International Journal of Medicinal Mushrooms, 21(1):1–11 (2019)

In Vitro and In Vivo Inhibition of Helicobacter pylori

by Ethanolic Extracts of Lion’s Mane Medicinal
Mushroom, Hericium erinaceus (Agaricomycetes)
Ge Wang,a Xiumin Zhang,b Susan E. Maier,a Liping Zhang,c & Robert J. Maiera,*
a
Department of Microbiology, University of Georgia, Athens, GA, USA; bCollege of Life Sciences, Hebei University,
Engineering Laboratory of Microbial Breeding and Preservation of Hebei Province, Key Disciplines of Bioengineering
in Hebei Province, Hebei-Baoding, China; cBaoding Baienjie Biotechnology Co. Ltd., Hebei-Baoding, China

*Address all correspondence to: Robert J. Maier, Department of Microbiology, University of Georgia, Athens, GA 30602, USA; Tel.: 706-542-
2323; Fax: 706-542-2674; rmaier@uga.edu

ABSTRACT: Natural products are sources for exploratory development of new agents to combat the gastric pathogen
Helicobacter pylori. Some edible fungi, such as the lion’s mane mushroom, have been used for several thousand years
to treat digestive diseases. Ethanol-based extractions to prepare Hericium erinaceus extracts were tested for growth
inhibition ability of six different H. pylori strains at an extract concentration that did not inhibit Escherichia coli
growth, and further for dose-dependent antibactericidal capacity on H. pylori. H. erinaceus extract exhibited similar
growth inhibitory effects on all H. pylori strains tested, with a minimum inhibitory concentration of about 2 mg/mL.
H. pylori survival in phosphate-buffered saline (PBS) was decreased 3 logs by 2 mg/mL extract addition. H. erinaceus
extract inhibited H. pylori adhesion capacity to human gastric epithelial cell line (ATCC CRL-1739) (AGS), even when
H. erinaceus extract was added at a concentration that affected neither H. pylori nor AGS viability. Interleukin-8 (IL-8,
representing an immune response factor) in supernatants from AGS and 8-oxo-guanine (8-oxoG, a marker for oxidative
DNA damage among the total host cell DNA) were measured from AGS cells exposed to H. erinaceus extract before
H. pylori addition. The subsequent H. pylori-mediated immune response (IL-8 production) was significantly (P <
0.01) decreased by H. erinaceus extract; at 1.0 mg/mL extract addition, IL-8 expression returned to nearly background
level (no H. pylori added). H. pylori infection of AGS caused a 3-fold increase in host 8-oxoG, but this increase was
abolished by including 2 mg/mL H. erinaceus extract. Mouse colonization assays of C57BL mice were performed on
homogenized stomachs 3 weeks after inoculating H. pylori into the animals; mice receiving the H. erinaceus extract
had a mean H. pylori load of 6 × 104 CFU/g of stomach, about 1 log lower than the control (no extract) animals.

KEY WORDS: Hericium erinaceus, Helicobacter pylori, cell adhesion, interleukin-8, 8-oxo-guanine, mouse colo-
nization, medicinal mushrooms

ABBREVIATIONS: 8-oxoG, 8-oxo-guanine; AGS, human gastric epithelial cell line (ATCC CRL-1739); BA, Brucella agar;
CagPAI, Cag pathogenesis island; FITC, fluorescein isothiocyanate; HE extract, ethanol extract of H. erinaceus; IL-8, interleu-
kin-8; MIC, minimum inhibitory concentration; MOI, multiplicity of infection; PBS, phosphate-buffered saline; PI, propidium
iodide; RONS, reactive oxygen and nitrogen species; ROS, reactive oxygen species

I. INTRODUCTION

Helicobacter pylori infection in the human stomach causes chronic gastric inflammation and tissue damage,
leading to alterations that could evolve into severe gastric diseases such as ulcers and gastric cancer.1,2 Current
therapies for H. pylori infection usually consist of a proton pump inhibitor in combination with two or three
antibiotics. These treatments are systemically toxic and drug resistance has limited their success.3,4 There is
a growing need to find new chemical compounds with bactericidal or bacteriostatic effects against H. pylori.
Natural products are potential sources for the discovery and development of new effective agents against
H. pylori infection with less toxic side effects. Some edible mushrooms, such as Hericium erinaceus (Bull.:
Fr.) Pers. (Hericiaceae, Agaricomycetes), have been extensively used in traditional Chinese medicine to treat

