The optimum environmental conditions for culture growth and polysaccharides
production of mushrooms in liquid cultures are dependent on strains. Initially, the
growth conditions of the culture were optimized that showed a stationary phase after
14 days. It was observed that L. edodes produced most polysaccharides and showed
highest immuno-stimulating activity in the stationary phase. The biomass of the CBF
extracts of all strains was estimated to be in the range 0.35 to 0.40 g/50 ml and pH
between 2.5 and 2.8 for 14 days (Fig. 2 and Fig. 3). These values would be useful as
references for optimization and pilot plant production studies in the future. Recently,
the submerged cultivation of mushroom has received much attention in Asian regions
as a promising alternative for efficient production of its valuable metabolites,
especially polysaccharides and ganoderic acids (38, 88). It usually takes several
months to cultivate the fruiting body of the mushroom, and it is also difficult to control
product quality during soil cultivation. There is a great need to supply the market with
a large amount of high-quality mushroom products. Therefore, submerged cultivation
of mushroom could eventually supplement the need and proves useful over fruiting
body cultivation.
Strain typing/phylogenetic mapping.
The ten isolates of L. edodes were grouped into three distinct clusters by AFLP:
(1) L24 and L25 isolates from China; (2) L1 and L4 isolates from Taiwan; (3) L6, L10,
L11, L15, L21 and L23 isolates from Japan. The DNA material and adaptors were
digested with restriction enzymes, Mse I and Eco RI, to prepare the AFLP template for
sequence analysis. Two primer sets, Eco RI-AC-FAM/Mse I-CAA and Eco
RI-AA-FAM/Mse I-CAC, were employed for selective amplification. The band
positions of selected primers were used to construct a similarity index and an attempt
was made to match the regionally different strains. A dendrogram of the similarity
index based on the bands obtained for selected two primer sets was plotted to
distinguish the closely related strains (Fig. 4). A very close genetic homogeneity
among cultivated strains of Japanese mushrooms, L11, L15 and L10 was seen.
Similarly, for the Taiwanese and Chinese mushrooms, the obtained AFLP fingerprints
point out close resemblance to the genetic homogeneity. However, L24 and L25
mushrooms from China were quite different from the Japanese and Taiwanese
mushrooms. The mushrooms, L1 and L4 were cultivated heterogeneous strains
comprising of Japanese strains SL-19 and 271.
The dendrogram obtained using AFLP (fingerprinting) analysis of L. edodes
provides new insight into the population structure of this mushroom species and
proves useful for phylogenetic type studies. The results appear promising and are well
supported by results obtained using random amplified polymorphic DNA (RAPD)
assay (fingerprinting) for the same species (89). Therefore, application of AFLP
fingerprint assay for phylogenetic studies of mushrooms can now be included among
other reported species in population studies.
Immuno-modulating and anti-cancer activities.
The molecular mass fractions and chemical compositions of polysaccharides
produced are strongly dependent on strain variations, extraction methods and culture
conditions (56). Therefore, a comparative study of immuno-modulating and
anti-cancer properties using CBF deserved further investigation. The CBF of all
mushroom strains were used to treat macrophage (RAW 264.7 cell line) to test the
immuno-stimulating activity (Fig. 5A). The strains with increasing NBT % reduction
follow the order: (L15>L23>L10)> (L21>L6)> (L11> L24)> (L1>L25>L4). Based on the
order of highest immuno-stimulating activity exhibits, the CBF of L15 and L23 strains
were further tested for its ability to produce TNF-α (Fig. 5B). When
lipo-polysaccharide or glucan extracts alone were used, TNF-α release was not
impressive. But adding them together resulted in synergistically increased production
of TNF-α. Similar behavior was observed previously among different strains of fungi
and it was suggested that lipo-polysaccharide triggers a so-called ‘priming effect’ on
polysaccharides (40). Thus, it was necessary to conduct further indirect anti-cancer
activity experiments using CBF. Indirect anti-cancer activity tests were conducted on
all the selected cancer cell lines with CBF of L15, L23, L10 and L21 strains (Fig. 5C).
The cancer cell survival rate was moderate (<60% for AGS and MCF-7) with L15 and
very high with L23 (for all selected cancer cell lines). Direct anti-cancer activity
experimental results did not show effective inhibition of tumors (Fig. 5D). Among all
the cell lines, gastric cancer cell line (AGS) responded to some extent better than
others. Normal cell line (MRC-5) responded in a similar range to all other cancer cell
lines.
