Anti-Nosemosis Activity of Aster scaber and Artemisia dubia Aqueous Extracts

Abstract

In our previous study, we demonstrated that the ethanol extracts of Artemisia dubia ( A. dubia ) and Aster scaber ( A. scaber ) have anti-nosemosis activity. In our present study, we intend to establish the anti-nosemosis activity of aqueous, ethyl acetate (EA), and butanol (BuOH) extracts of A. dubia and A. scaber . In order to determine the optimal dose, we performed both in vitro and in vivo toxicity for all the extracts and also carried out anti-nosemosis experiments. Although all of the extracts (aqueous, EA, and BuOH) showed in vitro and in vivo anti-nosemosis activity in a dose-dependent manner, the aqueous extracts of A. dubia and A. scaber showed more potent anti-nosemosis activity than the EA and BuOH extracts. Moreover, an aqueous extract of A. dubia + A. scaber demonstrated stronger anti-nosemosis activity compared with the aqueous extracts of either A. dubia or A. scaber alone. Although the main ingredients in A. dubia and A. scaber remain unclear, our results suggest that the active components of A. dubia and A. scaber could dissolve in the aqueous fraction.

Full text

J. APIC. SCI. Vol. 62 No. 1 2018
1
ANTI-NOSEMOSIS ACTIVITY OF ASTER SCABER
AND ARTEMISIA DUBIA AQUEOUS EXTRACTS
Jae Kwon Lee1*
Jeong Hwa Kim1
Mina Jo1
Balamurugan Rangachari2
Jin Kyu Park2
Abstract
In our previous study, we demonstrated that the ethanol extracts of
Artemisia dubia
(
A. dubia
) and
Aster scaber
(
A. scaber
) have anti-nosemosis activity. In our present study,
we intend to establish the anti-nosemosis activity of aqueous, ethyl acetate (EA), and
butanol (BuOH) extracts of
A. dubia
and
A. scaber
. In order to determine the optimal
dose, we performed both
in vitro
and
in vivo
toxicity for all the extracts and also carried
out anti-nosemosis experiments. Although all of the extracts (aqueous, EA, and BuOH)
showed
in vitro
and
in vivo
anti-nosemosis activity in a dose-dependent manner, the
aqueous extracts of
A. dubia
and
A. scaber
showed more potent anti-nosemosis activity
than the EA and BuOH extracts. Moreover, an aqueous extract of
A. dubia
+
A. scaber
demonstrated stronger anti-nosemosis activity compared with the aqueous extracts of
either
A. dubia
or
A. scaber
alone. Although the main ingredients in
A. dubia
and
A. scaber
remain unclear, our results suggest that the active components of
A. dubia
and
A. scaber
could dissolve in the aqueous fraction.
Keywords:
Artemisia dubia
,
Aster scaber
,
Nosema ceranae,
nosemosis
1 Department of Biology Education, College of Education, Chungbuk National
University, Cheongju, 361-763, Republic of Korea
2 Beesen Co., Ltd., Bioventure Town, Yuseong Daero 1662, Dae Jeon, Republic of
Korea
INTRODUCTION
Nosemosis is a disease of adult bees caused by
Nosema
species which belongs to the class of
Microsporidia of the fungal kingdom (Sprague
& Becnel, 1998; Sprague & Becnel, 1999). The
genus
Nosema
contains 322 species including
the sub-species prevalent in honey bees
Nosema
ceranae
(
N. ceranae)
and
Nosema apis
(
N.
apis)
(http://www.indexfungorum.org).
N. apis
is a parasite of the western honey bee (
Apis
mellifera
) and
N. ceranae
is a parasite of the
eastern honey bee (
Apis ceranae
). Honey bees
are important pollinators and crucial to the food
supply (Calderone, 2012), and those affected by
Nosema
species can be found across the world.
Generally, a spore of
N. apis
reproduces in the
midgut epithelium of the honey bee. However,
this is not the only anatomical location where
it can survive, as; it also appears in fat body,
the alimentary canal, malpighian tubules, hy-
popharyngeal glands and salivary glands;
(Chen & Huang, 2010; Ptaszyńska et al., 2012).
