology, 25:205230, 2005
Copyright
c Taylor & Francis Inc.
ISSN: 0738-8551 print / 1549-7801 online
DOI: 10.1080/07388550500376166
Natural and Modified (13)--D-Glucans
in Health Promotion and Disease Alleviation
Djordje B. Zekovic and
Stefan Kwiatkowski
Alltechs North American
Bioscience Center,
Nicholasville, KY, USA
Miroslav M. Vrvic
Department of Chemistry,
Institute of Chemistry,
Technology and Metallurgy,
Belgrade, Njegoseva 12 and
Faculty of Chemistry,
Studentski trg 16,
University of Belgrade,
Belgrade, Serbia and
Montenegro
Dragica Jakovljevic
Department of Chemistry,
Institute of Chemistry,
Technology and Metallurgy,
Belgrade, Njegoseva 12
Colm A. Moran
Alltechs North American
Bioscience Center,
Nicholasville, KY, USA
Address correspondence to Dj. B.
Zekovic, Alltechs North American
Bioscience Center, 3031 Catnip Hill
Pike, Nicholasville, KY 40356, USA.
E-mail: dzekovic@alltech.com
ABSTRACT A number of polysaccharides with -glycosidic linkage are
widespread in nature in a variety of sources. All have a common structure
and the (13)--D-glucan backbone is essential. They have attracted attention
over the years because of their bioactive and medicinal properties. In many
cases their functional role is a mystery, in others it is well established. Because
of their insoluble chemical nature, particulate (13)--D-glucans are not suit-
able for many medical applications. Various methods of changing or modifying
the -D-glucan chemical structure and transforming it to a soluble form have
been published. The -D-glucan bioactive properties can be affected positively
or negatively by such modifications. This review examines -glucan sources in
nature, health effects and structure-activity relationships. It presents the current
state of -D-glucan solubilization methods and discusses their effectiveness and
application possibilities for the future.
KEYWORDS glucan, soluble, derivative, solubilization, application, Saccharomyces.
1. INTRODUCTION
1.1. Overview
Glucans are natural polysaccharides comprised of D-glucopyranosyl units
and can be found in a wide variety of cereal, plant, algae, bacteria, fungi and
yeast sources. The macromolecular structure of the glucan depends on both the
source and method of isolation. These glucans consist of linear (13)--linked
backbones with either (16) or (14)-linked side chains of varying length and
distribution, which form complex tertiary structures stabilized by interchain
hydrogen bonds. The natural (13)--D-glucan characterizing parameters in-
clude: primary structure, solubility, degree of branching (DB), and molecular
weight (MW), as well as the polymer charge and/or solution conformation (triple
helix, single helix or random coil conformation). All these factors play a role in
glucan-associated biological activity.
This heterogeneous group of -linked polyglucoses is attracting the increasing
attention of the pharmaceutical and functional food industry because of its posi-
tive effects on human and animal health. Examples include bioactive and medic-
inal properties, such as immune-stimulation, anti-inflammatory, antimicro-
bial, antitumoral, hepatoprotective, cholesterol-lowering as well as antifibrotic,
205
antidiabetic and hypoglycemic activity.21,105,169,193,202
However, particulate (13)--D-glucans have been
found to be unsuitable for many human medical appli-
cations and responsible for significant adverse effects
such as granuloma formation, microembolization, in-
flammation, and pain. In order to improve (13)--
D-glucan solubility, several derivatization procedures
such as sulfation, phosphation, and carboxymethyla-
tion have been applied. In addition, many different
methods including acid and alkaline hydrolysis, enzy-
matic digestion, and ultrasound irradiation have been
applied to depolymerize the insoluble macromolecular
structure to produce underivatized soluble (13)--D-
glucans. Chemical modifications of (13)--D-glucan
can strongly influence biological activity and this review
will discuss past research as well as recent advances in
this area.
It was not our goal to review all of the literature re-
sources treating -D-glucans, separation, modification
and medicinal application, but rather to focus on the
specific areas identified above. It was also an arbitrary
decision not to include information from patents as the
data was too often not standardized for making compar-
isons on health effects. Excellent reviews on the wider
subject of -D-glucans are available.16,17,2022,101
1.2. Sources and Chemical Structure
of -Glucans Found in Nature
1.2.1. Prokaryotic Organisms
Prokaryotic organisms such as bacteria and blue
green algae produce a variety of polysaccharides as
components of capsules and other extracellular prod-
ucts. These polysaccharides include (13)--glucans,
(12)--glucans and cellulose. For example, two capsu-
lar (13)--glucans from Streptococcus pneumoniae con-
tribute to the antigenic specificity and virulence of the
parent organism.67,68,86,94,116
The known occurrences of (13)--glucans from
fermentation are limited to species of soil bacteria be-
longing to the genera Alcaligenes (Achromobacteriaceae),
Agrobacterium, and Rhizobium (Rhizobacteriaceae). The
curdlan, produced by fermentation of Agrobacterium
biovar 1 (identified as Alkaligenes faecalis var myxogenes
at the time it was discovered), is a non-ionic gel-forming
polysaccharide first described by Harada et al.65 and
given the name curdlan due to its ability to curdle
when heated. This (13)--D-glucan was approved as
D. B. Zekovic et al.
a food additive by the U.S. Food and Drug Adminis-
tration (FDA) in 1996.54 Due to its unique properties it
has been the subject of considerable investigation.166
Curdlan consists of (13)--linked glucose residues
and has the unusual property of forming an elastic gel
when the aqueous suspension is heated. The gels are
very stable over a wide pH range and under severe pro-
cessing conditions. The unique heat gelling and water
binding properties of curdlan are of great interest to the
food and pharmaceutical industries.87
1.2.2. Algae
The major storage polysaccharides in the eu-
glenoids (paramylon), brown algae (laminarin), diatoms
(chrysolaminarin), and chrysophites (leucosin, chryso-
laminarin) are glucans containing (13)--glucosidic
linkages.
Laminaran is a carbohydrate extract, roughly equiv-
alent to starch found in plants, obtained from brown
algae (Laminaria species). These laminarans (also called
laminarins) are a class of low-molecular-weight stor-
age -glucans consisting of (13)--D-glucopyranose
chains in which some 6-O-branches are present. The
structure of the laminarans extracted from the kelps, as
a by-product from production of alginic acid, has been
well investigated. The majority of laminarans contain
polymeric chains of two types: one type is built only
of Glcp residues (G-chains), whereas the other type is
terminated with 1-O-substituted D-mannitol residues
(M-chains).127,158,192 There is considerable variation in
the composition of laminarans extracted from different
species, for example, in the M:G ratio (in some cases M-
chains are absent), in the degree of branching (DB) and
degree of polymerization (DP) (up to 50 carbohydrate
residues, usually ca. 25), and in the ratio of (13)--
and (16)--glycosidic bonds. Several laminarans have
been shown to have structures quite distinct from those
originally suggested for the commercially available
preparations from Laminaria sp.127,192,207 Some lami-
naran derivatives have been shown to have interesting
biological activities. For example, a fluorescent-tagged
heptasaccharide prepared from Eisenia bicyclis laminaran
has been demonstrated to have elicitor activity,100 and
a glucan with immunostimulating activity was obtained
by transglycosylation of laminaran from Laminaria ci-
chorioides.245 Laminarans were very important in the
development of classical chemical methods of polysac-
charide structural analysis.158
206
 Although (13)--xylans and (13),(14)--
xylans are major wall components of green and red
algae, the (13)--glucans are reported to be only mi-
nor wall components of a few species of green algae. Of
the three functional groups of algal polysaccharides (cy-
toplasmic storage materials, fibrillar wall components
and the wall matrix and related mucilages), both the
storage and wall polysaccharides are (13)--glucan in
nature.192
1.2.3. Fungi, Yeast and Lichen
The (13, 16)--glucans are important surface
components of fungi due to their involvement in mor-
phogenetic changes as well as in establishing pathogenic
and symbiotic relationships with both animals and
higher plants.8,56,69,102,108,147,206 They play the role of
structural frame for the fungal cell and define its shape
and rigidity. Some (13, 16)--glucans can also
serve as fungal storage carbohydrates, especially in the
fungi Mastigomycotina (Oomycetes) and Basidomy-
cotina. As major cell wall components, these glucans
and the appropriate (13)--glucan hydrolases and
synthases, are involved in the cell wall modifications,
which occur during growth and morphogenesis. A num-
ber of biologically active glucans have been isolated, for
research purposes, from a variety of fungi (Table 1). The
U.S. Food and Drug Administration (FDA) has given
the -glucan derived from bakers yeast extract a GRAS
rating (Generally Recognized as Safe).53,54
The fungal and yeast glucans share a common struc-
ture: primary backbone chains of (13)-linked -D-
TABLE 1 Some fungal origins of biologically active glucans.
Fungal origin
Auricularia auricula-judae (kikurage, an edible mushroom)
Cryptoporus volvatus (the golfball fungus)
Glomerella cingulata (phytopathogenic fungus)
Grifola frondosa (Maitake mushroom)
Lentinus edodes (Shiitake mushroom)
Monilinia fructicola (brown rot fungus)
Monilinia fructigena (apple rot fungus)
Pleurotus tuber-regium (an edible mushroom-tiger milk mushroom)
Pythium aphanidermatum (phytopathogenic fungus-pythium blight)
Saccharomyces cerevisiae (bakers yeast)
Saccharomyces cerevisiae (genetically engineered yeast)
Schizophyllum commune (wood-rotting fungus)
Sclerotinia sclerotiorum (white mold or stem rot fungus)
Sclerotium glucanicum (filamentous fungus)
Sclerotium rolfsii
207 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
glucopyranosyl units, along which are randomly dis-
persed side chains of -D-glucopyranosyl units attached
by (16) linkages.107,128,225,244 These (13, 16)--
glucans are usually highly branched; often present as an
inner wall layer and are sometimes covalently associated
with other cell wall polymers, particularly polysaccha-
rides such as chitin. 1 H and 13 C NMR spectroscopy
are especially powerful for structural characterization of
natural (13)--D-glucans and can provide informa-
tion on degree of polymerization, degree of branching
and the structural features of the polysaccharides.96
Molecular analyses, using wide-angle X-ray diffrac-
tion, on purified -D-glucan fractions, from curdlan
and paramylon, indicate that these glucans are spatially
organized as a cluster of four triple helix chains of
(13)--D-glucans.27 The helical chains were 1.56 nm
apart, with a fiber period of 0.60 nm, consequently
defining six -D-glucopyranose units per turn of helix.