Both CBF and BWE of L15 strain show better performance in tests for
immuno-modulating properties. The BWE of L15 was further fractionated to obtain
four different molecular mass fractions (Fig. 6). The polysaccharide content of the
fractions was in the range 0.2-0.7 mg/ml (Fig. 7A). They were further tested for
immuno-stimulating activity and roles in the direct anticancer activity. The
immunity-enhancing properties of BWE’s were similar to those of CBF. The results of
immuno-stimulating activity assays of mixtures of all fractions were similar to
individual high molecular mass fractions ‘B’ (Fig. 7B, Fig. 8). However, in the direct
anti-cancer activity assays, low molecular mass fractions gave better results
confirming their vital role in immune-stimulating pathways (Fig. 7C).
Cell-cell communication assay.
The scrape loading/dye transfer were used to demonstrate the macrophage
cellular communication. The phase contrast and fluorescent images (Fig. 9A) shows
the normal macrophage unable to transfer dye to neighboring cells. The LPS or L15
polysaccharides-treated macrophage, the results shows the lack of Lucifer yellow dye
transfer to cells (Fig. 9B and Fig. 9C). The macrophage only treated with L15
polysaccharides or LPS can not exhibition cell-cell communication. Interestingly, an
additive effect of the macrophage first treated with L15 polysaccharides and then
treated with LPS on dye transfer was found in Fig. 9D. The Fig. 9D (L15
polysaccharides plus LPS) shows the dye transfer ability higher than the Fig. 9E (LPS
plus L15 polysaccharides).
Molecular mass and monosaccharide composition.
To deduce the structure-activity relationship, it is important to determine the
molecular mass distribution and monosaccharide composition of the various
polysaccharides isolates. Gel permeation chromatographic studies showed that all
isolated polysaccharides had up to four similar molecular mass fractions (designated
as A, B, C, D) with different distribution in the range 1x102 kDa and 3x103 kDa (Fig.
10). A thorough examination revealed that the molecular mass fractions A and D were
present in all strains while fractions B and C showed some disparity.
The monosaccharide composition was determined from the standard calibration
curves plotted for individual monosaccharide. The data revealed differences in the
distribution of glucose, mannose, xylose, galactose, fucose, rhamnose and arabinose
in the CBF of all mushrooms (Table 1). For example, the L15 polysaccharides
contained mainly glucose and mannose and the contents of glucose in PS of L1, L4,
L6, L10, L11, L21, L23, L24 and L25 were 55.4%, 23.1%, 51%, 56.2%, 47.1%, 47.4%,
56.4%, 88.8% and 55.6%, respectively. The nature of these compositions and
differences remain unclear at this moment and would need further investigation when
more relevant knowledge and analytical skills are available.
Structures.
The structural features of the crude polysaccharides/BWE extracted from strain
L15 were elucidated by using the FT-IR, NMR and GC-MS spectroscopic techniques
to establish structure-immuno-stimulating and anti-cancer activity relationships. In the
FT-IR spectra of the crude PS (all strains), the bands corresponding to the ν (C=O)
vibration in the carboxyl group at 1650 cm-1 indicate that this carboxyl group was
hydrogen bonded (Fig. 11). The absence of carbonyl bands at 1535 cm-1 and 1700
cm-1 indicates that these strains contain neither proteins nor uronic acids, respectively.
In addition to the characteristic bands of glucans in the 1000-1100 cm-1 range, FT-IR
spectra showed a weak band at 850 cm-1 (Fig. 11) that revealed the ‘α’ configuration
of the main glucan linkages (79).
The chemical shifts of individual proton and carbon peaks are shown in the NMR
spectra of L15 BWE (Fig. 12). The two groups of anomeric proton signals centered at
δ 5.29 and 4.93 ppm were assigned to (1→4)-D-Glcp and (1→6)-D-Glcp, respetively,
Fig. 12A (71). The two major carbon peaks at δ 99.68 and 102.43 ppm were assigned
to α-(1→4)-D-Glcp and β-(1→6)-D-Glcp residues, respectively (Fig. 12B). The
anomeric carbon signals at δ 71.55, 71.28, 76.06, 70.09, and 60.86 ppm were
assigned, respectively, to C-2, C-3, C-4, C-5, and C-6 of α-(1→4)-D-Glcp, due to their
relatively higher peak intensities. The other carbon signals assigned for
β-(1→6)-D-Glcp are C-2 (73.16), C-3 (76.79), C-4 (69.75), C-5 (73.45) and C6 (66.96).