N. ceranae
is considered a more dangerous mi-
crosporidian than
N. apis
due to its potential to
infect the entire body of
A. mellifera
(Williams
et al., 2014). Nosemosis is most common during
the spring and autumn than summer, which
could be due to the excess energy consumed
by the bees to enhance the immune defense
for combatting any microbial attack. Addition-
ally, higher humidity and colder temperatures
during spring was also one of the reasons that
decreased the rate of nosemosis in summer
(Ptaszyńska, Paleolog, & Borsuk, 2016). During
these seasons, brown feces due to dysentery,
a common sign of nosemosis, were found in the
comb and around the hive (Klee et al., 2007).
*corresponding author: chemokine@cbnu.ac.kr
Received: 1 July 2017; accepted 8 January 2018
DOI: 10.2478/JAS-2018-0003
Original Article
J. APIC. SCI. VOL. 62 NO. 1 2018
Unauthenticated
Download Date | 4/12/18 2:25 PM

lee et AL.
2
Anti-nosemosis activities
A beekeeper can recognize the nosemosis
infection when there are weakened and dead
bees found around the hive, and then the
prognosis at this point is severe.
There have been a few attempts to develop
nosemosis therapy. The traditional approach
is to remove the
Nosema
infection through
the sterilization of the hives (with boiling
water, 6% soda, and blue ame) and destruc-
tion of the combs after the infected bees are
killed. Since the discovery of its anti-nosemosis
effects, fumagillin has been considered the
rst treatment choice (Whittington & Winston,
2003). Williams et al. (2008) reported that the
appropriate administration of fumagillin ef-
ciently combats
N. apis
, but its activity against
N. ceranae
is not very promising (Williams et al.,
2008). Thus, there is a need to nd a suitable
substance to combat
N. ceranae
infection of
A. mellifera
. Anti-nosemosis effects has been
showed for caffeine (Strachecka et al., 2014a),
curcumin (Strachecka, Olszewski, & Paleolog,
2015), coenzyme Q10 (Strachecka et al., 2014b)
and also ethanol extracts of
Aster scaber
(
A.
scaber
) and
Artemisia dubia
(
A. dubia
)
(Kim et al.,
2016).
In our previous study (Kim et al., 2016) we had
reported the anti-nosemosis activity of
A. scaber
and
A.
dubia
in ethanol solvent, since it is a polar
solvent whose extract consists of both polar
and non-polar compounds. Thus in our current
study, we sequentially extracted the
A. scaber
and
A. dubia
, based on the polarity of solvents
(water, butanol (BuOH) and ethyl acetate (EA))
and they were screened for
in vitro
and
in vivo
anti-nosemosis activity. IPL cell culture is a
perfect tool to screen the activity of substances
against
Nosema
species, so we employed these
cells for
in vitro
anti-nosemosis analysis (Gisder
et al., 2010; Kim et al., 2016).
MATERIAL AND METHODS
Plant material
A. scaber
and
A. dubia
were purchased from
the Kyungdong Oriental Herbal Market in Seoul,
Korea in April 2016. The plants were identied
at the Wild Vegetable Experiment Station,
Gangwon ARES, and the voucher specimen was
deposited at Chungbuk National University,
Korea.
Plant extraction preparation
Whole plants of
A. dubia
and
A. scaber
were
shade-dried
for one week and powdered by
using a blender. To obtain aqeous extract, about
20 g of
A. dubia
and
A. scaber
were soaked in
400 mL of water and reux extraction was
carried out at 100ºC for three hours. The con-
centrated aqueous extracts of both plants
were lyophilized separately to obtain nal
powdered form. The lyophilized plant powders
were soaked in water and EA at 1:1 ratio in a
separating funnel, where the EA portion was
separated and evaporated
in vacuo
to yield 8-13
g of extract. The same procedure was followed
to obtain BuOH extract, where EA was replaced
with BuOH, and the nal yield of the BuOH
extract was 8-13 g. These extracts were then
dissolved in dimethylsulfoxid (Sigma-Aldrich, St.
Louis, MO, USA) for further estimation.
Reagents and maintenance of cells
The IPL-LD-65Y cell line (IPL cell) was obtained
from the Deutsche Sammlung von Mikroorgan-
ismen und Zellkulturen (DSMZ, Braunschweig,
Germany) and maintained for routine culture in
a TC-100 medium (Sigma-Aldrich) with 11% fetal
calf serum (FCS, Hyclone Laboratories). The
cells were seeded at an initial concentration of
2×10 5 cells/mL in tissue culture asks (Nunc,
Roskilde, Denmark) and incubated at 27°C in a
cooling incubator. The cell pass was carried out
on every seventh day.