The molecular structure of lentinan has been
extensively studied using NMR,96 atomic force
microscopy242,243 and X-ray spectroscopy.15 The X-ray
spectroscopy data confirmed that the triple helical
structure of native lentinan consists of three right-
handed helices.15 They proved that lentinan polysac-
charide is composed of a hexagonal unit cell with di-
mensions a = b = 15.8 A and c (fiber repeat) = 6 A
that is stabilized by interstrand hydrogen bonds.
The cell wall glucans of S. cerevisiae form two struc-
turally distinct macromolecular components compris-
ing of consecutively, (13)-linked -D-glucopyranosyl
residues with small numbers of (16)-linked branches
Glucan common names
Glucan
Glucan
Glomerellan
Grifolan
Lentinan
MFL-glucan
MFN-glucan
Glucan
Glucan
Zymosan and Glucan phosphate
PGG-glucan, triple helix-glucan, TH-glucan
or BetafectinTM
Schizophyllan
SSG Glucan
Scleroglucan
Scleroglucan

FIGURE 1 Structure of yeast -glucan showing (13, 16)-
glucan linkages.
(Figure 1), and a minor component with consec-
utive (16)-linkages and (13)-branches.32 -D-
glucan can be found as random coils or more orga-
nized conformations that constitute a network com-
posed of single helix chains associated in triple he-
lices stabilized by inter- and intra-hydrogen bonds
(Figure 2).101 Yiannikouris and co-workers229,230 inves-
tigated the (13, 16)--glucan of Saccharomyces cere-
visiae based on molecular mechanics techniques and
confirmed that the most stable confirmation for the
(13)--D-glucan single helical chain was found for
a glycosidic linkage of [, ] = (100 , 140 ) with
six -D-glucopyranose units per turn of helix. In ad-
dition, two distinct low-energy populated conforma-
tions of (16)--D-glucans were found for the dihy-
FIGURE 2 Triple helix structural conformation of (13)--D-
glucan forming the backbone of the yeast cell wall.
D. B. Zekovic et al.
dral angles [, , ] = (87.7 , +179.9 , 79.6 ) and
[, , ] = (85.5 , +179.7 , 177.9 ), underlining the
high flexibility of (16)--D-glucan side chains. How-
ever, it should be noted, in water solution this single
helical chain forms a trimer-triple helical structure that
is associated through hydrogen bonds between strands
lowering total energy.35,172,234
Frequently, three classes of -glucan are described in
the literature based on their solubility properties:49,186
r alkali-insolubleacetic acid insoluble (13)--
glucan;
r alkali-soluble (13)--glucan; and
r highly branched (16)--glucan.
The alkali insoluble-acetic acid insoluble (13)--
glucan is believed to be involved in maintaining wall
mechanical strength and shape; and the alkali soluble
(13)--glucan has been proposed to confer flexibil-
ity on the cell wall. The (16)--glucan plays a central
role in cell wall organization, interconnecting with the
(13)--glucan, mannoprotein and chitin.40,66,106,140
Lichens are another source of (13)--glucans hav-
ing both (13)-- and (14)--glucosidic linkages.
Olafsdottir and Ingolfsdottir154 have reviewed the cur-
rent knowledge on the structural characteristics and bi-
ological activity of lichen polysaccharides.
1.2.4. Cereal Grains and Higher Plants
The (13), (14)--glucans appear to be restricted
to members of the Gramineae (grasses), amongst An-
giosperms, where they are major components of en-
dosperm walls of commercially important cereals such
as barley, rye, sorghum, rice, and wheat. They are poly-
disperse, linear molecules containing both (13)- and
(14)--glucosidic linkages and form extremely vis-
cous solutions. The (13), (14)--glucans are present
in vegetative tissues as well as endosperms of the
Gramineae and may have a role in extension growth.
2. Glucan Health Effects
The -glucans have been reported to exhibit
bioactive and medicinal properties, such as immune-
stimulation, anti-inflammatory, antimicrobial, anti-
tumoral, hepatoprotective, cholesterol-lowering as
well as antifibrotic, antidiabetic, and hypoglycemic
activity.21,76,169,193,202 More than 1000 publications
have reported that (13)--glucans, either soluble
208
or particulate, exhibit immunomodulatory
properties.16,22,210 However, there is often confu-
sion in the literature concerning the precise properties
of -glucan preparations and this often stems from
the fact that different -glucans were used during
studies, which vary in origin, molecular structure,
and purity, all of which are known to influence their
activity.16,75
2.1. Glucans and Immune Response
Hot water extracts from various mushrooms and tree
fungi have been used for centuries as folk remedies
in Japan, China, and in Eastern Russia. Today, they
are used for general healthstimulating properties and
for specific therapies.101 In Europe during the 1940s,
zymosan, a crude yeast cell wall extract from S. cere-
visiae, was investigated for its purported drug-like ac-
tivities. Research conducted by Pillemer and Ecker160
demonstrated that zymosan could non-specifically po-
tentiate and modulate the immune system, regardless
of the type of invader or pathogen48,161 and the bac-
tericidal and virucidal activity of serum was raised af-
ter zymosan administration.13,97,155 Since these early
discoveries42 there have been literally hundreds of pa-
pers and patents describing the properties of fungal
(13)--glucans. Yeast (13)--glucans belong to a rable ligand specificity for the -glucans commonly
class of drugs known as biological response modifiers
(BRMs), which modify the hosts biological response
by stimulation of the immune system.16,101,109,174,204
By stimulating the hosts defense mechanisms against
disease challenge rather than attacking the infective
or tumor-causing agent, (13)--glucan remains non-
toxic to the cells of the host organism. This is often not
the case with synthetic or semi-synthetic therapeutics.
Various insoluble and soluble preparations of (13)-
-glucan differ significantly in both biological speci-
ficity and potency. Effective dosages vary from 2 to 500
mg/kg intravenously or intraperitoneally in models for
protection against infection and for hemopoiesis.
The main immunopharmacological activities of -
glucan include: increase of the host resistance to viral,
bacterial, fungal, and parasitic infections, an anti-tumor
effect and prevention of carcinogenesis, radioprotectiv-
ity, and an adjuvant effect.16,42 The protective action of
(13)--glucans has been described as non-specific im-
munomodulation due to the involvement of a number
of different immune pathways including, macrophage
activation, T-cell stimulation, stimulation of reticulo-
209 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
endothelial system (RES), activation of natural killer
(NK) cells, activation of the classical and alternative
complement pathways, and increased antibody produc-
tion. Among these, the macrophages are the best char-
acterized targets for the (13)--glucans.31
Activation of macrophages with glucan increases
their size and number, stimulates secretion of lysozyme
and tumor necrosis factor (TNF), and increases the
phagocytosis of antigens.132 Macrophage activation is
mediated via Toll-Like Receptor 2 (TLR2).163,205 Upon
stimulation with glucan, TLR2 is recruited to the phago-
some and signals the production of Tumor Necrosis
Factor- (TNF-) through the nuclear factor-B (NF-
B) pathway. TLR2 cooperates with the CD14 receptor
in response to glucan, as suggested by the high levels
of NF-B observed in TLR2+/CD14+ macrophages ex-
posed to the ligand compared to the levels obtained in
TLR2+/CD14-macrophages. Furthermore, TLR6 and
TLR2 were reported to coordinate macrophage activa-
tion by glucan.205
-Glucan receptor activity has been reported on
monocytes, macrophages, neutrophils, eosinophils,
and natural killer cells (NK cells), as well as on
non-immune cells such as endothelial cells, alveo-
lar epithelial cells, and fibroblasts.21 Human alveolar
macrophages possess phagocytic receptors of compa-
present in yeast and fungi. The Dectin-1 receptor,
from the surface of mammalian macrophages and neu-
trophils, which determines specificity for -glucan, was
recently isolated and characterized.20,23 Dectin-1, a
type II transmembrane glycoprotein, recognizes solu-
ble and particulate (13)--glucans and/or (16)--
glucans resulting in a variety of cellular responses in-
cluding phagocytosis, endocytosis, and the oxidative
burst.21,58 Of importance, the synergistic interplay be-
tween Dectin-1 and TLRs can induce the production of
pro-inflammatory cytokines and chemokines including
TNF, MIP-2 and IL-12 in macrophages and dendritic
cells.58
A number of in vivo studies, involving produc-
tion animal livestock, have demonstrated the ability of
-glucan to influence animal performance and health
through modulation of immune function. Soluble -
glucan, but not insoluble particulate -glucan, was
found to enhance the frequency of interferon (IFN )-
producing peripheral blood monocytes (PBMC) in a
dose-dependent manner in swine that had been infected
with porcine reproductive and respiratory syndrome
virus (PRRSV). This study found that soluble -glucan
had the potential to stimulate innate immune responses
that may elicit more robust immunity to PRRSV infec-
tion since it increases IFN production.224 In another
study, a purified -glucan feed additive significantly
decreased the incidence of Salmonella enterica subsp. en-
terica serovar Enteritidis organ invasion in immature
chickens and up-regulated the functional abilities of het-
erophils isolated from immature chickens against the
invading pathogens.119
2.2. Glucans and Anti-tumor Activity
The anti-tumor effect of (13)--glucans is often
treated separately to immune activity within the litera-
ture despite major similarities in mode of action. In vitro
studies using normal and tumor cells co-incubated with
particulate glucan have demonstrated that glucan exerts
a direct cytostatic effect on sarcoma and melanoma cells
and a proliferative effect on normal spleen and bone
marrow cells.215 The anti-tumor cytotoxic activity of
peritoneal macrophages, derived from control and par-
ticulate glucan-treated mice, has been studied to evalu-
ate the cellular basis of the anti-tumor activity of the par-
ticulate (13)--glucan.129 Moreover, Zymosan (crude
yeast cell wall extract), yeast glucan and other (13)-
-glucans have significant anti-tumor activity against a
variety of spontaneous and transplantable experimen-
tal animal tumors, and have been tested clinically in
man. Tumor growth has been reported to be inhibited
and host survival time increased either by simultaneous
glucan administration at the time of transplantation, or
by intravenous glucan administration prior to tumor
cell transplantation.25,39,43,124,176,191
Bakers yeast soluble and particulate (13)--
glucans have been shown to inhibit tumor growth
of the syngeneic anaplastic mammary carcinoma and
melanoma B-16 in mice.40,41 This work indicated that
peritoneal macrophages from glucan-treated mice pro-
duced a significant cytotoxic response compared to nor-
mal macrophages. These studies indicate that glucan, if
used therapeutically, could inhibit either hepatic metas-
tases or primary tumor growth and enhance survival.