Further confirmation was based on analysis for alditol acetates using GC-MS in crude
polysaccharides of L15 (Fig. 13). The resulting chromatograms demonstrated the
presence of α-(1→4)-linked-D-glucopyranosyl and β-(1→6)-linked-D-glucopyranosyl
moieties in the glucan, i.e. peak a: 1,4,5-tri-O-acetyl-1-deuterio-2,3,6-tri-O-
methyl-D-glucitol (m/z: 43, 59, 71, 87, 102, 118, 129, 142, 162, 173, 233; α-(1→4))
and peak b: 1,5,6-tri-O-acetyl-1-deuterio-2,3,4 -tri-O-methyl-D-glucitol (m/z: 43, 59, 71,
87, 102, 118, 129, 143, 162, 173, 189, 233; β-(1→6)). The 1,4,5,6-tetra-O-acetyl-1-
deuterio-2,3-di-O-methyl-glucitol (m/z: 43, 59, 74, 85, 102, 118, 127, 142, 162, 201,
261) indicates (1→4) and (1→6) linkage (71).
Linkage assignment was further confirmed by the 1H-13C HSQC 2D-NMR
spectrum that shows the cross relationships of the proton and carbon peaks that
define glycosidic linkages (71, 90, Fig. 14). Note that the chemical shifts of C-4 and
C’6 typify (1→4)-linked and (1→6)-linked glucose residues, respectively (91). Also
note that the relative positions of the anomeric signals for both constituents (denoted
H-1 and H’-1) are typical for an α-anomeric and a β-anomeric configuration. Although
this linkage has been found for other mushrooms, it is new for the BWE from L.
edodes.
Analysis of the L15 BWE monosaccharide composition revealed that the one with
the highest content is glucose (68.9%), the next is mannose (20%), and the remaining
five constitute a total of 11%. This composition is rare, particularly for the presence of
a total of seven different monosaccharides, and has not been reported in the literature.
The tentative 13C NMR peak assignments for the mannopyranosyl residual are: C1
(96.02), C2 (69.65), C3 (70.35), C4 (78.5), C5 (74.25), C6 (61.2) (37, 92).
Discussion
The procedures adopted to test the immuno-modulating and anti-cancer
activities were similar to the experiments described by several authors (34, 80). The
common protocol accepted is the use of fruiting bodies, extractions using aqueous or
non-aqueous phases and structural characterization of the isolated products. Some
experimental modifications were introduced including the method of isolation of the
polysaccharides and the experimental steps involving treatment of cancer cell lines
with polysaccharides. An important result of this study was the ability of the
polysaccharides to stimulate immune cells irrespective of method of isolation, i.e.
either using CBF or BWE or fractionated BWE. The polysaccharides from both BWE
and crude polysaccharide seem to contain similar backbone structures.
Ohno et al. (40) reported that the release of TNF-α by macrophages could be
induced by β-glucans with specific molecular weights and lower branching ratios. The
mechanisms for the recognition of β-glucans by macrophage were proposed to be
fairly complex and the β-glucans were assumed to be broken down to lower molecular
weight fragments through various cellular functions (40, 43). Also, the addition of
lipo-polysaccharide resulted in a “priming effect” and increased the TNF-α production
by various β-glucans (40, 93). Our results showed that structurally different α-glucans
extracted from different mushroom strains could also stimulate RAW 264.7 cells to
secret TNF-α. A synergistic effect on the TNF-α release by adding
lipo-polysaccharide was also observed in the in vitro studies. It would be of interest to
see if the same effect is observed in the in vivo studies.
In the indirect anti-cancer activity, the cytotoxic response of peritoneal
macrophages and enhanced release of TNF-α are accounted for increased
immuno-stimulating activity. Among the regional strains classified, the mushroom
isolates from Japan showed the highest immuno-stimulating activity, followed by
isolates from China and Taiwan, respectively. ploysaccharides in different strains of
mushroom have anti-cancer activities that differ greatly in their chemical compositions
and configurations. Although it is difficult to correlate the structure and anti-cancer
activity, particularly due to the difficulty in determining the three dimensional
structures, some correlations can be inferred.