Cell viability test
The cell viability test was conducted by using
the IPL-LD-65Y cell line, where the cell line was
treated with various concentrations (0.625-10
μg/mL) of the plant extracts. The cell viability
was measured using the Wst-8 based colorimet-
ric assay (Dojindo, Japan), which was based on
the ability of live cells to reduce tetrazolium salt
into a soluble colored formazan product. The cell
suspension with 5×104 cells/well was cultured in
triplicate in a at-bottomed 96-well plate for 96
hours. The Wst-8 reagent was added to both the
cells and the blank samples, which were then
incubated for three hours at 37°C and 5% CO2.
Unauthenticated
Download Date | 4/12/18 2:25 PM

J. APIC. SCI. Vol. 62 No. 1 2018
3
The level of the dye formed was the measured
using a spectrophotometer (Bio-Rad, Hercules,
CA) at a wavelength of 450 nm. The blank values
without cells were subtracted from each experi-
mental value. Cell viability was expressed by
the percentage of live cells compared with that
found in the negative controls. The percentage
of cell viability was calculated as follows: cell
viability (%) = (OD level of experimental group/
OD level of negative control) × 100.
Isolation of Nosema spores
Nosema
spores were isolated from a naturally
infected hive located in the experimental apiary
of BEESEN CO., LTD., in Chungnam, Republic of
Korea.
Nosema
spores were isolated from the
honey bee midguts as previously described
(Gisder et al., 2011). Briey, after dissection the
midgut contents were collected, and macerated
in phosphate-buffered saline (PBS) with the
use of a tissue grinder, and the suspension was
ltered with a 70-μm mesh lter. Then, the
suspension was centrifuged at a range of 1500
to 12000 rpm to remove large particles, and the
mixture was resuspended in distilled water to
calibrate the number of spores using a hemocy-
to meter.
Identication of Nosema spores
A qualitative microscopic diagnosis of the
spores was performed to detect
Nosema
-in-
fected bee colonies. To differentiate the spore
species,
molecular species differentiation was
analyzed with the use of a polymerase chain
reaction (PCR); according to the procedure of
previous reports (Genersch et al., 2010; Gisder
et al., 2010). The DNA was isolated using GeneAll
Exgene (GeneAll, Seoul, Korea) per the manufac-
turer’s instructions. Briey, DNA was extracted
fr om 1×10 4
Nosema
spores and then amplied
with specic primers for
N. ceranae
or
N. apis
and a universal primer (
Nosema
species) for
N.
ceranae
and
N. apis
. The PCR primers used in
this study are listed below and were purchased
from Bioneer (Daejeon, Korea):
sense strand
Nosema
5’-GGCAGTTATGGGAAG-
TAACA-3’,
anti-sense strand
Nosema
5’-GGTCGTCA-
CATTTCATCTCT-3’;
sense strand
N. ceranae
5’- CGGATAAAAGAGTC-
CG T TACC-3 ’,
anti-sense strand
N. ceranae
5’-TGAGCAGGGTTCTAGGGAT-3’;
sense strand
N. apis
5’- CCATTGCCGGATAAGA-
GAGT-3 ’,
ant-sense strand
N. apis
5’-CACGCATTGCTGCAT-
CATTGAC-3’.
Each PCR was preheated to 94ºC for 2 minutes
followed by 94ºC for 15 seconds, 60ºC for 30
seconds, and 72ºC for 45 seconds, with a nal
extension phase at 72ºC for 7 minutes. A
variable number of cycles were used to ensure
that amplication occurred in the linear phase.
The PCR products were separated on a 1.5%
agarose gel and visualized by ethidium bromide
staining and ultraviolet irradiation.
Infection of IPL cells and application of test
extracts
Previously described methods were followed
to infect the IPL cells with
Nosema
(Gisder et
al., 2010; Williams et al., 2008). To induce spore
germination in every sample approximately
1×10 8
Nosema
spores were suspended in 200
μL of freshly prepared germination buffer (0.5
M sodium chloride, 0.5 M sodium hydrogen
carbonate, pH to 6.0 with orthophosphoric acid)
followed by 15-minute incubation at 37ºC to
allow spore germination (de Graaf et al., 1993).