1521-9437/19/$35.00 © 2019 Begell House, Inc. www.begellhouse.com 1

2 Wang et al.

chronic superficial gastritis and gastric ulcers, although the underlying pharmaceutical mechanism is not
understood.5–7 H. erinaceus is known as lion’s mane mushroom and monkey’s head mushroom (Hou Tou
Gu in Chinese). H. erinaceus contains important pharmacologic constituents such as polysaccharides,
bioactive proteins, terpenoids, and hericenone. Recently, a polysaccharide component from H. erinaceus
was shown to have antioxidant activity in gastric epithelial cells.8 The ethanol extract of H. erinaceus (HE
extract) was shown to have anti-H. pylori activity in vitro9; some bioactive components for the anti-H. pylori
activity were subsequently isolated from H. erinaceus.10 Human trials with the anti-Helicobacter medici-
nal mushroom Tremella mesenterica were not conclusive regarding the efficacy of a 10-day mushroom
administration, but T. mesenterica-treated patients had fewer adverse effects than experienced with standard
antibiotic treatments, and longer-term studies are warranted.11
During the process of colonizing the host, H. pylori induces a strong inflammatory response medi-
ated by a variety of host immune cells, leading to the generation of a number of reactive oxygen species
(ROS).12,13 H. pylori-induced ROS and DNA damage in host cells play an important role in the formation
and development of gastritis and related diseases such as gastric cancer.14,15 In this study, we examined
the bacteriostatic and bactericidal effects of H. erinaceus ethanol extract against H. pylori in vitro and
in vivo (using a mouse infection model), and we investigated the extract’s inhibitory effects on H. pylori-
induced immune response and DNA damage in human gastric epithelial cells.

II. MATERIALS AND METHODS

A. Preparation of H. erinaceus Extracts

The fruit bodies of H. erinaceus were purchased from Beijing Tong Ren Tang Co., Ltd. (Baoding, China).
The methods for preparation of H. erinaceus extracts were adopted and modified from Shang et al.9 The
fruit bodies of H. erinaceus were blended into a powder and baked in a drying oven for 24 hours at 60°C.
About 2 kg of the powders were extracted with 2 L of 95% ethanol for 24 hours at room temperature.
After centrifugation, the supernatant was concentrated using vacuum rotary evaporation to yield the crude
extract, and precipitates were extracted three times as above. The extracts were combined and maintained
without movement for 30 minutes. Then the oil layer was removed using a separating funnel, and the
precipitates were removed using vacuum suction. The liquid was extracted with ethyl acetate three times
and concentrated.

B. H. pylori Culture Conditions

H. pylori was cultured on Brucella agar (BA; Difco) plates supplemented with 10% defibrinated sheep
blood or in BHI-YE liquid medium (plus 5% serum).16,17 Cultures of H. pylori were grown microaerobi-
cally at 37oC in an incubator under controlled levels of oxygen (4% partial pressure O2, 5% CO2, and the
balance was N2).18

C. Determination of Bactericidal Effect of H. erinaceus Extract on H. pylori

Phosphate-buffered saline (PBS) suspensions were prepared from late log phase H. pylori cells. Different
concentrations of H. erinaceus extract were added, and the mixtures were incubated for 4 hours in a
microaerobic incubator with occasional manual shaking. Serial 10-fold dilutions of the cell suspensions
were plated on BA plates and incubated for 3 to 5 days at 37°C under microaerobic conditions to count
the number of H. pylori colonies.18,19

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Helicobacter Inhibition by Hericium erinaceus 3

D. Coculture of AGS Cells with H. pylori to Test Effects of H. erinaceus Extracts

AGS human gastric cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (ATCC) with 10%
fetal bovine serum for 24 hours to subconfluent stage.20 Various concentrations of H. erinaceus extracts
were added to the cell culture, followed by the addition of H. pylori cells at a bacterium:cell ratio of 100:1.
The mixed cell cultures were further incubated at 37°C in the presence of 5% CO2.

E. AGS Cell Viability Assay

Assays were performed to assess the cell viability using trypan blue staining21 of untreated and HE extract-
treated AGS cells.

F. Bacterial Adherence Assay

The bacterial adherence assay was as described in previous work.20 AGS–H. pylori cocultures in the pres-
ence of various concentrations of H. erinaceus extracts were incubated for 4 hours at 37°C in the presence
of 5% CO2. The cell cocultures were washed with PBS three times to remove nonadherent bacteria. The
AGS cells were lysed with ice-cold PBS plus 0.1% saponin for 5 minutes. Serial 10-fold dilutions of cell
lysates were cultured on BA plates for 3 to 5 days at 37°C under microaerobic conditions18 to count the
number of H. pylori colonies.