Earlier investigations revealed that the enhanced immuno-stimulating activity of
the mushrooms might be due to the presence of α-glucans (94). Not only mushrooms,
α-glucan in the polysaccharides was also identified in several medicinal plant extracts
(94-96). All of these active polysaccharides, regardless of their origin, were obtained
by different extraction methods and collected at different fractions and were found to
vary in polymer structures. In some of the studies, extracted glucans with varying
substituted monosaccharides in the side chain have shown enhanced
immuno-modulating activity (75), e.g. Bao et al. employing hot water extracted
polysaccharides from Ganoderma lucidum. (73). Ukawa et al. found out that the
neutral monosaccharide composition, i.e. fucose, xylose., mannose., galactose, and
glucose, in the polysaccharides from isolates of fruiting bodies of Lyophyllum
decastes Sing. were different from other species and this along with various protein
content exerted increased anti-cancer activity (97). Tomati et al. (77) discovered that
the proportion of the monosaccharides present in the hot water extract of CBF of L.
edodes, glucose content was the highest and xylose second, with a 7:1 ratio. Other monosaccharides were ribose, arabinose and mannose and the total content was less
than 1% (77). Similar phenomena were observed for some medicinal plant extracts,
used as anti-cancer compounds (98).
Conclusions
Natural polysaccharides with various monosaccharide compositions are difficult
to synthesize in the laboratory, yet they are quite efficacious in many biological events
and pharmacological treatment. We have established an experimental platform by
adopting several classical and modern chemical and molecular biological protocols to
prepare these materials and to use them for further studies and biotechnological
applications. These include submerged liquid phase culture, separation by extraction,
precipitation and chromatographic purification, molecular mass and structural
characterization by various chromatographic and spectroscopic techniques such as
FT-IR, NMR and GC-MS, and various biological activity assays. This report reveals
that the polysaccharide fractions in the molecular mass range 1x102 and 3x103 kDa of
the culture broth filtrate (CBF) and boiling water extract (BWE) from L. edodes mycelia
are able to show macrophage-stimulating and indirect anti-cancer activities. A new
form of polysaccharide linkage with a backbone of α-(1→4)-glucan and side chains of
β-(1→6)-glucan has been identified. It is important to further identify the key structural
features at lower molecular mass fractions necessary to maintain similar or better
biological activities to establish a clearer structure-activity relationship and for more
convenient handling of future pharmacological and nutraceutical applications. The
detailed mechanisms as well as related signal transductions involved with the
biological activity such as macrophage-stimulation and subsequent release of TNF-α
and/or other biochemicals to inhibit tumor growth need further clarification. The effects
of these polysaccharides on other biological events such as cell-cell interactions are
also worth exploring. We are currently in the process to do so.
Tables
PartⅠ
Table 1. Monosaccharide composition of fractionated polysaccharide from different strains of L. edodes.
Lentinula edodes L1 L4 L6 L10 L11 L15 L21 L23 L24 L25
Arabinose % 6.03 11.19 8.25 7.77 6.76 5.41 8.42 7.73 2.22 6.45 Xylose % 4.82 10.65 5.89 5.58 5.21 3.83 5.88 5.49 1.13 4.55 Mannose % 32.28 49.22 31.83 26.89 36.24 20.02 33.5 26.72 7.23 30.91 Galactose % 1.44 5.09 2.59 3.56 4.3 1.42 3.73 3.19 0.03 1.87 Glucose % 55.44 23.1 50.95 56.2 47.12 68.93 47.36 56.36 88.82 55.58 Rhamnose % 0 0.22 0.24 0 0.14 0.28 0.56 0.32 0.45 0.4 Fucose % 0 0.52 0.24 0 0.24 0.11 0.56 0.19 0.12 0.24 Exopolysaccharide
Content (mg/ml)
0.58 0.61 0.53 0.31 0.48 0.44 0.2 0.15 0.59 0.59
Figure Captions
PartⅠ
Fig. 1: Step-wise experimental protocol adopted to isolate CBF and crude polysaccharide from L. edodes mycelia.
Fig. 2: Time course of the mycelium growth of L. edodes ‘L11’ strain submerged liquid culture.
Fig. 3: Dry cell weight of mushroom strains.
Fig. 4: Dendogram of L. edodes constructed using AFLP assay.
Fig. 5: (A) Macrophage stimulatory activity assay (% NBT reduction) using CBF of all strains. (B) TNF-α release activity using CBF of L15 and L23. (C) Indirect
anti-cancer assay (% MTT reduction) using CBF of 4 different strains. (D)
Direct anti-cancer assay using CBF of all strains. The CBF was first treated
with immune cell lines-RAW 264.7 (% NBT reduction) and J45.01 (% MTT
reduction) and then added to other different cancer cell lines (% MTT
reduction).