After incubation, the IPL cells were harvested
by centrifugation at 210×g for ve minutes.
The cell pellet was then washed twice with 1
mL of freshly prepared 0.1 M sucrose in 1×PBS
buffer and resuspended in a sucrose buffer at
a concentration of 2.5×107 cells/mL. The ger-
minating spores (1×108) were resuspended in
100 μL of the IPL cell suspension (2.5×106 cells),
and the cell-spore suspension was incubated
for ve minutes at room temperature. Infected
cells were resuspended in 9.5 mL of a TC-100
cell culture medium supplemented with 11%
FCS, 250 μg/mL penicillin/streptomycin, and
250 μL antibiotic/antimycotic-solution (Sigma
Aldrich). Finally, 100 μL of the cell suspension
(2.5×104 infected cells) was carefully trans-
ferred into each well of a 96-well microplate. For
evaluation, 1 μL of EA and BuOH extract of
A.
dubia
and
A. scaber
were added to the mixture
of germinating spores and IPL cell suspension
Unauthenticated
Download Date | 4/12/18 2:25 PM

lee et AL.
4
Anti-nosemosis activities
in a TC-100 medium to achieve the desired nal
concentrations (0.625-10 μg/mL). The cells were
then incubated for 72 hours at 27°C, and their
infection status was subsequently determined
via microscopic and PCR analysis.
To perform microscopic analysis, infected cells
and oating spores were harvested from each
well and the number of spores ware counted
using a hemocytometer. PCR analysis was
performed after the isolation of DNA from
harvested infected cells and oating spores. The
percentage of inhibition by microscopic analysis
was calculated as follows: Inhibition rate (%) =
[((number of
Nosema
spores in the treated cells
at the initial stage - number of
Nosema
spores
in the treated cells after 72 hours) / (number
of
Nosema
spores in the untreated cells at
initial stage - number of
Nosema
spores in the
untreated cells after 72 h) × 100)-100] × -1.
Experimental design of the in vivo study
At least 300 healthy bees were collected from
each of the three different source colonies and
they were carefully transferred to two mesh
cages (16.5×16.5×48 inches), and stored at 33
± 1°C. Microscopic analysis was carried out to
identify the normal bees and included in our
experiment.
Prior to the anti-nosemosis experiment with live
bees, an
in vivo
toxicity test was performed in
which about twenty honey bees per cage were
employed and usually treated in the morning
by 10 am. While normal control was fed with
50% sugar solution alone; cage 2 was fed with
fumagilin (fumidil B) (20 mg/mL), a commercial
reference drug. The rest of the cages were
treated with
A. dubia
water extract 0.125 - 10
(μg/mL);
A. scaber
water extract 0.125 - 10
(μg/
mL);
A. dubia
BuOH extract 0.125 - 10 (μg/mL);
A. scaber
BuOH
extract 0.125 - 10
(μg/mL);
A.
dubia
EA extract 0.125 - 10 (μg/mL);
A. scaber
extract 0.125 - 10 (μg/mL). The live bees were
counted at 0, 24, 48, 72, 96 and 120 hrs. The
results were expressed in percentage.
In vivo
activity tests of
A. scaber
and
A. dubia
were performed with uninfected (ie, healthy)
bees. The study lasted one week (2 day for
Nosema
infection induction and 5 days for
treatment) and on day 0 the bees were split into
two experimental groups each of twenty bees.
On the same day, group 1 was fed with only
50% sucrose solution and considered normal
control and group 2 was fed with
Nosema
spores 1x107 in 50% sucrose solution for 48
hrs to induce nosemosis. Afterwards, cage 1
was considered normal control and fed with
DMSO dissolved in 50% sucrose solution only,
and cage 2 was
Nosema
infection control. The
rest of the cages of the
Nosema
infected honey
bees were treated with
A. dubia
water extract
(0.125, 0.250, 0.50, 1 μg/mL);
A. dubia
BuOH
extract (0.125, 0.250, 0.50, 1 μg/mL);
A. dubia
EA extract (0.125, 0.250, 0.50, 1 μg/mL) and
A.
scaber
water extract (0.125, 0.250, 0.50, 1 μg/
mL);
A. scaber
BuOH extract (0.125, 0.250, 0.50,
1 μg/mL);
A. scaber
EA extract (0.125, 0.250,
0.50, 1 μg/mL) and fumagillin (20mg/mL). The
treatment usually occurred in the morning by 10
am. After one weak of the experiment, all the
normal infection control and treated honey-bee
cages were anesthetized with CO2 to facilitate
handling. To assess the anti-nosemosis activity,
the three honey-bee midguts were dissected
and introduced into antiseptic micro tubes lled
with 200 μL distilled water. After thorough
grinding, the spores were counted using a hemo-
cytometer under a phase-contrast microscope.
