Antimicrobial effects of β-glucans and pectin and of the agaricus. Blazei-based mushroom extract, andosan™ examples of mouse models for pneumococcal-, fecal bacterial-, and mycobacterial infections

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Antimicrobial effects of β-glucans and pectin and of the Agaricus blazei-
based mushroom extract, AndoSanTM. Examples of mouse models for
pneumococcal-, fecal bacterial-, and mycobacterial infections.
G. Hetland1, E. Johnson2,3, D.M. Eide4, B. Grinde5, A.B.C. Samuelsen6 and H. G. Wiker7,8
1Depts of Immunology and Transfusion Medicine 2Gastroenterological and Pediatric Surgery, Oslo University Hospital,
3Inst of Clinical Medicine, Medical Faculty, and 6School of Pharmacy, Faculty of Mathematics and Natural Sciences,
University of Oslo, 5Depts of 4Environmental Medicine and 5Mental Health, Norwegian Inst of Public Health, Oslo, and
7Dept of Microbiology, Haukeland University Hospital, Bergen and 8The Gade Research Group for Infection and
Immunity, Dept of Clinical Science, Faculty of Medicine and Dentistry, University of Bergen, Norway.
The increasing occurrence of multi drug-resistant (MDR) pathogenic microbes is a threat to the public health and prompts
a call for novel antimicrobial strategies. In Eastern traditional medicine edible mushrooms have been used for over 3000
years against a range of diseases including infections. β-glucans from yeast and mushrooms and pectin from Plantago
major L. have anti-infectious properties in rodent models against different microbes, including mycobacteria. The
medicinal mushroom Agaricus blazei Murill, used traditionally against cancer and hepatitis, has been found to have anti-
tumor effects in mouse models. An Agaricus extract, AndosanTM, also containing two related mushrooms has been shown
to protect against both Gram-positive and -negative sepsis in mice and has been tested against viral infections, as reviewed
here. Thus, in the future, biologically active substances isolated from medicinal mushrooms and plants, may prove useful
alternatives in the fight against serious infections by MDR pathogens.
Keywords MDR microbes, pneumococci, fecal bacteria, mycobacteria, β-glucan; Plantago major L., Agaricus blazei
Murill, AndosanTM
1. Introduction
Besides the well-known hospital tormentor, methicillin-resistant Staphylococcus aureus (MRSA), life-threatening
bacteria such as Mycobacterium tuberculosis, Streptococcus pneumoniae and others are becoming MDR, suggesting
that the microbes may win the battle over antibiotics. That would not be surprising because microbes have been on this
planet far longer than humans and have survived huge climate changes and are today adopted to life in quite different
habitats, e.g. thermic bacteria in Yellowstone geysers and in volcanoes that live on sulphur, pressure (>1000x
atmospheric pressure)-resistant bacteria in the sub-sea Mariana depression in the Pacific ocean near Guam [1],
anaerobic bacteria in the gut etc. The mechanisms for development of MDR in microbes under repression of
bacteriostatical or bactericide drugs, include lateral transfection of other strains and species of bacteria by plasmids
containing genes for drug resistance. One reason is unwise over-use of antibiotics for infections, also most probably
viral ones such as those causing otitis media in small children, or under-use - too low doses or shortened antibiotics
administration. The MRD infection epidemic is of great concern to the public health and new strategies are called for to
regain the upper hand in this battle.