G. Interleukin-8 Measurement

AGS cell cultures20 were added with various concentrations of HE extracts followed by H. pylori infec-
tion (multiplicity of infection [MOI] of 1:100) and further incubation for 6 hours. After centrifugation,
the supernatants were collected and stored at −80°C before analysis. The level of interleukin-8 (IL-8) in
supernatants from AGS cell cultures was determined16 by using a sandwich enzyme-linked immunosorbent
assay kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

H. Fluorescent Staining of AGS Cells and Quantification of 8-Oxo-Guanine

AGS cell cultures were added with various concentrations of H. erinaceus extract followed by
H. pylori infection (MOI of 1:100) for 12 hours. The cell cocultures were washed with PBS three times
to remove nonadherent bacteria. To determine the level of 8-oxo-guanine (8-oxoG), the AGS cells were
immunofluorescent-stained with fluorescein isothiocyanate (FITC)-avidin.22,23 Eight sets of the immuno-
fluorescent images were examined for the luminosity of FITC and propidium iodide (PI), and the average
ratio of the luminosity of FITC to PI was calculated, which represents the level of 8-oxoG.23

I. Mouse Colonization Assay and Ethics Approval for Animal Studies

Mouse colonization assays were performed essentially as described previously.16,18,24 All the procedures
were approved by the University of Georgia Institutional Animal Use and Care Committee, the committee
that has reviewed and approved our animal studies annually for 19 years. Wild-type H. pylori X47 cells were
harvested after 48 hours of growth on BA plates (37oC, 4% oxygen) and suspended in PBS to an optical den-
sity at 600 nm of 1.7. The headspace in the tube was sparged with argon gas to minimize oxygen exposure,18
and the tube was tightly sealed. The bacterial suspensions were administered to C57BL/6NCr mice (3 × 108
H. pylori cells/mouse). Three weeks after the inoculation, the mice were sacrified and the stomachs were

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4 Wang et al.

removed, weighed, and homogenized in argon-sparged PBS to avoid O2 exposure.18 Stomach homogenate
dilutions (dilutions were conducted in sealed tubes containing argon-sparged buffer) were plated on
BA plates supplemented with bacitracin (100 mg/mL), vancomycin (10 mg/mL), and amphotericin B
(10 mg/mL). The plates were rapidly transported into an incubator containing sustained 4% partial pres-
sure O2.16,18 After incubation for 5 to 7 days, H. pylori colonies were counted and the data were expressed
as CFU per gram of stomach.16,18

III. RESULTS

A. Growth Inhibition of H. pylori by Ethanol Extract of H. erinaceus

To test the anti-H. pylori activity of the ethanol extracts of H. erinaceus, we used different strains of
H. pylori, including ATCC type strains, mouse-colonizing strains, and the strains used in the gerbil model
to study cancer-causing abilities. B128 is a noncarcinogenic strain, and 7.13 is the (gerbil model) cancer-
associated strain.25 H. erinaceus extract exhibited similar inhibitory effects on different H. pylori strains
(Table 1). According to the data, the minimum inhibitory concentration (MIC) of H. erinaceus extract is
2 mg/mL. Up to 4 mg/mL H. erinaceus extract had no inhibitory effect on growth of E. coli in vitro (Table 1).
As a positive control, addition of 4 mg/mL metronidazole in agar plates completely inhibited growth of
both H. pylori and E. coli (data not shown).

B. H. erinaceus Extract Has a Dose-Dependent Bactericidal Effect on H. pylori

To determine the bactericidal effect, suspensions of cells in PBS were prepared from the grown (late log
phase) H. pylori cells. Different concentrations of H. erinaceus extract were added to the cell suspen-
sions and the mixtures were incubated for 4 hours; the surviving cell numbers were determined by plate
counting. As shown in Fig. 1, H. erinaceus extract has a dose-dependent bactericidal effect on H. pylori.
Treatment with 0.25 or 0.5 mg/mL H. erinaceus extract for 4 hours had no significant killing effect, while
the surviving number of H. pylori cells decreased several-fold by treatment with 1.0 mg/mL H. erinaceus
extract for 4 hours. The surviving number of H. pylori cells decreased almost 3 logs by treatment with
2.0 mg/mL H. erinaceus extract for 4 hours.