RESULTS
Identication of Nosema species
The spindled shaped spores isolated from
naturally infected bees were nosema (Fig 1A).
To differentiate the spore species,
molecular
species differentiation was performed with PCR
following the procedure of previous reports
(Gisder et al., 2011; Whittington & Winston,
2003). As shown in Fig. 1B, DNA from the
isolated spores was amplied with a universal
Nosema
primer and
N. ceranae
primer. However,
DNA was not amplied using an
N. apis
primer.
Viability of IPL cells with plant extracts
Since toxicity studies for this assay were
paramount, we evaluated the cytotoxicity of all
the extracts in a broad range of concentrations
(0.625-10 μg/mL) using Wst-8 assays. Concen-
trations below 1% DMSO did not inuence the
Unauthenticated
Download Date | 4/12/18 2:25 PM

J. APIC. SCI. Vol. 62 No. 1 2018
5
viability of the IPL cells. As shown in Fig. 2, each
extract of
A. dubia
(A) and
A. scaber
(B) showed
a concentration-dependent cytotoxicity, but
there was no effect on cell viability, even at high
concentrations.
In vitro screening of anti-nosemosis extracts
Fig. 3 exhibits the
in vitro
anti-nosemosis
activity of the extracts of
A. dubia and A. scaber
.
Although the level of activity varied, all of the
extracts of
A. dubia
(Fig. 3A) and
A. scaber
(Fig.
3B) showed anti-nosemosis activity in a dose-
dependent manner. Interestingly, the anti-nose-
mosis activity of both plants increased in the
order of aqueous, BuOH and EA. The aqueous
extract of
A. dubia
and
A. scaber
reduced the
population of
N. ceranae
to 33-34%, the BuOH
extract to 53-58%, and the EA extract to
64-76% of the original population. There was
no signicant difference in the anti-nosemosis
activity of
A. dubia
and
A. scaber
at the highest
concentration.
To conrm the anti-nosemosis activity of
A.
dubia
and
A. scaber
, PCR analysis was performed
in the highest concentration of each extract. As
shown in Fig. 4, the band intensities of the
N.
ceranae
DNA decreased after treatment with
aqueous extracts of
A. dubia
(Fig. 4A) or
A.
scaber
(Fig. 4B). However, the extracts of BuOH
and EA did not signicantly inuence the DNA
band intensity of
N. ceranae
.
Fig. 1. Identication of
Nosema
species. A
Nosema
spore was isolated from the midgut of the honey
bees and conrmed by microscope (A). Representa-
tive pictures of
Nosema
spores are shown. DNA was
extracted from 1×104 spores, and then the amplied
region indicated the specic primer. PCR analyses
using a specic primer were performed in triplicate,
and all ndings showed similar results (B).
Fig. 2.
In vitro
toxicity of water, BuOH and EA
extracts of
A. dubia
(A) and
A. scaber
(B). The IPL
cells were treated with various concentrations of
the plant extracts, and cell viability was measured
using the Wst-8 based colorimetric. The values that
are shown are the means ± SDs of the three inde-
pendent experiments.
Fig. 3. Effect of water, BuOH and EA extracts of
A.
dubia
(A) and
A. scaber
(B) on the development of
Nosema
spore. Germinated spores of
N. ceranae
were mixed with IPL cell line suspension and then
treated with plant extracts. Inhibition rate of spore
population was determined by microscopic analysis
with hemocytometer. The values that are shown
are the means ± SDs of the three independent ex-
periments.
Unauthenticated
Download Date | 4/12/18 2:25 PM

lee et AL.