In Eastern and African traditional medicine edible mushrooms and medicinal herbs have been used for thousands of
years against a range of diseases including infections, which is still the major health threat in Africa. This empiric
knowledge is very little tapped into by Western medicine and it is therefore pertinent to exploit this field in search of
drugs novel to Western societies that may supplement or adjuvate our current hospital therapies for infectious diseases
and also in other disciplines. Especially, it is of interest to activate the innate arm of the immune system because it is
evolutionary old and shared with sea urchin, sebra fish and banana fly, and has thus been successfully exploited in other
and more ancient species than ours. Many fungi and mushrooms are lethal to insects, animals and humans and we have
therefore developed specific receptors, e.g. toll-like receptor 2 (TLR2), on immune cells for the common fungal
signature molecule, β-1,3-D-glucan, that is the main structural ingredient of the cell wall of fungi and mushrooms. β-
glucan are also found in some bacteria and plants [2]. We share TLR with the banana fly, Drosophila melanogaster, in
which these receptors were detected in 1985 [3] and with rodents and other mammals. Other non-TLR receptors that
may be involved are dectin-1 and the lectin-binding site in CD11b/18 (complement receptor 3). Most probably fungi
and mushrooms also contain other such so-called pathogen-associated molecular pattern substances that may similarly
activate a native immune response in the host against potential danger. Therefore, β-glucans and other
immunomodulating molecules in fungi and mushrooms represent danger signals that trigger cells of the innate immune
system against potentially lethal attack from the outer world. β-glucans are D-glucose polymers linked by β-glycosidic
bonds. The structures of two different types of β-glucans are shown in Fig. 1. SSG (scleroglucan) is a soluble, although
viscous, gel-forming and highly branched β-glucan with high molecular weight (>5x106 kD) from the culture broth of
the fungus Sclerotinia sclerotiorum [4]. In Japan similar β-1,3-glucans from mushrooms such as lentinan, have been
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used in combination with chemotherapy to treat cancer patients for more than 30 years [5]. MacroGard® is a β-1,3-
glucan extracted from baker’s yeast with less frequent side-chains but that contain more than two glucose molecules. It
is a potent immuno-stimultant produced in both a soluble and particular form [6]. Others and we have found that
harmless substances such as β-glucans and the edible Bacidiomycetes mushroom, Agaricus bM, can be exploited to
enhance the immune response against invading and dangerous microbes both of extracellular, e.g. E.coli [7] and
pneumococci [8,9], and obligate intracellular nature, e.g. mycobacteria. The anti-mycobacterial properties of β-glucans
have been proven both for M. tuberculosis-infected macrophages in vitro [10] and in a M. bovis, BCG model in mice
[11]. Similar to β-glucans, Agaricus bM is also found to stimulate TLR2 [12].
Actions of innate immunity are swift, powerful and general and may thus be effective against different infections -
MDR ones or not. β-glucan is a structural polyglucose in the cell wall of yeast and mushrooms with known
immunomodulatory, antitumor and anti-infection effects in rodent models. Plantago major L. is a plant used
traditionally for wound healing world-wide [13], and we have shown that isolated biologically active pectin
polysaccharides from it have anti-infection effects in a mouse model for pneumococcal sepsis [14]. The medicinal
Basidiomycetes mushroom Agaricus bM (Fig. 2) is a.k.a. A. brasiliensis because of its Brazilian origin, but has also
been designated A. rufotegulis and A. subrufescens, already described in 1893 by CH Peck, [15]. It is closely related to
the common champignon, A. bisporus, which has also been found to have benefial health effects [16]. Since the
mushroom was used in traditional medicine in Brazil against cancer, chronic hepatitis and other serious conditions, it
was taken to Japan in the mid 1960-ties and cultivated commercially as health food. Scientists have since then
documented immunomodulatory and antitumor effects of Agaricus bM in mouse models and we have found that an
extract of it, AndosanTM, that also contains two other related Basidiomycetes mushrooms from Japan, i.e. Hericium
erinaceus and Grifola frondosa, can protect against both Gram positive and Gram negative coliform sepsis in mice
[9,17]. This was basis for Dr. SV Bernardshaw’s PhD thesis in 2007 at the University of Oslo, Norway. Effect of
Andosan was also examined against influenza infection in mice as well as against chronic hepatitic C virus infection in
humans. Here, we review antimicrobial findings with bioactive polysaccharides such as β-glucans and Plantago major
L. pectin and a combined extract of medicinal Bacidiomycetes mushrooms in in vitro systems and in examples of
infection models in the mouse.
Fig. 1 Structure of two different types of β-1,3-glucan: Structural composition of SSG (scleroglucan) and MacroGard® showing the
β-1,3-linked backbone and its β-1,6-attached side-chains, which are responsible for the binding to glucan receptors (CD11b/18,
TLR2, dectin-1) and the resulting immunomodulatory effect. The figure, which also shows another SSG-like β-1,3-glucan, lentinan,
is modified from an illustration in the publication: “MacroGard®: structural aspects and basic mode of action on phagocytes” from
Biotec Pharmacon ASA, Tromsø, Norway by R.E. Engstad. Also see [6].
Fig. 2 Agaricus blazei Murill. Photo NutriCon.