TABLE 1: In Vitro Growth Inhibition of Helicobacter pylori by Hericium erinaceus Ethanol Extract

Concentration H. pylori Strains Escherichia coli

of HE (mg/mL) DH10B
SS1 43504 26695 X47 B128 7.13
0 +++ +++ +++ +++ +++ +++ +++
0.25 +++ +++ +++ +++ +++ +++ +++
0.5 ++ ++ ++ ++ ++ ++ +++
1.0 + + + + + + +++
2.0 − − − − − − +++
4.0 − − − − − − +++

The data show the growth of H. pylori on Brucella agar (plus 10% blood) plates containing the indicated concentrations of
the ethanol extracts of H. erinaceus. −, no growth; +, slight growth; ++, moderate growth; +++, complete growth. All plates
contain ethanol at a final concentration of 4% (v/v), which has no inhibitory effect on growth of H. pylori. HE, ethanol extract
of H. erinaceus.

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Helicobacter Inhibition by Hericium erinaceus 5

FIG. 1: Killing effect of Hericium erinaceus extract on Helicobacter pylori. H. pylori X47 cell suspensions in PBS
were treated with different concentrations (as indicated on the x-axis) of H. erinaceus extract for 4 hours. The CFU
(i.e., survivors) were determined by plate counts after 4 days of plate incubations under a microaerobic condition.18
The data are the means of three experiments with standard deviations as indicated.

C. Treatment with H. erinaceus Extract Does Not Influence AGS Cell Viability

After showing the bacteriostatic and bactericidal effects of H. erinaceus extract on H. pylori in vitro, we
sought to investigate the inhibitory effects against H. pylori when cocultured with human gastric epithelial
AGS cells. First, to test if H. erinaceus extract has any effect on AGS cell viability, assays were performed
to assess the cell viability using trypan blue staining21 of untreated and H. erinaceus extract-treated AGS
cells. At the concentrations (0.5, 1, and 2 mg/mL H. erinaceus) and conditions (4 and 6 hours) tested, the
H. erinaceus extract showed no significant effect on AGS cell viability (data not shown).

D. H. erinaceus Extract Has Antiadhesive Activity

To investigate whether H. erinaceus extract has any inhibitory effect on H. pylori adhesion to epithelial
cells, we performed bacterial adherence assay. AGS–H. pylori cocultures in the presence of various
concentrations of H. erinaceus extract were incubated for 4 hours at 37°C in the presence of 5% CO2.
We chose the concentrations of H. erinaceus extract that have no significant killing effect on H. pylori
(0.25 and 0.5 mg/mL, Fig. 1). Compared with the untreated control (no HE added), H. pylori adherence
activity decreased 35% and 63%, respectively (Fig. 2). At the H. erinaceus extract concentration of 1.0
mg/mL, H. pylori lost approximately 90% adherence capacity, but this result is compromised by the fact
that significant amount of H. pylori cells were killed at this condition (Fig. 1).

E. H. erinaceus Extract Has Inhibitory Effect on H. pylori-Induced IL-8 Expression

by AGS Cells

It is well documented that several cytokines are expressed in human gastric epithelial cells in response
to H. pylori infection. We performed experiments to investigate whether H. erinaceus extract has an
effect on H. pylori-induced IL-8 expression by AGS cells. AGS cell cultures were added with various
concentrations (0.25, 0.5, and 1.0 mg/mL) of H. erinaceus extract, then infected by H. pylori (MOI of

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6 Wang et al.

FIG. 2: Effects of Hericium erinaceus extract treatment on Helicobacter pylori adhesion to AGS cells.
AGS–H. pylori cocultures in the presence of indicated concentrations of H. erinaceus extracts were incubated
for 4 hours followed by bacterial adhesion assay. Numbers shown are the relative adhesion (percentage, related
to the untreated control). Data are means from three independent experiments with standard deviations. The 0
(no addition) indicates positive adhesion was observed, as seen in previous experiments.20

1:100), and further incubated for 6 hours, followed by IL-8 measurement. In this experiment, we used
H. pylori B128, as it is a Cag pathogenesis island (CagPAI)-positive strain. As shown in Fig. 3, AGS
cells alone produced a lower level of IL-8 (mean, 92 pg/mL), and the expression of IL-8 was induced by
H. pylori to a higher level (mean, 163 pg/mL). IL-8 levels significantly (P < 0.01) decreased with the

FIG. 3: Effect of Hericium erinaceus extract on Helicobacter pylori-induced IL-8 expression by AGS cells. AGS
cell cultures were added with various concentrations of H. erinaceus extract as indicated and H. pylori B128 cell
suspensions and further incubation for 6 hours, followed by IL-8 measurement. The AGS cells alone served as a
control for the background level of IL-8 expression. The no H. erinaceus (0) but plus H. pylori indicates the posi-
tive bacterial-mediated effect on IL-8 expression, as seen in previous experiments.16 Data are means from three
independent experiments with standard deviations.