This observation has been confirmed in a separate in-
vestigation by using glucan as adjuvant in a L1210 tu-
mor vaccine model as it was shown to be a strong
stimulator of antitumor immunity. Antitumor immu-
nity was maximally expressed when vaccine and adju-
D. B. Zekovic et al.
vant were co-administered intraperitoneally.26 Notwith-
standing these biological properties, intravenous appli-
cation of particulate glucan led to granuloma formation
in the liver and therefore soluble preparations are more
favorable.40,41
An alternative clinical practice may be found in the
combined therapy of soluble glucan with a cyclophos-
phamide, which has been demonstrated to result in a
reduction in number of experimental hepatic metas-
tases and in a prolonged life span in a group of treated
mice. A significant primary tumor weight reduction was
also observed in this study. It was suggested that solu-
ble glucan exerted antitumor activity by activation of
Kupffer cell cytolytic activity as well as enhancement of
cell-mediated immunity.182,183,216
2.3. Glucans and Mycotoxin
Adsorption
Mycotoxins are secondary metabolites produced by
fungi of various genera before or after harvest, during
transportation or during storage. Some of the agricul-
tural commodities affected are cereal grains, soybeans,
peanuts, and forage crops. It has been estimated that
there are at least 300 mycotoxins known to induce
signs of toxicity in mammalian and avian species, with
more being discovered as our analytical capabilities
develop.38 The most significant mycotoxins in naturally
contaminated foods and feeds are aflatoxins, ochratox-
ins, zearalenone, T-2 toxin, vomitoxin, and the fumon-
isins. Biochemically diverse with many pharmacologi-
cal effects, these toxins can have a deleterious impact
on animal and human health at extremely low con-
centrations. Various strategies have been employed to
control the effects of mycotoxin contamination, in-
cluding mycotoxin adsorption to nutritionally inert sor-
bents to decrease bioavailability.162 The formation of a
sorbent-mycotoxin complex reduces the availability of
the toxin to be absorbed across the gut barrier and re-
sults in the eventual excretion of the toxin. Numerous
inorganic sorbent materials have been tested includ-
ing hydrated sodium calcium aluminosilicate (HSCAS),
zeolites, bentonites, clays, and activated carbons.
There are a number of inert problems associated
with their inclusion in the diet, namely high inclu-
sion levels, narrow range of binding efficiency, se-
lect number of toxins absorbed, and risk of dioxin
contamination.38
210
 In recent years, research into the generation of bi-
ological products to reduce the bioavailability of my-
cotoxins has gained momentum. Early investigations
involved the addition of a live yeast culture (Yea-Sacc
1026
, Alltech Inc., KY, USA) to suppress the effects
of aflatoxicosis in poultry. An improvement in hatcha-
bility was observed,131 improvement in broiler body
weight gain36,37,189 and an enhancement in the im-
mune response.37 In vitro studies have confirmed dose-
dependent binding of aflatoxin to purified yeast cell
wall.36 Other in vitro studies have demonstrated that the
cell wall of Saccharomyces cerevisiae is capable of binding
a wide range of toxins; including zearalenone (66.7%),
fumonisin (67.0%), DON (12.6%), ochratoxin (12.5%),
citrinin (18.4%), T-2 toxin (33.4%) and DAS (12.7%).38
Dawson et al.33 have investigated the use of adsorp-
tion isotherms to further understand the mycotoxin-
cell wall fraction interactions, including the adsorption
affinity (Kd ), saturation point (Ksat ) and absorption
capacity (Qmax ) of the material. The use of ad-
sorption isotherms recognizes the fact that myco-
toxin adsorption in biological systems is a reversible
process that can be characterized as a chemical equi-
librium. Consequently, adsorption is a concentration-
dependent process influenced by mycotoxin concentra-
tion, the amount of adsorbent, and the relative affinity
of the adsorbent for the mycotoxin. Moreover, adsorp-
tion isotherms can be used to compare the relative bind-
ing capacity and affinity of different adsorbents. Using
this method, Dawson et al.33 compared the efficiency of
aflatoxin B1 adsorption by a yeast cell wall preparation
(Mycosorb
, Alltech, Inc., KY, USA) to two commer-
cial mycotoxin clay-based adsorbents. Mycosorb
was
demonstrated to be a more effective adsorbent at low
aflatoxin concentrations, had a higher affinity for afla-
toxin and possessed an overall greater capacity for the
toxin.
Researchers in France have recently proposed
an alternative ligand-toxin model based on Hills
equation.227 They modeled data from an experiment
studying the interaction between zearalenone and yeast
cell walls and found an improved fit of the experi-
mental data compared with an isothermal saturation
curve. Furthermore, they found that the new model
provided clues for the biological interpretation of toxin-
cell wall interaction, an interpretation not possible us-
ing an adsorption isotherm model. The data showed
that interaction between zearalenone and the cell wall is
co-operative, supporting the hypothesis that the three-
211 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
dimensional mobility of the yeast cell wall is probably
important in the adsorption event. This finding is in
contrast to that found with inorganic binders that dis-
play isothermal behavior (not co-operative), which can
be ascribed to their rigid structure.62,227
In a separate study, Hills equation was used to com-
pare zearalenone adsorption by four yeasts differing in
cell wall composition (glucans, mannans and chitin).228
Additionally, the four yeasts were fractionated to pre-
pare three components (total cell wall, alkali-soluble
glucan, alkali-insoluble glucan) to aid in the elucida-
tion of the adsorption process and identify the yeast
cell wall component involved in binding toxin. The re-
sults clearly demonstrated that there were differences
between yeast strains in their capacity to absorb zear-
alenone and that the -D-glucan concentration in the
cell wall can explain 83.6% of the adsorption. The sig-
moid shape of binding curves, analyzed according to
Hills model, indicated a co-operative association ex-
isting between zearalenone and the yeast cell wall at
low concentrations. Interestingly, there was a negative
correlation between chitin content in the cell wall and
the adsorptive capacity of glucan. Cooperativity and
the three-dimensional structure of -D-glucans indi-
cated that weak noncovalent bonds are involved in
the complex-forming mechanisms associated with zear-
alenone. It was stated that chemical interactions be-
tween -D-glucans and zearalenone are therefore more
of an adsorption type than a binding type.229
Yiannikouris and co-workers230 studied various lin-
ear or branched soluble or insoluble -D-glucans in
order to identify which form of glucan was responsible
for the toxin adsorption effect:
r laminarina soluble linear (13)--D-glucan with
some branching of (16)--D-glucan (extracted
from Laminaria digitata); MW of 5 to 8 kDa;
r curdlanan insoluble linear (13)--D-glucan
(extracted from Alcaligenes faecalis); MW of 50 to
80 kDa;
r pachymanan insoluble branched (13, 16)--
D-glucan (extracted from Poria cocos); MW of 80 to
120 kDa);
r pustulanan insoluble linear (16)--D-glucan (ex-
tracted from Umbiliaria papulosa); MW of 20 kDa.
These compounds were evaluated to elucidate their
roles in the adsorption mechanisms under three pH
conditions (3.0, 6.0 and 8.0) found in the monogastric
digestive tract. A constant quantity of each -D-glucan
(1 mg/ml) was mixed at 39 C with increasing
amounts of zearalenone (2 to 100 g/ml), and the
amount of bound toxin was measured. Acidic and
neutral conditions gave the highest affinity rates
(64 to 77%) by (13)--D-glucans, whereas alkaline
conditions decreased adsorption except when (16)-
-D-glucans side chains were branched on (13)--
D-glucans. Alkaline conditions appear to impede the
active three-dimensional conformation of -D-glucans
and favor single helix and/or random coil structures.
Studies of the equilibrium between -D-glucan-bound
and free toxins revealed that two types of chemical in-
teractions occur during toxin complexation with -D-
glucans, identified as weak chemical linkages such as
hydrogen and van der Waals interactions.230
The mycotoxin adsorption properties of the yeast
cell wall and its derivatives have been confirmed in
numerous in vivo studies involving poultry,93,164,196,198
swine,195,197,198,201 and cattle.2,214
2.4. Glucans and Functional
Food Ingredients
The relationship between health and nutrition is now
becoming increasingly important as medical and pub-
lic health services in the wealthier nations have largely
removed the risk of infectious and microbial medi-
ated diseases. Today, we are now more concerned with
non-infectious diseases such as diabetes, various can-
cers, and inflammatory diseases including heart dis-
ease. Diet is playing an increasing important role in
health maintenance and non-infectious disease preven-
tion. Functional foods can be defined as non-nutritional
components of foods, which have specific properties
to maintain optimum health, contributing to the pre-
vention or the delay of the onset of chronic illnesses
associated with advancing age. Medical experts advo-
cate dietary changes to reduce the risk of heart disease.