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2. Infection models in which β-glucans and pectin have been used:
2.1. β-glucans and M. tuberculosis – infected macrophage cultures
Since β-glucans stimulate innate immune cells such as monocytes and macrophages via binding to TLR2 and other
receptors, it was pertinent to examine whether these host cells for obligate intra-cellular pathogens such as
mycobacteria, could be activated to intracellular killing of these parasites. We used both a highly virulent strain of M.
tuberculosis, the culprit of the cardinal bacterial infection, tuberculosis, and its attenuated live vaccine; M. bovis,
Bacillus Calmette-Guerin (BCG).
Peritoneal macrophages were harvested from Balb/c mice and cultured in vitro. The cells were then infected with the
highly virulent M. tuberculosis strain H37Rv in presence or absence of PBS control, scleroglucan (SSG) or particulate
yeast β-glucan MacroGard®(MG). After 24 h of co-incubation extracellular bacteria were washed away, the
macrophages lysed and the lysate with the intracellular bacteria cultured for 3 weeks on Løwenstein-Jensen egg
medium and examined by immunofluorescence microscopy after auramin O-staining of the acid-fast bacilli. Although
SSG at 0.5 mg/ml gave a significant 40% reduction in the number of M. tuberculosis colony-forming units (CFU),
particulate MG (but not soluble MG; not shown) was 50x more efficient at inhibiting M. tuberculosis growth dose-
dependently (Fig. 3) [10].
Fig. 3 Effect on M. tuberculosis growth of yeast β-glucan (MacroGard® =MG) and scleroglucan (SSG) incubated simultaneously for
24 h with the tubercle bacteria in macrophage cell cultures, previously published [10].
2.2. β-glucan and M. bovis, BCG– infected Balb/c mice
Instead of hazardous animal studies with highly virulent M. tuberculosis bacteria, we chose to establish a model for M.
bovis, BCG, in the susceptible Balb/c mice by i.v. injection of viable bacteria into the tail vein. The animals were
injected i.v. either 100 µg of soluble β-glucan from barley (G-6513, from Sigma) or vehicle (PBS). At the peek of
infection 4 weeks after challenge, the mice were sacrificed and major organs homogenized and cultured or stained for
immunofluorescence microscopy. We found significantly lower bacterial counts in the spleen (p=0.01) of β-glucan-
treated than of PBS-treated mice when given pre-challenge (Fig . 4). Similar findings were done in liver homogenates
(not shown). When the β-glucan was injected post-challenge there was also a significantly lower bacterial load
(p<0.05) in the spleen (Fig. 4), but not in the liver [11].
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Fig. 4 Immunofluorescence microscopy of M. bovis, BCG, bacteria (formaldehyde-fixed and treated with auramin O) in spleen of
Balb/c mice (n=8) sacrificed 4 weeks after challenge. Animals were given 100 µg of barley β-glucan i.v. 3 days pre- or 7 days post-
challenge. P<0.05, **P<0.001. Adapted from figure in [11].
2.3. β-glucan and pneumococcal sepsis
NIH/OlaHsd mice infected i.p. with S. pneumoniae serotype 6B were also injected i.p. with SSG (4 µg (low dose)-200
µg (high dose)) or PBS either 3 days before or 3 h, 24 h and or 72 h after bacterial challenge. Tiny blood samples were
collected daily from the lateral femoral vein and plated, and the number of bacteria (CFU) in the animals’ blood and
their survival were recorded. Pre-challenge SSG administration protected against S. pneumoniae sepsis as shown by a
dose-dependent inhibition of bacteremia and increased survival rates up to 50% with 200 µg of SSG as compared with
10% survival after 14 days of the PBS-treated mice (P=0.005) [8] (figs not shown). This high dose of SSG injected once
post-challenge after 24 h had curative effect against S. pneumoniae 6B as demonstrated by 40% survival at end of
experiment compared with none of the PBS controls (P=0.02) (Fig. 5).
Fig. 5 Survival (median values, n=8) from peritonitis and sepsis of NIH/OlaHsd mice challenged with S. pneumoniae serotype 6B
and treated with PBS or SSG β-glucan (L=low and h= high dose) 3 h, 24 h and/or 72 h later (adapted from [8]).