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Helicobacter Inhibition by Hericium erinaceus 7

addition of H. erinaceus extract. IL-8 expression reached nearly background level with addition of
1.0 mg/mL H. erinaceus extract. These results indicate that treatment of AGS cells with HE extract attenu-
ated at least a portion of the H. pylori-induced immune response.

F. H. erinaceus Extract Prevents AGS Cells from Susceptibility to H. pylori-Induced

Oxidative DNA Damage

H. pylori infection leads to the release of reactive oxygen and nitrogen species (RONS) from inflammatory
cells, and RONS can cause a number of different types of DNA damage.26 One mechanism of H. pylori-
induced carcinogenesis is believed to be dependent on cumulative oxidative DNA damage.27,28 8-OxoG
is a major biomarker for oxidative DNA damage.22
We thus determined the effect of HE treatment in preventing H. pylori-induced 8-oxoG within AGS
cells. AGS cell cultures were added with various concentrations (0.25, 0.5, 1.0, and 2.0 mg/mL) of
H. erinaceus extract, then infected by H. pylori (MOI of 1:100), with further incubation for 12 hours. After
removing nonadherent bacteria, the level of 8-oxoG in AGS cells was determined by immunofluorescent
staining with FITC-avidin (Table 2). As expected, H. pylori infection caused a marked (3-fold) increase of
8-oxoG levels in AGS cells, as seen for the negative (AGS cells only) and positive (AGS + Hp, without
H. erinaceus) controls. The treatment with various concentrations of H. erinaceus extract lowered 8-oxoG
levels in a dose-dependent manner. According to statistical analysis with the Student’s t-test, the data
for 1.0 and 2.0 mg/mL H. erinaceus are significantly lower than the positive control (P < 0.001). In the
presence of 2.0 mg/mL H. erinaceus extract, the 8-oxoG level was similar to the negative control level.
These results indicated that treatment of AGS cells with H. erinaceus extract protected AGS cells from
H. pylori-induced oxidative DNA damages.

G. H. erinaceus Extract Supplement in Drinking Water for Mice Attenuated H. pylori

Colonization in the Stomach

Next, we sought to examine the effect of the H. erinaceus extract on H. pylori pathogenesis using the
mouse infection model. H. erinaceus extract was given to eight mice in drinking water (50 mg/mouse
per day) for 4 days before and for 20 days after H. pylori infection, followed by examination of H. pylori
colonization in mouse stomachs (Fig. 4). As negative controls, a group of four mice did not receive
H. pylori inoculation; no H. pylori were detected in the stomachs of these mice (data not shown). A group
of eight mice received the H. pylori inoculant, but no H. erinaceus extract was added to their drinking
water; this group thus served as a positive control. H. pylori was recovered from all mice of this group,

TABLE 2: Levels of 8-Oxo-Guanine in AGS Cells Cocultured with Helicobacter pylori in the Presence of
Various Concentrations of Hericium erinaceus Extracts

Coculture H. erinaceus (mg/mL) 8-Oxo-Guanine Level (FITC/PI Ratio)

AGS cells only 0 0.43 ± 0.08
AGS cells plus H. pylori 0 1.31 ± 0.18
0.25 1.28 ± 0.16
0.5 1.08 ± 0.15
1.0 0.75 ± 0.12
2.0 0.47 ± 0.06

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FIG. 4: Effect of Hericium erinaceus extract on Helicobacter pylori colonization in mice. A group of eight mice
were given HE extract in their drinking water (50 mg/mouse per day) for 4 days and then were inoculated with
H. pylori strain X47 with a dose of 3 × 108 viable cells administered per animal. H. erinaceus extract in drinking water
(50 mg/mouse/day) was further provided for 20 days, followed by examination of H. pylori colonization in mouse
stomachs. Another group of eight mice served as control, with no H. erinaceus extract added to the drinking water.
Data are presented as a scatter plot (at log scale) of CFU per gram of stomach as determined by plate counts. Each
point represents the CFU count from one mouse stomach, and the solid lines represent the geometric means of the
colonization numbers for each group (negative control or + H. erinaceus extract). The baseline [log10 (CFU/g) = 2.7]
is the detection limit of the assay, which represents a count below 500 CFU/g stomach. A negative colonization
control group of uninoculated mice contained no H. pylori.