Ingestion of soluble -glucan has been shown to im-
prove the pattern of lipids, in humans and experimental
animals, with elevated serum cholesterol.11 In clinical
studies, the reversal of hypercholesterolemia has been
demonstrated with dietary supplementation of oats or
oat bran and purified -glucans from yeast. Four serv-
ings of oat-based foods containing greater than 0.75 g
(per serving) of -glucan are required to meet the FDAs
claim of reducing the risk of heart disease.11,55,115 Nutri-
tional effects of oat and barley extrudates were evaluated
in vitro as well as in feeding experiments in rats and
D. B. Zekovic et al.
in human nutritional experiments. Beneficial effects
were seen in both cases. In addition to the known ef-
fect on serum cholesterol levels in hypercholesterolemic
persons, it may also prevent inflammatory bowel dis-
eases (higher short chain fatty acid (SCFA) levels during
fermentation).44
In a groundwork study, the effect of yeast -glucan
on blood serum lipids in 15 obese hypercholesterolemic
(>240 mg/dL) men was evaluated.146 After a 3-week
period in which subjects ate their usual diet and their
baseline blood samples were determined, all men re-
ceived 7.5 g of -glucan dissolved in orange juice twice
daily for eight weeks. Blood analyses were conducted
at weeks 7 and 8, and again at week 12 (4 weeks
post treatment). The results of the study demonstrated
that supplementation of the diet with 15-g -glucan
from yeast per day significantly lowered total and LDL
cholesterol and improved HDL cholesterol by 16%.
However, further work needs to be conducted to op-
timize the -glucan dose and investigate long-term ef-
fects of supplementation on blood lipid chemistry. The
eventual goal would be to combine -glucan supple-
mentation with a special diet and thus eliminate the
necessity for cholesterol-lowering drugs in many hyper-
cholesterolemic patients.146
Functional properties such as fat binding capacity,
emulsion capacity, and control of foaming are of ma-
jor importance in the production of certain processed
foods.170 The use of natural ingredients is often difficult
to justify based on high cost or the high inclusion level
required. High inclusion levels may also have a nega-
tive impact on the final product. Yeast -glucan is one
ingredient that has demonstrated potential in improv-
ing the physical properties of food products; being used
as a thickening, water-holding, emulsifying stabilizer or
oil-binding agent.200
2.5. Adverse Side Effects of
Intravenous -Glucans
2.5.1. Insoluble (Particulate) Glucan
Preparations
Insoluble preparations have demonstrated undesir-
able toxicological properties manifested by hepato-
splenomegaly and granuloma formation.40,220 Glucan-
induced granuloma formation exhibits a characteristic
temporal pattern that involves the local synthesis of
chemotactic cytokines and recruitment of leucocytes
212
into sites of glucan deposition.51,52,89 Due to the
particulate nature of the glucan preparation (13 m),
it is difficult to administer it via an intravenous route.
The intravenous infusion of particulate yeast cell glucan
into rats results in the rapid, synchronous development
of foreign body-type lung granulomas composed almost
entirely of monocytes and macrophages.51,89 A number
of systemic diseases are characterized by the presence of
monocyte granulomas that resemble those found in the
rats after glucan infusion such as sarcoidosis, berylliosis,
talcosis, and Wegeners granulomatosis.95,120,181,208
An increased susceptibility to gram-negative infec-
tions and endotoxins has been reported when insolu-
ble (particulate) (13)--glucan was administered.18,29
This is the form most often found in environ-
mental settings.217 Pathogens such as Candida and
Aspergilli contain glucan cell wall components consist-
ing of branched homopolymers of -D-glucose with
1,3-consecutive and 1,6-crosslinked chains and proto-
typic of Saccharomyces cerevisiae.32 An anti--glucan an-
tibody has been detected in normal human and nor-
mal mouse sera. This antibody showed reactivity to
pathogenic fungal Aspergillus and Candida cell wall
glucan and was able to bind the whole Candida cell. It
was suggested that the anti--glucan antibody formed
an antigenantibody complex and participated in the
immune response as a molecule recognizing pathogenic
fungi.80
A number of other side effects have become appar-
ent when insoluble (particulate) glucan is administered
in vivo to animals. Development of pulmonary granulo-
matous vasculitis, activation of complement (anaphy-
lyotoxin), enhanced endotoxin sensitivity and devel-
opment of microembolism when administered at high
concentrations have been reported.19,42,88,243 The ad-
ministration of zymosan to rats represents a new ex-
perimental shock model by inducing acute peritonitis,
severe hypotension, and signs of systemic illness. How-
ever, it has been proposed that the zymosan-induced
shock, like septic shock, may be mediated by overpro-
duction of nitric oxide.121
2.5.2. Slightly Soluble Glucan Preparations
Although slightly soluble neutral glucans are com-
mercially available, these preparations are not suit-
able for intravenous administration as these aqueous
solutions have a very high viscosity.42 Lentinan, a
213 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
high molecular weight and poorly soluble (13)--
and (16)--glucan obtained from Lentinus edodes, has
been studied following intravenous administration to a
Beagle dogs. It induced changes in the cytoplasm of
macrophagic cells in the liver, spleen, kidney, lungs,
lymph nodes, and small intestine. Electron-lucent or
filamentous inclusions were demonstrated in the liver,
kidney, and spleen. One animal showed circulatory col-
lapse upon the first injection at 8.0 mg/kg. However, a
dose level of 0.5 mg/kg/day was administered without
adverse effect.24 Additional toxicity studies were per-
formed in which a variety of doses of lentinan ranging
from 0.1 to 1.0 mg/kg/day were given intravenously to
rats for nine weeks. Toxicity was manifested by the de-
velopment of cutaneous lesions and discoloration of
the ears suggesting thromboembolic events.30
These adverse side effects of particulate and slightly
soluble glucans have made these compounds all but
useless in clinical medicine and have moved the focus of
the clinical interest to soluble preparations that preserve
their biological activity and generate negligible toxicity
when administrated systematically.
2.5.3. Soluble Glucan Preparations
Most of the reported inhibitory effects of (13)--
glucan on endotoxin were seen with soluble (13)--
glucans.187,209 The toxicity of water-soluble -glucans
and their lethal activity in mice when administrated
concomitantly with indomethacin (IND) or other non-
steroidal anti-inflammatory drugs (NSAIDs) has at-
tracted attention lately. It was concluded that IND/-
glucan modifies the cytokine network and causes acute
inflammation, resulting in systematic inflammatory re-
sponse syndrome (SIRS) and death.141,199,235 Although
-glucans were detected in the blood in low concentra-
tions at the fungal infection, it is difficult to under-
stand what types of effects these have on the body.
Since mammals do not possess enzymes that decom-
pose -glucans, their administration to mice over a long
period of time has been demonstrated to result in an
accumulation in the liver, spleen and other organs of
the reticuloendothelial system.139
3. -GLUCAN STRUCTURE
ACTIVITY RELATIONSHIP
A variety of biological, immunomodulating and
immuno-pharmacological activities are observed with
(13)--glucans. The most important factors for deter-
mining the biological activity are the degree of branch-
ing (DB), the molecular weight (MW), and higher-order
structure (i.e. conformation).
3.1. Degree of Branching (DB)
and Activity
Lentinan and schizophyllan are chemically pure
(13)--glucans with a backbone of (13)--glucan
and side chains of single (16)--linked D-glucose
residues, together with a few internal (16)--
linkages.192 These polysaccharides have a degree of
branching of 0.33. There are other biologically active
-glucans with a higher degree of branching such as
the glucan isolated from Auricularia auricula-judae (DB
0.75)135 and those with a lower degree of branching
such as the glucan isolated from Pythium aphaniderma-
tum (DB 0.04).12 Glucans with a degree of branching
between these values exist, but those with a degree of
branching between 0.20 and 0.33 appear to be the most
active.
Water-soluble (16)-branched (13)--D-glucans
isolated from a hot-water extract of the fruiting bodies of
the fungus, Cryptoporus volvatus (Basidiomycetes) (DB
0.25), exhibited antitumor activity against the sarcoma
180 tumor.99 The alkali-insoluble highly branched
(13)--D-glucan, that is a major constituent of the
fruiting body of Auricularia auricula-judae (kikurage,
an edible mushroom) (DB 0.75),135,188 and the extra-
cellular highly (16)-branched (13)- -D-glucan of
Pestalotia sp. 815 (DB 0.67),136 both exhibited moderate
activity against tumors.
Seljelid et al.178 examined 42 different glycans for
their ability to stimulate macrophages and observed no
detectable patterns relating chemical structure to stim-
ulatory activity. The majority of these glycans had lit-
tle or no branching. None of the water-soluble glycans
were shown to be markedly stimulatory, however some
but not all of the insoluble glycans tested showed a
potent stimulatory effect. The (13)--D-glucans and
block polymers with (13)-linked segments were the
strongest stimulants.
Laminaran, an unbranched water-soluble low molec-
ular weight (13)--glucan, had no stimulatory effect
but after crosslinking, became as stimulatory as yeast
glucan.178 It was speculated that cross-linking may have
generated the necessary branchtype structure to elicit
D. B. Zekovic et al.
a reaction or it may have generated a large enough poly-
mer for stimulatory activity.
3.2. Molecular Weight (MW)
and Activity
The activity of -glucans is related to size. Numerous
studies have attempted to correlate biological activ-
ity (predominantly anti-tumor activity) and molecu-
lar weight. Overall, the fractions with high molecu-
lar weight (MW 100200 kDa), which exist as single
and/or triple helix conformers,172 were shown to be the
most active, while a fraction from the same source with
a MW in the range 5 to 10 kDa showed no activity
or only limited activity.12,47,105 The smallest fragment
that competes for the glucan receptor on neutrophils85
is a (l3)-linked -glucanoheptasaccharide, which has
been studied and synthesized by Sharp et al.179 The im-
mune response against cell wall polysaccharide activity
may be in part non-specific and determined by size
rather than by chemical structure.13,16
Samples of -D-glucans having the same MW dif-
fered significantly in their biological potency depend-
ing on whether they were in single or triple-helical form.
The single helical conformation of (13)--D-glucan
was clearly demonstrated by Aketagawa et al.3 to be the
dominant contributor to the activation of limulus co-
agulation factor G. However, a study of correlation be-
tween antitumor activity, molecular weight and confor-
mation of lentinan by Zhang et al.238 demonstrated that
samples with a lower MW had higher activity in vitro,
while higher MW samples were more active in vivo.