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2.4. Pectin: Plantago major L. pectin PMII and pneumococcal infection in mice
Plantago major L., large plantain leaves, have been used as a wound healing remedy in traditional medicine for
centuries and in most parts of the World [13]. The purposed wound healing effect is not well documented. Never-the-
less, one might regard this plant as a potential source of immunomodulatory components triggering the healing process
or other processes involving the innate immune system. This was supported by Lithander [18] who reported
prophylactic effects of a P. major aqueous extract on mammary cancer in mice, indicating immunomodulatory
activities. A complex pectin fraction, PMII, that was isolated from the leaves of P. major showed anti-complementary
(complement-fixing) activity in vitro and was also shown to induce TNFα secretion after stimulation of human
monocytes [19,20,21].
Pectin polysaccharides are water soluble compounds found in the cell wall of dicotyledons. In general, pectins are
composed of unbranched homogalacturonan regions and regions with different types of side chains such as
arabinogalactans, galactans or arabinans linked to rhamnogalacturonan sequences of the backbone in so called
rhamnogalacturonan I (RGI) structures. In addition, single xylose residues and well defined side chains called
rhamnogalacturonan II (RGII) are found linked to the galacturonan backbone. For review, see [22]. The fine structures
of RGI vary with regard to monosaccharide composition, linkages, ramification and chain length. Structurally, PMII
was composed of galacturonic acid (71.7 %), rhamnose (4.2%), arabinose (8.8 %), galactose (8 %) and glucose and had
a molecular weight of 46-48 kDa. PMII was highly methylesterified (67 %), and contained both smooth and ramified
regions. Structure-activity studies revealed that the RGI-like structures of PMII containing 1,4- and 1,3,6-linked
galactose residues had the highest anti-complementary activity [23,20]. The fine structure and bioactivity of pectins
from different sources vary. For instance, cabbage (Brassica oleracea) leaves, which are also used to aid the healing of
wounds in folk medicine, were found to contain pectin fractions with lower complement-fixing activity than PMII [24]
even when the same isolation procedure was applied. Multivariate statistical analysis suggested that pectin activity is
enhanced by the content of 1,6- and 1,3,6- galactose side chains and low amounts of homogalacturonan regions [25]. It
was also found that isolated single side chains of white cabbage pectin did not affect the complement system, side
chains were only active when attached to the rhamnogalacturonan backbone [26]. The isolation procedure also affect
the structure of isolated pectins as well as their activity [27]. Due to modest activity in the complement system, Brassica
pectins were not subjected to further testing.
Due to its high complement-fixing activity, PMII was subjected to an in vivo study revealing a protective effect
against bacterial infection in mice. Inbred NIH/OlaHsd and Fox Chase SCID mice were pretreated with i.p. 12, 120 or
1200 µg PMII, 1.2 µg LPS or PBS 3 days before infection with S. pneumoniae serotype 6B. In PBS treated mice,
bacteremia levels increased after one day (see Fig. 6), and after 3 days none of the PBS treated mice were alive
compared to ≥ 50 % in the PMII and LPS-treated groups. In PMII treated mice bacteremia rose moderately until
reaching PBS levels at day 9, whereas bacteremia levels in LPS treated mice reached lethal levels after 4 days. PMII
had no effect after established infection, and there was not found any correlation between levels of anti-6B
pneumococcal IgM or IgG antibodies and the dose of PMII given indicating that the protective effect was due to
stimulation of the innate rather than the adaptive immune system [14].
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Fig. 6 Colony-forming units (CFU) in peripheral blood from NIH/OlaHsd female mice pretreated with PBS, PMII low dose (PM L):
12 µg, median dose (PM m) 120 µg, high dose (PM h) 1200 µg or E.coli LPS (1.2 µg) i.p. 3 days before challenge with 10
6
pneumococci 6B i.p. The data points represent median values from eight animals. Reprinted from [14] with permission from John
Wiley & Sons, Inc.
3. Infection models in which Agaricus blazei extract has been used:
3.1. Antimicrobial effects of the Agaricus blazei Murill-based mushroom extract AndoSanTM.
The potential anti-bacterial effect of the Agaricus blazei Murill (AbM) (82%)-based Basidiomycetes mushroom extract,
AndoSan™ (Immunopharma AS, Høvik, Norway), including Hericeum erinaceus (15%) and Grifola frondosa (3%),
was studied in mice given monobacterial or fecal polymicrobial peritonitis. AndoSan™ was introduced by orogastric
intubation to NIH/OlaHsd mice prior to (24 h or 2 h) or simultaneously with induction of peritonitis by intraperitoneal
inoculation with moderately virulent S. pneumoniae serotype 6B [9]. End points were bacteremia and survival rate.