with a mean number of 4.7 × 105 CFU/g stomach. Seven of eight mice that received H. erinaceus extract
were found to also harbor H. pylori, but with a mean bacterial load of 6.0 × 104 CFU/g stomach.
According to Wilcoxon rank test analysis, the range of colonization values for the H. erinaceus
extract group is significantly lower than that of the positive control group at the 99% confidence level
(P < 0.01). These results demonstrated the in vivo effects of H. erinaceus extract in attenuating H. pylori
survival/colonization, and suggested that H. erinaceus is a potential candidate against H. pylori-mediated
pathogenesis and carcinogenesis.

H. Chemical Composition of the H. erinaceus Extract

The ethanol extracts of H. erinaceus used in this study were analyzed with high-performance liquid chro-
matography. As shown in Fig. 5, the H. erinaceus extract was highly heterogeneous in content; it contains
polysaccharides, lipids, nucleosides, and diterpenoid alkaloids.

IV. DISCUSSION

Previous studies reported that the ethanol extracts of H. erinaceus have significant anti-H. pylori activ-
ity in vitro.9,10 With our samples, we confirmed those results (Table 1). By testing different strains of
H. pylori, our results indicated that the H. erinaceus extract has both bacteriostatic and bactericidal

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Helicobacter Inhibition by Hericium erinaceus 9

FIG. 5: High-performance liquid chromatography chromatograms of the Hericium erinaceus extract

effects on H. pylori. Adhesion of H. pylori to epithelial cells is important for its persistent infection in
the human stomach. Our results indicated that HE extract is not only able to kill H. pylori (at higher
doses), but also inhibits H. pylori adherence to epithelial cells (at lower doses). H. pylori adhesion to
epithelial cells is mediated mainly by its outer membrane proteins such as BabA, SabA, OipA, and
AlpA/B. The mechanisms underlying the antiadhesive activity of H. erinaceus extract against H. pylori
will need further investigation.
H. pylori infection in humans induces a robust proinflammatory response associated with gas-
tric inflammation, atrophy, epithelial hyperplasia, and dysplasia.26 For example, the gastric mucosa of
H. pylori-infected patients has increased levels of proinflammatory cytokines such as IL-8 and tumor
necrosis factor-alpha.27–30 IL-8 is known to be a key mediator of the inflammatory responses in H. pylori-
infected individuals, and it plays an important role in activating and recruiting neutrophils in response
to infection by H. pylori.30–32 The epithelium is the greatest source of IL-8 in the gastric mucosa, and the
coculture of H. pylori CagPAI-positive strains with epithelial cells stimulates secretion of IL-8.33 Our
results indicated that H. pylori-induced host immune response to the pathogen is inhibited when the extract
is added to gastric cells at a concentration below that affecting pathogen viability.
In its earliest stage, cancer development is associated with host cell genetic instability and activation
of DNA damage response.34,35 A hallmark of gastric cancer etiology is oxidative DNA damage and genetic
instability leading to frequent chromosome aberrations.36 The data in this report provided additional
evidence that certain components in the H. erinaceus extract have potential anticancer activity.37 It is
interesting that a recent study reported that a unique polysaccharide purified from H. erinaceus prevents
H2O2-induced oxidative stress in human gastric epithelial cells.8

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10 Wang et al.

V. CONCLUSIONS

Both in vitro and in vivo attenuation of H. pylori are indicated by an extract from a mushroom that has been
used for many generations in Chinese medicine to abrogate H. pylori-mediated diseases. Not only is the
pathogen’s cell viability affected by the H. erinaceus extract, but H. pylori-mediated host DNA damage
and normal host immune responses are inhibited when the extract is used at lower concentrations—ones
not affecting pathogen viability. It is important that the stomach colonization capacity of the pathogen is
significantly inhibited by the mushroom extract. Identification of the relative importance of the (many)
specific molecules/factors in the extract that are inhibitory will require intense investigation; it will take a
combined in vitro approach along with animal colonization studies initially using subfractions of the extract.