6
Anti-nosemosis activities
In vivo toxicity of each extract of A. dubia
and A. scaber
In the untreated control group, more than 90%
of the bees survived during the experimental
period (Fig. 5). At the same time, the Fumagi-
lin-treated group showed intermediate toxicity
after 120 hours of incubation. Interestingly, all
extracts up to a concentration of 1 μg/mL in 50%
sucrose solution showed the survival rate of
bees between 81.3-98.1%. However, increasing
the concentrations of extracts to 2-10 μg/mL
caused a slight decrease in the percentage of
live bees to 63-79% of the original population.
In particular, feeding 10 μg/mL of the
A. dubia
BuOH extract showed the lowest percent of live
bees (63%). Concentrations from 0.125-10 μg/
mL of each of the extracts showed the following
live bee viability: aqueous extract of
A. dubia
75-90%, BuOH extract of
A. dubia
63-98%, EA
extract of
A. dubia
68-89%, aqueous extract of
A. scaber
68-88%, BuOH extract of
A. scaber
64-95%, and EA extract of
A. scaber
65-86%.
The aqueous extract showed lower toxicity
than either the BuOH or EA extracts.
In vivo activity of three different kinds of
extract
Fig. 6 shows the anti-nosemosis activity of
different extracts of
A. dubia and A. scaber,
where the DMSO-only control group had the
highest number of
Nosema
spores compared
with the groups treated with extracts. Among
the three kinds of solvents used for extraction
Fig. 4. Anti-nosemosis screening by PCR analysis. In
the same condition with Fig. 3, PCR analyses using a
specic primer were performed in triplicate, and all
of them produced similar results.
Fig. 5.
In vivo
toxicity of each extracts. Healthy bees
were divided into nine groups and then treated with
various concentrations of each extract (
A. dubia
:
AD,
A. scaber
: AS) with 50% sucrose solution for 5
days. The survival rate was calculated on the fth
day as follows: Survival rate (%) = [(number of live
bees)/(total number of bees)] x 100. The values that
are shown are the means ± SDs of the three inde-
pendent experiments.
Fig. 6.
In vivo
activity of each extract. Infected bees
by
N. ceramae
were treated with 0.125 - 1 μg/ml
extracts of
A. dubia
(A) and
A. scaber
(B) in a 50%
sucrose solution for 5 days. The spores, which were
derived from the midgut, were counted using a
hemocytometer. The values that are shown are the
means ± SDs of the three independent experiments.
∗∗p < 0.01 as compared to the untreated bees.
Unauthenticated
Download Date | 4/12/18 2:25 PM

J. APIC. SCI. Vol. 62 No. 1 2018
7
(aqueous, BuOH, and EA), the highest inhibition
of spore proliferation was observed in the
groups treated with aqueous extracts. Notably,
1 μg/mL of the aqueous extract of
A. dubia
reduced the number of spores by around 75%.
The aqueous extract of
A. scaber
also reduced
the number of spore by almost 72% at the 1
μg/mL concentration. The positive control drug,
fumagillin (20 mg/mL), showed less anti-nose-
mosis activity (27-33%), whereas the BuOH and
EA extracts of
A. dubia
reduced the number
of spores by 57% and 52%, respectively. The
BuOH and EA extracts of
A. scaber
reduced the
number of spores by 55% and 54%, respectively.
In vivo activity of aqueous extract of A.
dubia + A. scaber
In order to evaluate the anti-nosemosis activity
of the aqueous extracts, the aqueous extract of
A. scaber
+
A. dubia
was prepared in addition to
the
A. scaber
and
A. dubia
extracts. As shown in
Fig. 7, 85% of the untreated, uninfected control
bees survived for 120 hours, but only 70% of
the fumagillin-treated bees were survived. In-
terestingly, none of the three extracts tested
showed toxicity at any concentration. Even in
bees treated with
A. scaber
1 μg/mL
,
showed a
less toxicity with
85% survival rate.
The anti-nosemosis activity of each extract
was analyzed using the same methods depicted
in Fig. 6. Fumagillin (20 mg/mL) was used as a
positive control drug. As shown in Fig. 8, all of
the aqueous extracts exhibited anti-nosemosis
activity. Among the three aqueous extracts,
those treated with both
A. scaber
+
A. dubia
showed the highest inhibition of spore prolifera-
tion. The aqueous extracts of
A. dubia
and
A.
scaber
alone also reduced the number of spores
by almost 58% and 64%, respectively at 1 μg/
mL. However, the aqueous extract of
A. dubia
+
A. scaber
reduced the number of spores by
almost 76% at 1 μg/mL.