The antitumor activity of glomerellan, a (13)--
glucan with a high molecular weight (670 kDa) iso-
lated from Glomerella cingulata, against Sarcoma 180,
appeared to be independent of the presence of a highly
ordered structure.59 However, there are no set rules and
a polysaccharide fraction from a Pythium aphaniderma-
tum glucan, with a molecular weight between 1020 kDa
and with a low degree of branching and a single helical
conformation, has been reported to exhibit antitumor
activity.12,59,112
Hot alkali extract (HAE) fractions, from the edi-
ble mushroom Pleurotus tuber-regium molecular weight
ranging from 58171 kDa, were administered intraperi-
toneally to mice implanted with solid tumor Sarcoma
180.240 The fractions were found to be effective in in-
hibiting tumor proliferation with an inhibition ratio
214
of 50%. In vitro experiments with human tumor cell
lines HL-60 and HepG2 showed that hot alkali extract
fractions with molecular weights of 58 kDa to 422 kDa
showed antiproliferative activity toward the tumor cell
lines tested. However, the hot alkali extract (HAE) frac-
tions did not inhibit the growth of a normal monkey
kidney cell line (Vero). Zhang et al.240 postulated that
the antitumor effects of the (13)--D-glucan fractions
from the sclerotia of Pleurotus tuber-regium were probably
host-mediated and cytocidal.
A number of methods have been employed to re-
duce the -glucan molecular weight, including: acid
and alkaline hydrolysis, enzymatic digestion and ul-
trasound irradiation. Of these methods ultrasonication
has an advantage, as it does not change the chemical
nature of the polymer, while still reducing its molec-
ular weight by splitting the most susceptible chemical
bonds. It has been used in isolation, purification and
derivatization work to obtain highly biologically active
compounds.175
3.3. Higher-Order Structure
and Activity
The conformation of (13)--D-glucans, which in-
cludes triple helix, single helix, and random coil con-
formers, is recognized as an important contributing fac-
tor of biological activity.231 Structural studies, using
solid-state nuclear magnetic resonance on intact cell
walls, have determined that (13)--glucan possesses
a helical conformation.111 Such helices are composed
of a single polysaccharide chain or three hydrogen-
bonded chains (a triple helix). X-ray diffraction studies
have demonstrated that particulate glucans exist in the
form of triple-stranded helices and an analogy would
be three spiral stairways running side by side, creating
a very strong structure.57
To date, the question relating to the biologically ac-
tive form of the glucan has not been resolved. Sev-
eral studies suggest that the triple-helix is the most
biologically active conformation64,104,105,126,148,149,226
but other reports suggest that the single helix is more
potent.3,171,172 The triple helix form of (13)--D-
glucan is stabilized by hydrogen bonds between -OH
groups of glucan strands.27,35 It is possible to convert
the triple helix rich form of glucan into the single he-
lix form by treating glucan with sodium hydroxide fol-
lowed by neutralization and dialysis against water.152,172
215 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
Hydrogen bonds, which are responsible for the thermo-
dynamic stability of the triple helix form of glucan, can
be easily broken at high temperature, high pH value or
by using dipolar solvents, e.g. DMSO. Therefore, single
helix forms of glucan in solution tend to revert into the
more stable triple helix conformation. This may play a
role in the inconsistency of experimental results con-
ducted under non-standardized conditions.
Yoshioka et al.234 applied carbon-13 nuclear mag-
netic resonance spectroscopy to follow conformational
changes in the -D-glucan higher order structures and
tested their anti-tumor activity. They found that the
(13)--D-glucans with the triple helical conforma-
tion were ineffective in the inhibition of tumor growth.
They also found that the change in the glucan struc-
ture from the triple helix to the single helix, caused
by their lyophilization in DMSO followed by dialysis
against water thereby removing DMSO, restored their
anti-tumor growth properties. The same glucans were
inactive when present in the DMSO solution without
further treatment. In the same study it was discovered
that the highly branched (13, 16)--D-glucans
were effective when used in saline or DMSO solutions
and that the triple helix form did not diminish their
biological activity.
The descriptors wormlike chain and dense ran-
dom coil were suggested by Zhang et al.239 for water-
insoluble glucan from the sclerotium of Pleurotus tuber-
regium extracted with hot sodium hydroxide solution.
The chemical structure consisted of a (13)--D-
glucan chain with (16)--D-glucan branches on ev-
ery third unit as deducted from 13 C NMR, FT-IR spec-
tra and mass spectrometry. Various molecular weight
fractions ranging from 10.7774 kDa were screened and
showed potent anti-tumor activity. A mechanism of ac-
tion, that included mediation by the immune system
and direct cytotoxicity, was postulated.240
Sonifilan (SPG), prepared by the ultrasonic partial
degradation of schizophyllan, is thought to be a triple
helix conformer.237 Alkaline treatment changes its triple
helix conformer to a random coil, which can concomi-
tantly be transformed by neutralization to the single
helix rich (SPG-OH) conformer. The fluorescence in-
tensity of aniline blue bound to SPG can be used to
estimate the relative amount of single helix convert-
ing to triple helix during different stages of a denature-
renature cycle, as aniline blue is believed to bind only
to single helix forms of SPG.237 Using fluorescence
response energy transfer (FRET) spectroscopy obser-
vations, it was proposed, that treating the triple-helix
glucans with NaOH forms a partially opened triple he-
lix rather than a single helix. Both Limulus amoebocyte
lysate activity and nitric oxide production were related
to the degree of opening of the triple-helix and it was
shown that the open conformers were more biologi-
cally active than the intact triple-helix.236 Comparing
the clearance of SPG-OH from the blood of mice with
that of SPG, and using a limulus factor G test, it was
shown that SPG-OH was removed faster than SPG.138
Both SPG and SPG-OH showed a significant antitumor
effect on a solid form tumor in vivo and a hematopoietic
response on cyclophosphamide induced leucopenia.203
In vitro studies investigating the activities of the prim-
ing effect of lipopolysaccharide (LPS) triggered tumor
necrosis factor (TNF) synthesis, nitric oxide synthesis
of peritoneal macrophages in vivo and hydrogen per-
oxide synthesis of peritoneal macrophages in vivo was
stronger in mice treated with SPG-OH.152
The ability to stimulate production of tumor necro-
sis factor (TNF-) and superoxide-anion release from
human blood monocytes was studied using SR-glucan
from Sclerotium rolfsii, SG-glucan from Sclerotium glu-
canicum, MFN-glucan from Monilinia fructigena, and
MFL-glucan from Monilinia fructicola.114 These fungal
glucans had a degree of branching in the range of 0.31
to 0.39 and a MW of 280 kDa to 550 kDa. Results
showed that stimulatory biological activity was signifi-
cant, regardless of molar mass or solution structure. This
was contrary to the majority of the literature and sug-
gested that helical structures were not essential, or even
advantageous, for immunological activity.114 Recently
Kogan101 proposed that for the production of super-
oxide and for release of TNF-, neither helical confor-
mation nor the high molecular weights of -glucan are
important.
-Glucans, like endotoxins (e.g. lipopolysaccharides,
LPS) have the ability to cause activation of coagu-
lation factor G from Limulus amoebocyte lysate (LAL).
The Limulus test is very sensitive for LPS determina-
tion.81,101 Ohno et al.151 studied the structure-activity
relationship resulting from -D-glucan coagulation of
the amoebocyte lysates of the horseshoe crab (Limu-
lus), finding little to no coagulation in the presence of
glucans without (13) linkages. However, significant
coagulation was observed in the presence of curdlan,
an unbranched (13)--D-glucan. Also, grifolan and
schizophyllan (DB 0.33), lentinan (DB 0.4), SSG (DB
D. B. Zekovic et al.
0.5), and OL-2 (from Omphalia lapidescens) (DB 0.66)
gave positive reaction to the Limulust test. Optimum
concentration for coagulation was correlated with the
degree of branching. Higher reactivities were observed
for glucans with a higher molecular weight and a higher
population of single helix conformation.
4. SOLUBLE (13)--D-GLUCANS
AND (13)--D-GLUCAN
DERIVATIVES
4.1. Impact of Degree of
Polymerization and Degree of
Branching and Glucan Solubility
The (13)--glucans with a high degree of poly-
merization (DP > 100) are completely insoluble in wa-
ter. The conformation assumed by the (13)--glucan
chains allows stronger cooperative interactions and as-
sociations between chains than between the chains
and water molecules.16,101 The solubility increases as
the degree of polymerization of the (13)--glucan
is lowered. Thus, the (13)--glucan solubility appears
to depend both on degree of polymerization and on
the contents of the side substituted branches. The fre-
quency of side branches determines the solubility of
different (13)--glucans.50 The interactions between
(13)--glucan chains are disturbed by the presence of
single lateral glucose substituents on otherwise linear
molecules. Even a single (16)- linked glucose side
chain in a laminarin molecule can transform the glucan
into a more soluble form compared to its unbranched
molecule.145
The linear (13)--glucan core can be precipitated
by removal of the glucan side branches using Smith
degradation.159 The (13, 16)--glucans with a low
DP (DP 20) are soluble in water. Presumably the more
flexible intrachain (16) glucosidic linkages and pos-
sible side branches favor polysaccharide-solvent inter-
actions and consequent hydration of molecules. High
molecular weight branched (13, 16)--glucans
from yeast and fungal cell walls, which contain vari-
able but minor proportions of (16)--glucosidic in-
terchain linkages, are insoluble in water. Cellulose is
also a -D-glucan, but consists of only (14)--D-
linkages and is therefore stiff, highly crystalline and
non-soluble.6 The linear glucans with (13) and (14)
linkages and DP > 200 in the chain although soluble in
water, tend to precipitate on cooling.7 The placement
216
of two types of linkages within the same glucan chain
lowers the possibility of appearance of regular inter-
chain associations and allows better polysaccharide sol-
vent interactions. The same is true in the case of the
main component of soluble fiber in oats, which is
the mixed-linkage (13, 14)--D-glucan polysac-
charide. Most of the molecule consists of cellotriose
and cellotetraose oligosaccharides separated by (13)-
linkages with a minor participation of (14)-linkages
that are longer then the tetraose type.223
Uncharged polysaccharides are, in general, insolu-
ble in neutral aqueous solutions, but will dissolve in
strongly alkaline media due to ionization of the glu-
cosyl hydroxyl groups, which have pKs in the range
1214.165 Repulsion between ionized groups and better
hydration of ionic species helps molecular separation,
leads to disruption of the ordered structure and causes
dissolution. Some water-soluble (13)--glucans be-
have in this way when solubilized in 1 M NaOH.