REFERENCES

1. Polk DB, Peek RM Jr. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer. 2010;10:403–14.
2. Suerbaum C, Michetti P. Helicobacter pylori infection. N Engl J Med. 2002;347(15):1175–86.
3. Roesler BM, Costa S, Zeitune JM. Eradication treatment of Helicobacter pylori infection: its importance and possible
relationships in preventing the development of gastric cancer. ISRN Gastroenterol. 2012;2012:935410.
4. Thung I, Arain H, Vavinshaya V, Gupta S, Park JV. Crowe SE, Valasek MA. Review article: the global emergence of
Helicobacter pylori antibiotic resistance. Aliment Pharmacol Ther. 2016;43:514–33.
5. Lakshmanan J, Raman J, David P, Wong KH, Naidu M, Sabaratnam V. Haematological, biochemical and histopathological
aspects of Herimium erinaceus ingestion in a rodent model: a sub-chronic toxicological assessment. J Ethnopharmacol.
2016;194:1051–9.
6. Wang M, Konishi T, Gao Y, Xu D, Gao Q. Anti-gastric ulcer activity of polysaccharide fraction isolated from mycelium culture
of lion’s mane medicinal mushroom, Hericium erinaceus (higher Basidiomycetes). Int J Med Mushrooms. 2015;17:1055–60.
7. Wong JY, Abdulla MA, Raman J, Phan CW, Kuppusamy UR, Golbabapour S, Sabaratnam V. Gastroprotective effects of
lion’s mane mushroom Hericium erinaceus (Bull:Fr) Pers. (Aphyllophoromycetidae) extract against ethanol-induced ulcer
in rats. Evid Based Complement Alernat Med. 2013;2013:492976.
8. Wang M, Kanako N, Zhang Y, Ziao X, Gao Q, Tetsuya K. A unique polysaccharide purified from Hericium erinaceus myce-
lium prevents oxidative stress induced by H2O2 in human gastric mucosa epithelium cell. PLoS One. 2017;12:e0181546.
9. Shang X, TanQ, Liu R, Yu K, Li P, Zhao GP. In vitro anti-Helicobacter pylori effects of medicinal mushroom extracts,
with special emphasis on the lion’s mane mushroom, Hericium erinaceus (higher Basidiomycetes). Int J Med Mushrooms.
2013;15:165–74.
10. Liu JH, Li L, Shang XD, Zhang JL, Tan Q. Anti-Helicobacter pylori activity of bioactive components isolated from Hericium
erinaceus. J Ethnopharmacol. 2016;183:54–8.
11. Lachter JY, Yampolsky Y, Gafni-Schieber R, Wasser SP. Yellow brain culinary-medicinal mushroom, Tremella mesenterica
Ritz:Fr. (higher Basidiomycetes), is subjectively but not objectively effective for eradication of Helicobacter pylori: a pro-
spective controlled trial. Int J Med Mushrooms. 2012;14(1):55–63.
12. Bagchi D, Bhattacharya G, Stohs SJ. Production of reactive oxygen species by gastric cells in association with Helicobacter
pylori. Free Radic Res. 1996;24:439–50.
13. Ramarao N, Gray-Owen SD, Meyer TF. Helicobacter-pylori induces but survives the extracellular release of oxygen radicals
from professional phagocytes using its catalase activity. Mol Microbiol. 2000;38:103–13.
14. Gobert AP, Wilson KT. Polyamine- and NADPH-dependent generation of ROS during Helicobacter pylori infection: a
blessing in disguise. Free Radic Biol Med. 2017;105:16–27.
15. Hardbower DM, Peek, RM Jr, Wilson KT. At the bench: Helicobacter pylori, dysregulated host responses, DNA damage,
and gastric cancer. J Leukoc Biol. 2014;96:201–12.
16. Wang G, Maier SE, Lo LF, Maier G, Dosi S, Maier RJ. Peptidoglycan deacetylation in Helicobacter pylori contributes to
bacterial survival by mitigating host immune responses. Infect Immun. 2010;78:4660–6.
17. Benoit SL, Bayyareddy K, Mahawar M, Sharp J, Maier RJ. Alkyl hydroperoxide reductase repair by Helicobacter pylori
methionine sulfoxide reductase. J Bacteriol. 2014;195:5396–401.
18. Olczak AA, Seyler, RW Jr, Olson JW, Maier RJ. Association of Helicobacter pylori antioxidant activities with host coloniza-
tion proficiency. Infect Immun. 2003;71:580–3.
19. Kuhns LG, Benoit SL, Bayyareddy K, Johnson D, Orlando R, Evans AL, Waldrop GL, Maier RJ. Carbon fixation
driven by molecular hydrogen results in chemolithoautotrophically enhanced growth of Helicobacter pylori. J Bacteriol.
2016;198:1423–8.