Fig. 6: Molecular mass fractions of L15 BWE. (A) >2750 kDa (B) ~2700 kDa (C)
~534 kDa (D) ~11.7 kDa.
Fig. 7: (A) Polysaccharides content of different molecular weight fractions (A, B, C, D and E) extracted from L15 BWE and control sample from L15 BWE
mixture fractions. (B) Macrophage stimulatory activity (% NBT reduction) of
obtained weight fractions, and, (C) Direct anti-cancer assay (% MTT
reduction) of obtained weight fractions (PBS as control).
Fig. 8: Macrophage stimulatory activity morphological images by NBT reduction assay. (A), (B), (C), (D) and (E) show that macrophage was treated of
different molecular weight fractions (A, B, C, D and E) extracted from L15
BWE (L15 BWE mixture fractions as control sample and PBS as blank
control).
Fig. 9: Macrophage cell-cell communication measured by the Lucifer yellow scrape-loading/dye transfer technique. (A) Phase contrast and fluorescent
images of normal macrophage, (B) LPS treatment of macrophage, (C) L15
polysaccharides treatment of macrophage, (D) L15 polysaccharides (1.5
mg/ml) plus LPS (10 μl/ml) treatment macrophage, and (D) LPS (10 μl/ml)
plus L15 polysaccharides (1.5 mg/ml) of macrophage.
Fig. 10: GP chromatogram obtained from CBF of mushroom strains. Molecular mass fractions are indicated.
Fig. 11: A typical FTIR spectrum recorded from crude polysaccharides of L15.
Fig. 12: NMR spectra of crude polysaccharides boiling water extracted from L15. (A) The two anomeric proton signals are at δ 5.29 and 4.93 ppm that were
assigned as (1→4)-D-Glcp and (1→6)-D-Glcp (600 MHz), (B) The anomeric
carbon signals for the (1→4)-D-Glcp and (1→6)-D-Glcp residues were
assigned at δ 99.68 and 102.43 ppm, respectively, from 13C (150 MHz). The
carbon signals at δ 71.55, 71.28, 70.09, and 60.86 ppm correspond,
respectively, to C-2, C-3, C-5, and C-6 of (1→4)-D-Glcp. The other signals
for (1→6)-D-Glcp are C-2 (73.16), C-3 (76.79), C-4 (69.75), and C-5
(73.45),
Fig. 13: GC-MS data for the alditol acetates derived from the methylated polysaccharide BWE isolated from L15 L. edodes. The presence of (a)
1,4,5-tri-O acetyl-1-deuterio-2, 3, 6-tri-O-methyl-D-glucitol and, (b) 1,5,6-
tri-O acetyl-1-deuterio-2, 3, 4-tri-O-methyl-D-glucitol were detected. (c)
The1,4,5,6-tetra-O-acetyl-1-deuterio-2,3-di-O-methyl-glucitol indicates (1→
4) and (1→6) linkage.
Fig. 14: The 2D (HSQC) NMR spectrum of the L15 BWE in D2O.
PartⅠ
Fig. 1 Step-wise experimental protocol adopted to isolate CBF and crude polysaccharide from L. edodes mycelia.
Fig. 2 Time course of the mycelium growth of L. edodes ‘L11’ strain submerged liquid culture.
Fig. 3 Dry cell weight of mushroom strains.
Fig. 4 Dendogram of L. edodes constructed using AFLP assay.
0 5000 10000 15000 20000 25000 30000
Control LPS CBF CBF+Medium CBF+LPS
TNF-a production (pg/ml)
L15 L23
0 5000 10000 15000 20000 25000 30000
Control LPS CBF CBF+Medium CBF+LPS
TNF-a production (pg/ml)
L15 L23
Fig. 5 (A) Macrophage stimulatory activity assay (% NBT reduction) using CBF of all strains. (B) TNF-α release activity using CBF of L15 and L23. (C) Indirect anti-cancer
assay (% MTT reduction) using CBF of 4 different strains. (D) Direct anti-cancer assay
using CBF of all strains. The CBF was first treated with immune cell lines-RAW 264.7
(% NBT reduction) and J45.01 (% MTT reduction) and then added to other different
cancer cell lines (% MTT reduction).