In contrast, high molecular weight (13, 16)--
glucan from the yeast cell wall and similar fungal
cell wall glucans are not soluble, even in hot alkaline
solutions.
In addition to the alkali-insoluble glucans from fun-
gal and yeast cell walls, that are mostly composed
of (13)--glucan chains, there is a group of water-
insoluble, alkali-soluble fungal and yeast glucans. These
glucans are comparable in size to the alkali-insoluble
glucans but they have a much higher proportion of
(16) linked intrachain residues. Presence of these flex-
ible linkages is sufficient to allow their solubility in
alkaline solution.50 The solubility of (13, 16)--
glucans in 1M sodium hydroxide has been used to dif-
ferentiate them from the (14)--glucans (cellulose),
which are not soluble at this alkaline concentration.165
Dipolar solvents such as dimethylsufoxide (DMSO),
formamide and N-N-dimethylformamide are able to
dissolve -D-glucans due to formation of hydrogen
bonds with this polysaccharides hydroxyl groups.142
Dimethylsufoxide will dissolve lentinan, oat (13),
(14)--glucan, paramylon, pachyman, curdlan, and
yeast cell wall (13, 16)--glucans but only above
60 C.14,63,7779,130,134,239
4.2. Soluble Neutral -D-glucan
Preparations
Solubilization of natural -D-glucans by a mild,
acidic hydrolysis of the glycosidic bonds can pro-
217 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
duce neutral soluble glucan preparations with reduced
molecular weight in comparison with the native ma-
terial. Chromatography of this material on Bio-Gel P4
and P2 columns can provide different molecular weight
fractions.84 The -D-heptaglucoside separated from the
mixture was found to be the smallest active oligosac-
charide that stimulated production of phytoalexins in
plants.180
Underivatized, aqueous soluble (13)--D-glucan
(poly-1-6-glucotriosyl- 1-3-glucopyranose) also
known as PGG-glucan, TH-glucan or BetafectinTM )
is manufactured from the cell wall of a genetically
engineered Saccharomyces cerevisiae.83 The degree of
branching (DB) in this material is 0.5, significantly
higher than the DB (0.2) found in wild yeast glucan.
The structural difference produces a 35-fold higher
affinity of the PGG glucan for the -glucan receptor of
human monocytes and neutrophils.83,156 The primary
structure of PGG has been optimized for enhanced
macrophage stimulating properties via -glucan recep-
tors found on monocytes and neutrophils. This material
has been produced through a series of alkaline, acidic
and neutral treatments of the cell wall glucan to yield
a conformationally pure neutral, soluble glucan that
was re-annealed under controlled time, temperature
and pH conditions to rearrange the single helix confor-
mation to a new ordered triple helix conformation.82
PGG-glucan has been demonstrated to have im-
munomodulatory effects in cell cultures, animals and
humans.9,10,34,133,157,190
The effect of an endotoxin-free underivatized, sol-
uble yeast (13)--D-glucan and lipopolysaccharide
(LPS), either alone or in combination, on tumor necro-
sis factor- (TNF), interleukin-6 (IL-6), IL-8 and IL-
10 secretion and monocyte tissue factor (TF) expres-
sion in human whole blood was studied by Engstad
et al.45 These researchers described their yeast -glucan
as having a branched structure with (13)--linked
side chains anchored through a (16)--linkage to
the main (13)--chain for approximately every 10th
15th glucosyl unit of the main chain. Importantly, they
observed that (13)--D-glucan had a strong syner-
gistic effect on LPS-induced secretion of IL-8, IL-10,
and on monocyte TF activity, with no apparent effect
observed on TNF- and IL-6 production. However,
when pre-incubated in whole blood samples, the
(13)--D-glucan strongly primed LPS stimulation for
all parameters, including TNF- and IL-6 production.
Furthermore, independent of transcription, induction
of polymorphonuclear leukocyte degranulation could
be assigned to the inclusion of soluble -D-glucan. En-
gstad and co-workers45 postulated that this was most
probably due to aggregation of CD11b/CD18 on the
surface of monocytes.
The controlled periodate oxidation of natural -D-
glucans followed by the subsequent reduction of the
aldehyde groups with sodium borohydride has yielded
water-soluble glucan polyols, in which most of the side
chains have been modified.135,136 Their antitumor ac-
tivity was directly related to the content of polyol side
chains and activity ceased when these chains were re-
moved. The structure of the (16)--D-glucan side
chains impacts decisively the anitumor activity of -D-
glucans. Dehydration of the glucopyranose side chains
into the 3,6-anhydro--D-glucopyranosyl units, not
only resulted in the lost of antitumor activity, but in
some instances stimulated growth of Sarcoma 180 solid
tumor cells. Therefore, the removal or modification of
the D-glucosyl side chains from the conversion of the
branch units into the polyols might be related to the in-
creased water solubility of the glucan. In addition, the
presence of polyhydroxylated groups on the periphery
of a helical structure may be related to the enhancement
of the immunopotentiating activity. When curdlan, a
linear (13)--D-glucan with no side units, was treated
with epichlorochydrin, it produced epoxylated glucan
(presumably not cross-linked) without any antitumor
activity.98,137 However, when the epoxy-groups were
subjected to an alkaline hydrolysis to produce glycerol
derivated glucans, these derivatives showed enhanced
antitumor activity.
4.3. Charged (13)--D-glucan
Derivatives
The water insolubility of yeast glucan substantially
limits this products medicinal application; therefore
significant efforts have been directed towards chem-
ical derivatization of the natural -D-glucans to en-
hance their aqueous solubility. The methods include
preparation of sulfated,191,219 phosphorylated,40,217
and carboxymethylated150,177 derivatives of yeast and
other native -D-glucans.
4.3.1. Sulfated (13)--D-glucan
Derivatives
The three general methods for sulfonation/sulfation
have been developed. These include the use of SO3 -
D. B. Zekovic et al.
pyridine complex,104 chlorosulfonic acid in pyridine,28
and direct sulfonation with concentrated sulfuric
acid.218
For a series of studies, a curdlan sulfate was pre-
pared by the sulfonation of a natural linear (13)--
D-glucan. This material demonstrated potent anti-HIV
activity both in vitro and in vivo.232,233 During Phase
I/II clinical trials, increased numbers of CD4 and T4+
lymphocytes were observed in the blood of HIV carriers
upon curdlan sulfate administration.60,61
In a more recent study by Koumoto et al.,110 five sul-
fated curdlan samples with a range of degrees of substi-
tution (DS) from 0 to 76 mol% were prepared. Measure-
ments of the circular dichroism showed that the curdlan
sulfates with DS from 1.7 to 8.7mol% could form com-
plexes with polycytidylic acid in the same manner as
schizophyllan.173 The complexed polynucleotide chain
showed a significant resistance against enzymatic hy-
drolysis. It has been postulated that these water-soluble
sulfated curdlan-nucleotide complexes could find ap-
plication in gene technology.
In the homogenous sulfation of -D-glucan from
Saccharomyces cerevisiae in a DMSO-urea solution
at 100 C, substantial fragmentation of yeast glucan
occurred.219 On average, a single sulfate group was
introduced into every third glucose unit along the
polysaccharide chain. The 125 kDa fraction of the sul-
fated material accounted for only 1% of the product,
with 99% of MW of sulfated glucan being 14.5 kDa.
Interestingly, in this case the sulfated glucan contained
a significant quantity of nitrogen, which was probably
introduced with urea, working as a chaotropic agent.
The 13 C NMR confirmed the presence of (13)- in-
terchain linkage in the polysaccharide chain and in solu-
tion the chains were self-associated in a triple helix form.
Subsequent in vivo studies found this form of yeast glu-
can stimulated murine bone marrow proliferation after
intravenous administration.219
Williams et al.219 and Wang et al.212 both demon-
strated that -D-glucan sulfation could be carried
out under much milder conditions using a sulfuric
acid/n-propanol system at 10 C, but the degree
of sulfonation in this heterogenous system was only
37.4mol%. Sulfated (13)--D-glucan derivatives hav-
ing DS between 1.0 and 1.3 and molecular weight 38
kDa were soluble in water and existed in the solution in
the form of expanded chains. In the opinion of Wang
et al.211 the relative chain stiffness, moderate molecular
weight and good solubility in water has contributed to
218
the enhancement of antitumor activity of these sulfated
glucans against Sarcoma S-180 and gastric carcinoma.
Zhang et al.242 extracted six water-insoluble fractions
(TM8-1 to TM8-6) of (13)--D-glucan from the scle-
rotia of Pleurotus tuber-regium using a hot alkali extrac-
tion. They sulfated fractions with different molecular
weights (MW 58 to 774 kDa) to give their corresponding
water-soluble derivatives (S-TM8-1 to S-TM8-6) with the
degree of sulfation (DS) ranging from 1.14 to 1.74 and
MW 60 to 648 kDa. The in vitro anti-viral activities of
the native (13)--D-glucans and associated sulfated
derivatives were evaluated by the cytopathic effect as-
say (CPE) and the plaque reduction assay against four
kinds of viruses (herpes simplex virus type 1 (HSV-1),
herpes simplex virus type 2 (HSV-2), respiratory syncy-
tial virus (RSV), and influenza A virus (Flu A)). This
study found that the insoluble (13)--D-glucans did
not inhibit viral replication, irrespective of molecular
weight. However, the S-TM8 fractions had potent anti-
viral activity against HSV-1 and HSV-2. The plaque re-
duction assay results suggest that S-TM8 fractions pu-
tatively exert their anti-viral effect by binding to the
viral particles, thereby preventing viral infection of the
host cells. Moreover, the negative charges on the poly-
mer chain of the S-TM8 fractions may interact with
the positively charged glycoproteins on the surface of
HSV, minimizing the interaction between the HSV and
the negatively charged host cells. The anti-viral activity
of the S-TM8 fractions may also be explained by their
more extended chain conformation in solution, due to
an increase in one of their molecular parameters such as
persistence length (q), as compared to the native TM8
fractions.242
Sulfated glucans such as curdlan sulfate, dextran sul-
fate, lentinan sulfate and pullulan sulfate also exhibited
anti-HIV and anticoagulant activities.232 Anticoagulant
activity is strongly influenced by the degree of sulfation,
molecular weight and basic polysaccharide structure.