International Journal of Medicinal Mushrooms

Helicobacter Inhibition by Hericium erinaceus 11

20. Wang G, Romer-Gallo J, Benoit SL, Piazuelo MB, Dominguez RL, Morgan DR, Peek RM Jr, Maier RJ. Hydrogen metabolism
in Helicobacter pylori plays a role in gastric carcinogenesis through facilitating cagA translocation. mBio. 2016;7:e01022-16.
21. Strober W. Trypan blue-exclusion test of cell viability. Curr Protoc Immunol. 2001;Appendix 3:Appendix 3B.
22. Chen SK, Tsal MH, Hwang JJ, Chang WP. Determination of 8-oxoguanine in individual cell nucleus of gamma-irradiated
mammalian cells. Radiat Res. 2001;155:832–6.
23. Wang G, Conover RC, Olcsak AA, Alamuri P, Johnson MK, Maier RJ. Oxidative stress defense mechanisms to counter
iron-promoted DNA damage in Helicobacter pylori. Free Radic Res. 2005;39:1183–91.
24. Wang G, Maier RJ. A novel DNA-binding protein plays an important role in Helicobacter pylori stress tolerance and survival
in the host. J Bacteriol. 2015;197:973–82.
25. Franco AT, Israel DA, Washington MK, Krishna U, Fox JG, Rogers AB, Neish AS, Collier-Hyams L, Perez-Perez Gi,
Hatakeyama M, Whitehead R, Gaus K, O’Brian DP, Romero-Gallo J, Peek, RM Jr. Activation of beta-catenin by carcino-
genic Helicobacter pylori. Proc Natl Acad Sci U S A. 2005;102:10646–51.
26. Fox JG, Wang TC. Inflammation, atrophy, and gastric cancer. J Clin Invest. 2007;117:60–9.
27. Crabtree JE, Peichl P, Wyatt JI, Stachl U, Lindley IJ. Gastric interleukin-B and IgA IL-8 autoantibodies in Helicobacter
pylori infection. Scand J Immunol. 1993;37:65–70.
28. Garcia-Gonzales MA, Aisa MA, Strunk M, Benito R, Piazuelo E, Jimenez P, Sopena F, Lanas A. Relevance of IL-1 and
TNF gene polymorphisms on interleukin-1beta and tumor necrosis factor-alpha gastric mucosal production. Hum Immunol.
2009;70:935–45.
29. Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN. Mucosal tumor necrosis factor-alpha, interleukin-1
beta, and interleukin-8 production in patients with Helicobacter pylori infection. Scan J Gastroenterol. 1994;29(5):425–9.
30. Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J. Induction of various cytokines and development of severe
mucosal inflammation by cagA gene positive Helicobacter pylori strains. Gut. 1997;41:442–51.
31. Atherton JC. The pathogenesis of Helicobacter pylori-induced gastro-duodenal diseases. Annu Rev Pathol. 2006;1:63–96.
32. Crabtree JE, Covacci A, Farmery SM, Xiang Z, Tompkins DS, Perry S, Lindley IJ, Rappuoli R. Helicobacter pylori induced
interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype. J Clin Pathol. 1995;48:41–5.
33. Handa O, Naito Y, Yoshikawa T. Redox biology and gastric carcinogenesis: the role of Helicobacter pylori. Redox Rep.
2011;16:1–7.
34. Raza Y, Khan A, Farooqui A, Mubarak M, Facista A, Akhtar SS, Khn S, Kazi JI, Bernstein C, Kazmi SU. Oxidative DNA
damage as a potential early biomarker of Helicobacter pylori associated carcinogenesis. Pathol Oncol Res. 2014;20:839–46.
35. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas
J, Bartek J. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature. 2005;434:864–70.
36. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Lilogiou T, Venere M, Ditillio, RA Jr, Kastrinakis
NG, Levy B, Kletsas D, Yoneta A, Herlyn M, Kittas C, Halazonetis TD. Activation of the DNA damage checkpoint and
genomic instability in human precancerous lesions. Nature. 2005;434:907–13.
37. Li G, Yu K, Li F, Xu K, Li J, He S, Cao S, Tan S. Anticancer potential of Hericium erinaceus extracts against human gas-
trointestinal cancers. J Ethnopharmacol. 2014;153:521–30.

Volume 21, Issue 1, 2019