DISCUSSION
In our previous paper (Kim et al., 2016), we
prepared ethanol extracts of
A. scaber
,
A. dubia
and
A. scaber
+
A. dubia,
and demonstrated the
anti-nosemosis activity of both
A. scaber
and
A. dubia
at non-toxic concentrations. Although
both
A. scaber
and
A. dubia
separately had
showed anti-nosemosis activity, combined
A. scaber
+
A. dubia
exhibited even stronger
activity. Therefore in the present study, we
Fig. 7.
In vivo
toxicity of water extracts. Healthy
bees were treated with water extracts of
A. dubia
(AD),
A. scaber
(AS) and
A. dubia
+
A. scaber
(AD+AS)
as in Fig. 5. The survival rate was represented by
percent of live bees. The values that are shown are
the means ± SDs of the three independent experi-
ments.
Fig. 8.
In vivo
activity of water extracts. Infected
bees by
N. ceramae
were treated with water
extracts of
A. dubia
(AD),
A. scaber
(AS) and
A. dubia
+
A. scaber
(AD+AS) in a 50% sucrose solution for
5 days. The spores, which were derived from the
midgut, were counted using a hemocytometer.
The values that are shown are the means ± SDs of
the three independent experiments. ∗∗p < 0.01 as
compared to the untreated bees.
Unauthenticated
Download Date | 4/12/18 2:25 PM

lee et AL.
8
Anti-nosemosis activities
have tried to demonstrate the chemical char-
acteristics of the active compounds in each
extract. In our previous study, 100 μg/mL of
the ethanol extract of
A. scaber
+
A. dubia
showed 77% spore reduction without
in vivo
toxicity, but as expected, the aqueous extract
of
A. dubia
+
A. scaber
, in which BuOH and EA
soluble compounds were eliminated, reduced
the number of spores to almost 76% at 1 μg/mL.
As a result, the active molecule had to dissolve
in water. Both the BuOH and EA extracts also
showed anti-nosemosis activity albeit not as
strong as the aqueous extracts. These results
indicate that
A. dubia
and
A. scaber
have
more than one anti-nosemosis compound with
different solubility in each solvent (aqueous,
BuOH, and EA).
The anti-nosemosis effects of
A. dubia
and
A. scaber
have not been reported except in our
previous paper. As
A. dubia,
Artemisia absinthium
has also been tested against
Nosema
species
by two different research groups (Pohorecka,
2004; Porrini et al., 2011). Pohorecka (2004) rst
reported that an ethanol extract of
Artemisia
absinthium
inhibited
N. apis
, and
Porrini et al.,
(2011) reported the anti-nosemosis activity of
Artemisia absinthium
seven years later.
Unfortunately, the results of these two research
teams are inconsistent. While Pohorecka (2004)
reported that
Artemisia absinthium
had an an-
timicrobial effect, Porrini et al. (2011) reported
no such effect. Porrini and his colleagues (20 11)
reported that different susceptibilities of
N.
ceranae
and
N. apis
to the ethanol extract of
this herb or other factors that could inuence
the chemical composition (eg, the extraction
method) might explain this difference between
the two studies Ahameethunisa & Hopper,
(2010) and by Tariq et al., (2009). But in our
opinion,
A. scaber
which contains ve important
secondary metabolites namely, Caffeoyl quinic
acid, (-) 3, 5-dicaffeoyl-muco-quinic acid, (-) 3,
5-dicaffeoyl quinic acid, (-) 4, 5-dicaffeoyl quinic
acid, (-) 5-caffeoyl quinic acid with anti-fungal
property, and
A. dubia
composed of caffeic acid,
gallic acid, catechin, coumarin, and camphor could
act against fungus.
Nosema
’s place
in the fungi
kingdom lead us to speculate that both
A. scaber
and
A. dubia
could have anti nosemosis activity
(Kwon.et al., 2000; Rhimi et al., 2017; Sardi et al.,
2016; Li et al., 2017; Hirasawa & Takada, 2004;
Montagner et al., 2008; Mahilrajan et al., 2014;
Kiani et al., 2016).