The sulfated linear (13)--D-glucans with DS greater
than 1.0 and with a molecular weight between 18 kDa
and 50 kDa were shown to be suitable as a potential
heparin alternative.4,5
4.3.2. Phosphorylated (13)--D-glucan
Derivatives
The direct extraction of -D-glucan using orthophos-
phoric acid yields a phosphated -D-glucan with a
mean molecular weight of 34 kDa (DP 210) and DS
20mol% resulting from a partial hydrolysis of the
219 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
polysaccharide.96 Earlier reported values varied from
DP 137 to DP 450 and were proven to be inaccurate due
to aggregation of the phosphorylated glucan molecular
and other factors resulting from the initial preparation
of the -D-glucan.143,144,217 A milder method217 that
utilizes orthophosphoric acid in the presence of urea in
a DMSO solution can yield a product with the molecu-
lar weight of 110 kDa as the major fraction constituting
98% of the final yield. A detailed analysis of the site
specific phosphorylation of the Saccharomyces cerevisiae
yeast cell wall -D-glucan118 conducted with the use
of 13 C and 31 P 2D NMR spectroscopy revealed that
the phosphorylation was limited to C-6 and C-2 posi-
tions in the glucopyranose rings due to the steric fac-
tors. In solution, these glucan phosphate polymers are
self-associated in a triple helix structure.144
Rice et al.167 studied the pharmacokinetics following
intravenous administration of three highly purified and
characterized glucans (glucan phosphate, laminarin and
scleroglucan). They developed a method to covalently
label carbohydrates with a fluorophore on the reducing
terminus. The three glucans studied varied in molecu-
lar size, branching frequency and solution conforma-
tion. Elimination half-life was longer (3.8 +/ 0.8 vs.
2.6 +/ 0.2 and 3.1 +/ 0.6 h) and volume of dis-
tribution lower (350 +/ 88 ml/kg vs. 540 +/ 146
and 612 +/ 154 ml/kg) for glucan phosphate than
for laminarin and scleroglucan. Clearance was lower
for glucan phosphate (42 +/ 6 ml/kg h) than for lami-
narin (103 +/ 17 ml/kg h) and scleroglucan (117 +/
19 ml/kg h). An inverse relationship between plasma
levels at steady state and clearance was established,
thereby suggesting that the physicochemical properties
of different glucan preparations play a role in pharma-
cokinetics at higher blood concentrations. In a subse-
quent study Rice et al.168 examined the pharmacoki-
netics of the same three water-soluble glucans (glucan
phosphate, laminarin and scleroglucan) following oral
administration in rats. The maximum plasma concen-
trations for glucan phosphate occurred after 4 hours.
In contrast, laminarin and scleroglucan showed two
plasma peaks between 0.5 and 12 hours. At 24 hours
post-ingestion, 27 +/ 3 % of the glucan phosphate and
20+/7% of the laminarin remained in the serum. The
scleroglucan was rapidly absorbed and eliminated, with-
out significant contribution from the liver. Of impor-
tance, this study demonstrated that the soluble glucans
were bound and internalized by intestinal epithelial
cells and gut-associated lymphoid tissue cells, thereby
translocating the polysaccharides from the gastrointesti-
nal tract into the systemic circulation. Gut-associated
lymphoid tissue expression of Dectin-1 and TLR2, but
not TLR4, increased following oral administration of
glucan. The studies of Rice and co-workers167,168 are im-
portant as they clearly demonstrate that the pharmoki-
netic behavior of the glucan preparations is dependent
on method of administration (i.e., oral or intravenous).
Independently, Hong et al.72 also demonstrated the
ability of macrophages to orally take up ingested (13)-
-D-glucans and transport them to the reticuloen-
dothelial tissues and bone marrow. These researchers
suggested that in addition to a role for Dectin-1 for sol-
uble (13)--D-glucan uptake, membrane CR3 of ma-
ture bone marrow granulocytes served as a receptor for
(13)--D-glucan in vivo. They found that only CR3
with bound (13)--D-glucan triggered a cytotoxic de-
granulation response to iC3b-tumor cells. Furthermore,
the in vivo administration of soluble (13)--D-glucan
resulted in a partial priming of CR3 as evidenced by
the initial lower level of CR3-dependent tumoricidal
activity.
A substantial number of bioactivity studies have
been conducted with -D-glucan phosphate. This ma-
terial has been demonstrated to possess anti-infective,
anti-inflammatory, cardio-protective and immunomod-
ulating properties. Endotoxin-free lyophilized (13)-
-D-glucan phosphate (MW 156 kDa)118,217 improved
survival in experimental sepsis, and this correlated with
decreased tissue NFB activation.220,221 In a more re-
cent study by Williams et al.,222 glucan phosphate was
used to determine whether there was a correlation be-
tween induction of polymicrobial sepsis, modulation
of tissue Toll-Like Receptor (TLR) gene, and protein ex-
pression and survival outcome. They found that early
increases in TLR2/4 gene and TLR4 protein expres-
sion correlated with mortality. Hence, they postulated
that an early up-regulation of tissue TLR2/4 could play
a role in the pro-inflammatory response and patho-
physiology of polymicrobial sepsis. In a separate study,
the same research group found a cardio-protective ef-
fect from administration of glucan phosphate follow-
ing myocardial injury (ischemia/reperfusion) in rats.117
Their results highlight the importance of activation of
the TLR mediated MyD88-dependent NFB signaling
pathway by glucan phosphate whilst having a protective
role coming from stimulation of the PI3K/Akt path-
way. Sherwood et al.184 investigated the effect of glu-
can phosphate on IFN- expression in normal (con-
D. B. Zekovic et al.
trol), and in endotoxin (LPS)-tolerant mice. Glucan
phosphate potentiated IFN- expression in the con-
trol mice and attenuated suppression of IFN- expres-
sion in LPS-tolerant mice. Therefore, the authors high-
lighted the potential advantages of using glucan phos-
phate as a tool in fighting trauma and sepsis induced
immunosuppression.
The effects of treatments with the soluble polysac-
charide immunomodulator glucan phosphate on the
inflammatory response induced by burn injury and on
resistance to Pseudomonas aeruginosa burn wound infec-
tion in mice was evaluated by Lyuksutova et al.122 The
glucan phosphate treatment clearly attenuated burn-
induced inflammation and increased resistance to P.
aeruginosa burn wound infection. Improved survival
correlated with lower bacterial load in the burn wound,
attenuated production of proinflammatory cytokines,
and enhanced production of Th1 cytokines.122
4.3.3. Carboxymethylated
(13)--D-glucan
Derivatives of -D-glucan have been produced using
chloroacetic acid in either 2-propyl alcohol or water at a
pH greater than 12 (NaOH).74,150,153 The derivatives of
CM glucan that are formed following this general pro-
cedure possess improved solubility in water, in com-
parison with the native glucans that were used in the
derivatization. The degree of substitution can be con-
trolled by the ratio of sodium hydroxide to chloroacetic
acid, and increases with the addition of base46 and can
reach values above 1.0. The site of carboxymethylation
was established to be at C-2, C-4 and C-6. The structure
of the CM glucans in the solution depends upon the
degree of substitution and with the increase of the DS,
a transition from triple-helical structure through single
helical to random structure takes place.101
Carboxymethylated (13)--D-glucan from mush-
room sclerotia from Poria cocos (CM pachyman) with
DS 1.01.3 and molecular weight 189 kDa211 showed
good solubility in water and antitumor activity against
Sarcoma 18090,125 and gastric carcinoma in vivo and
in vitro.211 CM glucan isolated from Alkaligenes faecalis
var. myxogenes IFO 13140 retained potent antitumor ac-
tivities with DS up to 0.14 but activity decreased with
the higher DS values.177 CM glucan obtained from
highly branched (13)--D-glucan obtained from Scle-
rotinia sclerotiorum IFO 9395 with the DS between 0.04
and 0.49 possessed antitumor activity150 that appeared
not to be related to the inability of this CM glucan to
220
form helical structures. Another glucan demonstrating
antitumor properties isolated from Girifola frondosa lost
most of its activity after carboxymethylation. However,
antitumor activity was detected for the compound with
DS below 0.25 and when administered repeatedly at
high doses.1
Zhang et al.243 isolated six hot alkali-soluble -
glucan fractions from the sclerotium of Pleurotus tuber-
regium. These were carboxymethylated to produce six
water-soluble carboxymethylated fractions. The yield
of the chemical derivates was more then 70%, with
DS ranging from 0.37 to 0.68 as determined by ele-
mental analyses.241 All investigated water-soluble car-
boxymethylated fractions obtained larger molecular
weight then their corresponding native -glucan241,243
indicating the effectiveness of substitution of the hy-
droxyl groups in the glucose residues by the car-
boxymethyl groups and that no hydrolysis of the
polysaccharide chain had occurred. They showed that
the carboxymethylation was effective in improving wa-
ter solubility and enhancing both in vivo (Sarcoma
180 solid tumor implanted on BALB/c mice) and in
vitro (HL-60 tumor cell culture) anti-tumor activity as
well as the immunomodulatory effect of the native -
glucan from the sclerotia of Pleurotus tuber-regium.243
The importance of branching frequency in car-
boxymethylated glucans for human blood leucocyte
stimulation and cytokine production was demonstrated
by Kubala et al.113 Two (13)--D-glucans isolated
from two different sources, schizophyllan (SPG) pro-
duced by Schizophyllum commune (DB 0.25) and glu-
can from the cell wall of Saccharomyces cerevisiae (DB
0.11),were derivatized by the action of monochloracetic
acid under alkaline pH. The SPG derivative demon-
strated significantly higher potential to stimulate hu-
man blood leukocytes and pro-inflammatory cytokines
production. Kubala et al.113 suggested that the higher
potency of SPG to stimulate leucocytes could be caused
by factors such as higher branching frequencies (1:3),
higher polymer charge of SPG, or a different confor-
mation in solution (triple helix) compared with car-
boxymethyl glucan.