Controls were mice treated likewise but given PBS instead of AbM. The number of CFU was significantly reduced in
the AbM group compared to the PBS group (Fig. 7A). Furthermore, the effect was comparable and more pronounced
when given 24 h before relative to 2 h before or simultaneously with the induction of pneumococcal peritonitis. The
survival of the mice was improved in the three AbM treated groups of mice, but was also most pronounced (50%) after
treatment of AbM 24 h prior to induction of peritonitis (Fig. 7B). Since cultivation of the bacteria in the presence of
AbM on agar plates indicated no detectable reduction of number of CFU, AbM per se had no antimicrobial effect on the
pneumococci. Increases in the level of pro-inflammatory cytokines MIP-2 (murine equivalent to human IL-8) and
TNFα in the serum of mice receiving AbM once but more pronounced when received twice, indicated that the
protective effect of AbM was mediated by involvement of the native immune system. In order to study further the
potential protective effect of AbM in a more physiologically relevant setting for clinical and secondary aerobic
peritonitis, an experimental and reproducible model for induction of fecal peritonitis was developed in Balb/c mice [17].
Dilutions 1/4, 1/8 and 1/12 of mouse feces inoculated i.p. in the mice lead to severe, moderate and mild peritonitis,
respectively. In this model, using AbM compared with control (PBS) introduced orogastrically 24 h before bacterial
inoculation, a significant protection was revealed as measured by significantly improved overall survival for all degrees
of peritonitis (45% vs 28%) and particularly, severe peritonitis (25% vs 0%) (Fig. 8). Similarly, there was as significant
reduction of CFU in AbM-treated mice with severe and moderate peritonitis [17]. The temperature measurements
showed a negative correlation with the degrees of septicemia conditions and higher CFU, which is normal for septic
mice. These animal experiments took place at The Norwegian Institute of Public Health, Oslo. Quantitative and
qualitative characterization of the bacteremia revealed that both Gram-positive streptococci and Gram-negative
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coliform bacteria dominated. In both studies the protective effect of AbM was demonstrated by use of two different
strains of mice expressing either Th-1/Th-2 balanced immunity (NIH/OlaHsd) or pronation towards Th-2 immunity
(Balb/c). A moderately virulent S. pneumoniae 6B [9] or fecal bacterial flora [17] were used in these studies. Since
AndosanTM seems to inhibit TLR4 (the LPS receptor)-mediated cellular stimulation of NF-κB activation, this may
partly explain the observed protection against Gram negative sepsis in the mouse model [12].
Fig. 7A Number of CFU of S. pneumoniae serotype 6B in blood of NIH/OlaHsd mice after treatment intragastrically with AbM and
PBS before (24 h) or simultaneously (0 h) with intraperitoneal bacterial inoculation (from [9] with permission).
Fig. 7B Survival in NIH/OlaHsd mice given AbM or PBS intragastrically prior to (24 h) or simultaneously (0 h) with intraperitoneal
inoculation with S. pneumoniae serotype 6B (from [9] with permission).
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Fig. 8 Survival of severe fecal peritonitis in Balb/c mice after orogastric introduction of Andosan (AbM) or PBS control 24 h prior to
challenge. The results are based on 2 separate experiments with 8 mice in each group (from [17] with permission).
Table 1 Comparison of minimum inhibiting concentration (MIC) of the polysaccharide and mushroom products reviewed.
Polysaccharide or
mushroom product
Antimicrobial action in type
of infection model
MIC of compound given
pre-challenge or with
challenge*
MIC of compound
given post-
challenge
β
-glucan from barley
β-glucan from baker’s yeast
(S-2721)
M. bovis, BCG-infection in
mice
100 μg/ mouse (i.v.)
10 μg/ mouse (i.v.)
100 μg/ mouse (i.v.)
N.D.
SSG
MacroGard®(particulate)
MacroGard® (soluble)
M. tuberculosis (strain
H37Rv)-infected mouse
macrophage cultures
500 μg/ml* in vitro
10 μg/ml* ”
No effect* ”
No effect
N.D.
N.D.
SSG
PMII
S.pneumoniae (type 6B)-
infection in mice
4 μg/ mouse (i.p.)
12 μg/ mouse (i.p.)
4 μg/ mouse (i.p.)
(effect on
bacteremia)
200 μg/ mouse (i.p.)