Fig. 6 Molecular mass fractions of L15 BWE. (A) >2750 kDa (B) ~2700 kDa (C) ~534 kDa (D) ~11.7 kDa.
Fig. 7(A) Polysaccharides content of different molecular weight fractions (A, B, C, D
A
B C D
A E
B C D
E
and E) extracted from L15 BWE and control sample from L15 BWE mixture fractions.
(B) Macrophage stimulatory activity (% NBT reduction) of obtained weight fractions,
and, (C) Direct anti-cancer assay (% MTT reduction) of obtained weight fractions
(PBS as control).
Fig. 8 Macrophage stimulatory activity morphological images by NBT reduction assay.
(A), (B), (C), (D) and (E) show that macrophage was treated of different molecular
weight fractions (A, B, C, D and E) extracted from L15 BWE (L15 BWE mixture
fractions as control sample and PBS as blank control).
PBS Control (A)
(B) (C) (D) (E)
Fig. 9 Macrophage cell-cell communication measured by the Lucifer yellow scrape-loading/dye transfer technique. (A) Phase contrast and fluorescent images of
normal macrophage, (B) LPS treatment of macrophage, (C) L15 polysaccharides
treatment of macrophage, (D) L15 polysaccharides (1.5 mg/ml) plus LPS (10 μl/ml)
treatment macrophage, and (D) LPS (10 μl/ml) plus L15 polysaccharides (1.5 mg/ml)
of macrophage.
(D) L15 polysaccharides plus LPS
(A) Control (B) LPS
(C) L 15 polysaccharides
(E) LPS plus L15 polysaccharides
(D) L15 polysaccharides plus LPS
(A) Control (B) LPS
(C) L 15 polysaccharides
(E) LPS plus L15 polysaccharides
Fig. 10 GP chromatogram obtained from CBF of mushroom strains. Molecular mass fractions are indicated.
Fig. 11 A typical FTIR spectrum recorded from crude polysaccharides of L15.
A B
C A D
B
C D
(A)
(B)
Fig. 12 NMR spectra of crude polysaccharides boiling water extracted from L15. (A) The two anomeric proton signals are at δ 5.29 and 4.93 ppm that were assigned as
(1→4)-D-Glcp and (1→6)-D-Glcp (600 MHz), (B) The anomeric carbon signals for the
(1→4)-D-Glcp and (1→6)-D-Glcp residues were assigned at δ 99.68 and 102.43 ppm,
respectively, from 13C (150 MHz). The carbon signals at δ 71.55, 71.28, 70.09, and
60.86 ppm correspond, respectively, to C-2, C-3, C-5, and C-6 of (1→4)-D-Glcp. The
other signals for (1→6)-D-Glcp are C-2 (73.16), C-3 (76.79), C-4 (69.75), and C-5
Fig. 13 GC-MS data for the alditol acetates derived from the methylated polysaccharide BWE isolated from L15 L. edodes. The presence of (a) 1,4,5-tri-O
acetyl-1-deuterio-2, 3, 6-tri-O-methyl-D-glucitol and, (b) 1,5,6-tri-O acetyl-1-deuterio-2,
3, 4-tri-O-methyl-D-glucitol were detected. (c) The1,4,5,6-tetra-O-acetyl-1-deuterio-
2,3-di-O-methyl-glucitol indicates (1→4) and (1→6) linkage.
b
PARTⅡ
Pressurized Water Extraction of Polysaccharides as Secondary Metabolites from Lentinula edodes
Abstract
The suitability of pressurized water extraction (PWE) of crude polysaccharides
as secondary metabolites from L. edodes was investigated. A series of experiments
were carried out to examine the effects of extraction times and pressures. The results
indicated that the maximum recovery of polysaccharides was about 90% of the crude
polysaccharides from mycelia pellets when the pressure was at 10.1 MPa for 70 min
(28 °C). This was a drastic improvement over that of boiling water extraction (BWE) at
0.1 MPa for 40 min, which gave only 27.9% recovery. A nitroblue tetrazolium (NBT)
reduction assay was used to examine the macrophage stimulating activities (MSA),
and it was found that the PWE polysaccharides retained the MSA. The morphology of
the macrophage cells treated by PWE polysaccharides was also examined and found
to be similar to that of the positive control lipopolysaccharides treated. Finally, gel
chromatographic and NMR experiments revealed that both PWE and BWE
polysaccharides showed the presence of four similar molecular mass components
polysaccharides showed the presence of four similar molecular mass components