A variety of CM glucans with different DS (up to
1.0) and molecular weight (up to 366 kDa) have been
obtained by carboxymethylation of (13)--D-glucan
from Saccharomyces cerevisiae.73,123,174 Sandula et al.174
examined the immunomodulatory activity of various
derivatives in mitogenic and co-mitogenic tests on rat
thymocytes and found that the derivatives with DS 0.75
221 Natural and Modified (13)--D-Glucans in Health Promotion and Disease Alleviation
exhibited the highest activities. Ultrasonic treatment of
CM glucan of high molecular weight (300600 kDa)
produced derivatives with reduced molecular weight
(90100 kDa), which showed high mitogenic, radiopro-
tective and antimutagenic properties.175
Hofer et al.70,71 investigated the enhancement of
haemopoiesis ability by carboxymethylated-glucan in
gamma-irradiated mice. When this preparation was
given to mice after gamma-radiation it enhanced the
number of granulocytes in the peripheral blood and
the number of granulocyte-macrophage progenitor cells
in bone marrow, together with an increase in spleen
weight. These beneficial effects were, unfortunately,
challenged by an increase in liver weight and the devel-
opment of anemia during long-term administration of
the glucan preparation, which suggested the occurrence
of undesirable inflammatory and hepatic side effects.
Soluble CM-glucan with DS 0.56 prepared accord-
ing to the modified procedure of Machova et al.123
was administered together with cyclophosamide dur-
ing chemotherapy of Lewis lung carcinoma. The mix-
ture inhibited growth of intra-muscular tumor implants
by 7590% whilst the treatment with cyclophosamide
alone yielded only 57% inhibition. Similar synergism
was observed in inhibition of lung cancer metastases
(up to 9294%). In this case the effect of CM-glucan
was attributed to an increased level in tumor tissue of
the intracellular inhibitors of two proteases: cysteine
proteases-stefin A and cystatin C.102
Slamenova et al.185 investigated the protective ef-
fect of three different water soluble glucan derivatives
from (i) carboxymethylated-glucan (DS 0.8 and MW
250 kDa), (ii) sulfoethyl glucan (DS 0.3 and MW 250
kDa), and (iii) carboxymethyl-chitin-glucan (CM-CG)
(DS 0.43 and MW 220 kDa) against oxidative DNA
damage induced by H2 O2 and visible light-excited
methylene blue in V79 hamster lung cells. They demon-
strated that all three tested glucan derivates reduced ox-
idative DNA damage. The ability to reduce genotoxic
activity increased in the order: carboxymethylated-
glucan < sulfoethyl glucan < carboxymethyl-chitin-
glucan. They suggested that the tested glucans from
Saccharomyces cerevisiae and a -glucan-chitin complex
from the mycelium of filamentous fungus Aspergillus
niger exhibited protective effects against oxidative dam-
age to DNA as a consequence of scavenging of both
OH radicals and singlet oxygen.
Recently, Kogan et al.103 investigated the radical-
scavenging activity of yeast cell wall carboxymethylated
-glucan (DS 0.8 and MW 250 kDa) using electron
paramagnetic resonance. In the in vivo experiments, car-
boxymethylated -glucan was administered to rats with
experimentally induced adjuvant arthritis. A substan-
tial decline in the level of plasmatic carbonyls was ob-
served. On the basis of these results it was assumed that
carboxymethylated -glucan radical-scavenging proper-
ties could be responsible for the antioxidant activity in
the adjuvant arthritis model. This brings the promise of
possible applications of this yeast glucan derivative in
arthritis treatment.
5. THE FUTURE
Glucans are attracting increasing attention because
of their positive effects on human and animal health
and because they are natural polysaccharides found in
microorganisms and plants. The natural (13)--D-
glucan parameters of primary structure including solu-
bility, degree of branching (DB) and molecular weight
(MW), as well as the polymer charge and/or solution
conformation (triple helix, single helix or random coil
conformation) all play a role in their biological activity.
Insoluble or hardly soluble -glucans can cause sig-
nificant adverse health effects such as microemboliza-
tion, granuloma formation, inflammation and pain. To
improve (13)--D-glucan solubility, several deriva-
tization (sulfation, phosphation and carboxymethyla-
tion) and molecular weight lowering (acid and alkaline
hydrolysis, enzymatic digestion and ultrasound irradia-
tion) procedures are being applied.
Chemical modifications of (13)--D-glucan can
strongly influence biological activity. In the future glu-
can derivatives will be increasingly used for a number
of health related benefits including their radioprotec-
tive and antimutagenic effects,175 as functional material
for gene technology,110 and in the treatment of diseases
such as arthritis,103 cancer and AIDS.91,92,210,232
Borchers et al.17 in their review of mush-
rooms/glucans and their effects on tumors and immu-
nity discussed the issue that in much of the published
experimental work, interpretation of data is very diffi-
cult due to lack of consistency in use of designations and
abbreviations for compounds. Parameters for growth,
such as time of harvest, storage conditions and extrac-
tion procedures, all influence the polysaccharide con-
tent of the fungi. Their comments can be expanded
to include the wider field of glucan research and other
sources of glucans. Details of glucan isolation and treat-
D. B. Zekovic et al.
ments before use in experimental work requires meticu-
lous documentation so that new research can effectively
build on past experimental work.
-D-glucans have been present in human diets from
the beginning of time, but only within the last 50 years
have their positive impact on human and animal health
been elucidated. They are attracting increasing attention
as new natural sources are discovered and genetically
modified ones are created. However, the glucans from
Saccharomyces cerevisiae with cost of production, simple
extraction technology and potential infinite supply will
dominate the market for the foreseeable future. Emerg-
ing technologies, revolving around glucan solubiliza-
tion and fractionation that do not alter their natural
structure, but improve their functional properties, will
be added to the established chemical and physical mod-
ifications leading to new applications in human and
animal health and nutrition.
GLOSSARY
BMRs, Biological Response Modifiers; CM-CG,
Carboxymethylated-Chitin-Glucan; CM-glucan, Car-
boxymethylated Glucan; CMGRN LE, Carboxymethy-
lated Grifolan; CMHAE, Carboxymethylated Hot
Alkali Extract; CPE assay, Cytopathic Effect Assay;
Curdlan, An insoluble linear (13)--D-glucan (ex-
tracted from Alcaligenes faecalis), MW of 50 to 80
kDa; DB, Degree of Branching; Dectin-1, A major
-glucan receptor on macrophages (immune cells);
DMSO, Dimethylsulfoxide; DP, Degree of Polymer-
ization; DS, Degree of Substitution/Sulfation; FDA,
Food and Drug Administration; Flu A, Influenza A
Virus; FRET, Fluorescence Response Energy Transfer;
Glomerellan, A (13)--glucan with a high molecu-
lar weight (670,000) isolated from Glomerella cingu-
lata; -Glucan, Complex polysaccharide derived from
sources such as the cell wall of yeast, oat and barley
fiber and many medicinal mushrooms; Glycans, Rela-
tively complex carbohydrates consisting of a number
of monosaccharides joined by glycosidic bonds; GRN-
grifolan, A -glucan isolated from Grifola frondosa; GRN
LE, grifolanAn antitumor glucan from Grifola frondosa;
HAE, Hot Alkali Extract; HDL and LDL, High Density
Lipoprotein and Low Density Lipoprotein; HSCAS,
Hydrated Sodium Calcium Aluminosilicate; HSV, Her-
pes Simplex Virus; I/R injury, Ischemia/Reperfusion
Injury; IL, Interleukin; IND, Indomethacin; INF- ,
Interferon- ; LAL, Limulus amoebocyte lysate; Laminarin
222
or Laminaran, A soluble linear (13)--D-glucan with
some branching of (16)--D-glucan (extracted from
brown seaweeds such as Laminaria digitata; Lentinan,
A purified -glucan from Lentinus (Lentinula) edode;
LPS, lipopolysaccharide; LR, Lactated Ringers So-
lution; MFL-Glucan, Glucan from Monilinia fructi-
cola.; MFN-Glucan, Glucan from Monilinia fructigena;
MIP-2, Macrophage Inflammatory Protein-2; MW,
Molecular Weight; NF-B, Nuclear Factor-B; NK
cells, Natural Killer Cells; NSAIDs, Non-Steroidal
Anti-Inflammatory Drugs; Pachyman, An insoluble
branched (13, 16)--D-glucan (extracted from Po-
ria cocos); MW of 80 to 120 kDa); Pachymanan, An
anti tumor polysaccharide derived from pachyman;
PBMC, Peripheral Blood Monocytes; PBS, Phosphate
Buffered Saline; PGG, Poly- 1-6-Glucotriosyl- 1-3-
Glucopyranose Glucan; Poly(C), Polycytidylic Acid;
PRA, Plaque Reduction Assay; PRRSV, Porcine Repro-
ductive and Respiratory Syndrome Virus; PTR, Glu-
can from Pleurotus tuberregium (sclerotia); Pustulan,
An insoluble linear (16)- -D-glucan (extracted from
Umbiliaria papulosa), MW of 20 kDa; RES, Reticulo
Endothelial System; RSV, Respiratory Syncytial Virus;
Scleroglucan, Glucan produced by the fermentation
of the fungus Sclerotium rolfsii. Main chain of (13)-
linked -D-glucopyranosyl units with every third unit
having a single -D-glucopyranosyl unit linked (16).
SE, Sulfoethyl Glucan; SG, Glucan from Sclerotium glu-
canicum; SIRS, Systematic Inflammatory Response Syn-
drome; SPG, -glucan - Schizophyllan (Sonifilan) from
Schizophyllum commune; SR, Glucan from Sclerotium rolf-
sii; SSG, A -glucan- from Sclerotinia sclerotiorum; TF,
Tissue Factor; TLR, Toll-Like Receptor; TNF, Tumor
Necrosis Factor; Zymosan, Name given in 1940s to a
crude yeast cell wall extract from S. cerevisiae. Zymosan
is a -glucan and mannan rich particle.
ACKNOWLEDGEMENTS
Thanks are given to Dr. T. P. Lyons, Alltech, Inc., for
his continuing support and generous funding.
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