(effect on survival)
No effect (i.p.)
AndoSanTM
S.pneumoniae (type 6B)-
infection in mice
Fecal (Gram negative)
bacteria-infection in mice
0.9 μg/ mouse (p.o.),
*also with challenge
0.9 μg/ mouse (p.o.)
N.D.
N.D.
There was no direct antimicrobial effect of AndoSanTM against S. pneumoniae serotype 6B in bacterial cultures. Each
mouse was given 200 μl of AndoSanTM by orogastric installation, which is equivalent to 0.9 μg according to the dry
weight of 4.5 mg/ml after lyophilation of the extract (Samuelsen, unpublished results). The table shows that AndoSanTM
Time after challenge (days)
876543210
Survival (%)
100
80
60
40
20
0
Ab M
PBS
Numbers at risk 0 d 1 d 2 d 3 d 4 d 5 d 6 d 7 d p* me an **
Seve re
peri tonitis
Ab M 16 9 5 5 4 4 4 4 0.005 3
PBS 16 2 1 0 0 0 0 0 1
* log- rank test ** mean su rvival time in d ays
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was the product with the lowest MIC even though it was given enterally, in contrast with the parenteral administartion
of β-glucans and PMII. Hence, it proved to be the product with the highest efficacy in the comparison above.
3.2. Viral infections
Agaricus blazei extracts have previously been reported to inhibit certain viruses. More specifically, in vitro studies on
cell cultures have found antiviral effects against Western equine encephalitis virus [28], poliovirus type 1 [29], and
bovine herpes virus 1 [30]. As the extract has traditionally been used in connection with liver diseases, including
chronic hepatitis, we were allowed to test the in vivo effect of AbM on five patients with chronic hepatitis C virus
infection [31].
The patients did not respond to interferon treatment and were not given other anti-viral therapy. Daily, oral doses of
AbM were administrated for one week. Blood samples were obtained before and after treatment. The viral load was
slightly, but not significantly, decreased after treatment (5.3 compared to 5.8 million copies of virus per ml plasma).
The experimental setup allowed us to examine changes in gene expression in leucocytes from the patients prior to and
after treatment [30]. As might be expected, the changes were less pronounced compared to previous studies looking at
similar effects on monocytes treated in vitro [32]. Moreover, the cytokine genes most strongly induced in vitro were not
induced in vivo. The more notable changes in mRNA levels were related to genes involved in the G-protein coupled
receptor signaling pathway, in cell cycling, and in transcriptional regulation. The results suggest that the β-glucans of
the extract, which presumably are responsible for cytokine induction, did not readily enter the blood; while other
components, such as substances proposed to have anticancer effects, were active. The treatment did, however,
upregulate the gene for IFNα-receptor. Consequently, a study examining AbM intake combined with regular IFNα
treatment, might have been of interest. However now, other antiviral treatment than IFNα is used against HCV
infection.
The AbM extract was also tested on a mice model for influenza. No antiviral effect was demonstrated (unpublished
results). The discrepancy between the previously published in vitro antiviral effects, and the in vivo results on hepatitis
C and influenza virus, may be explained by the antiviral ingredients in the extract not readily entering the blood upon
oral administration.
4. Conclusions
The time has come to exploit novel and alternative strategies to combat MDR resistant harmfull and potentially lethal
microbes. Since the mixed Basidiomycetes mushroom product AndoSanTM has proven to be the most efficient of the
polysaccharide and mushroom-related products tested and reviewed here, we recommend this extract or components
thereof for future investigative clinical studies in patients with hard-to-cure bacterial infections inflicted by MDR
microbes. One such attempt is a planned clinical study, albeit awaiting ethical approval and financing, in which
AndoSanTM can be used against MDR-tuberculosis in patients at Armauer Hansen Research Institute (AHRI) in Addis
Abeba, Ethiopia. Regarding viral infections, we have so far not observed any significant effects of Basidiomycetes
mushroom extracts on viral load.
Acknowledgements We thank prof Berit Smestad Paulsen, School of Pharmacy, University of Oslo, for valuable discussions and
help with lyophilization of AndoSanTM for preservation, prof Ketil K. Melby at Department of Microbiology, Oslo University
Hospital for microbial quality control of AndoSanTM batches. Sponsor: AndoSanTM was supplied by Immunopharma AS, Høvik,
Norway.
Conflict of interest GH is a stock holder of Immunopharma AS.
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