Fumagillin, isolated from the microbial organism
A. fumigatus,
has been used against a variety of
microsporidial parasites in both bee and human
medicine. It has also been shown to inhibit an-
giogenesis (Chung et al., 1993) and thus been
studied in cancer treatment research. Moreover,
it has been the most commonly used medicinal
product in the treatment of
Nosema
infection in
western honey bees,
A. mellifera
(Bai ley, 1953;
Higes et al., 2011).
Although the most valuable compound in
human medicine and apiculture, fumagillin is not
free from such side effects as gastrointestinal
cramping, diarrhea, and signicant weight loss,
and has limited application in humans (Chung
et al., 1993; Molina et al., 2000; Molina et al.,
2002; Yanase et al., 1993). Moreover, chro-
mosomal aberrations and genotoxic potential
have been observed in mice (Kulić et al., 2009;
Stanimirović, 2010). Enhanced
Nosema
species
infection leads to increased fumagillin sales,
and residues of fumagillin have been detected
in harvested apicultural products (Lopez et al.,
2008). Therefore, the potential fumagillin con-
tamination of apicultural products intended for
human consumption could be eliminated by the
development of alternative treatments against
Nosema
species.
Fumagillin is used as a primary treatment for
Nosema
infection, but the latest report has
shown it to be ineffective against
N. ceranae
(Huang et al., 2013).
Some reports state that
N. ceranae
can regrow six months after a
treatment is terminated, despite some evidence
that
N. apis
has never developed a resistance
to fumagillin (Higes et al., 2011; Pajuelo, Torres,
& Bermejo, 2008). Neither susceptibility nor
quick recuperation after fumagillin treatment
could account for the replacement of
N. apis
by
N. ceranae
, which has seemingly occurred in
North America and elsewhere (Chen et al., 2009;
Huang et al., 2008; Klee et al., 2007).
Therefore, in this study, we suggested two anti-
Unauthenticated
Download Date | 4/12/18 2:25 PM

J. APIC. SCI. Vol. 62 No. 1 2018
9
nosemosis plants,
A. scaber
and
A. dubia
, which
reduced the spore development of
N. ceranae
in both
in vitro
and
in vivo
experiments. In
particular, the mixture of
A. scaber
and
A. dubia
showed stronger activity than treatment with
only a single plant, which could be due to the
synergetic activity of secondary metabolites
present in
A. scaber
and
A. dubia
. Moreover,
the anti-nosemosis effects are conrmed to be
better when the extracts are dissolved in water
compared with butanol or ethyl acetate. The
mechanisms of action of
A. scaber
and
A. dubia
against
N. ceranae
remains unclear, and further
research is necessary to identify these active
compounds. In conclusion, our results suggest
new possibilities for controlling
N. ceranae
infection in honey bees.
ACKNOWLEDGEMENTS
This research was nancially supported by
the Ministry of Trade, Industry and Energy
(MOTIE) and Korea Institute for Advancement
of Technology (KIAT) through the Research and
Development for Regional Industry.
REFERENCES
Ahameethunisa, A.R., & Hopper, W. (2010). Antibac-
terial activity of
Artemisia nilagirica
leaf extracts
against clinical and phytopathogenic bacteria
. BMC
Complementary and Alternative Medicine, 10
, 6.
DOI:10.1186/1472- 6 882-10-6
Bailey, L. (1953). Effect of Fumagillin upon
Nosema
apis
(Zander).
Nature 171
: 212-213.
Calderone, N.W. (2012). Insect pollinated crops, in-
sect pollinators and US agriculture: trend analysis of
aggregate data for the period 1992-2009.
PloS One
7
, e37235. DOI:10.1371/journal.pone.0037235
Chen, Y., Evans, J.D., Zhou, L., Boncristiani, H., Kimura, K.,
Xiao, T., Litkowski, A.M., Pettis, J.S. (2009). Asymmet-
rical coexistence of
Nosema ceranae
and
Nosema
apis
in honey bees. J
ournal of Invertebrate Pathol-
ogy, 101
, 204-209. DOI:10.1016/j.jip.2009.05.012
Chen, Y.P., & Huang, Z.Y. (2010).
Nosema ceranae
, a
newly identied pathogen of
Apis mellifera
in the
USA and Asia.
Apidologie, 41
, 364-374. DOI: 10.1051/
apido/2010021
Chung, J.W., Im, J.G., Park, J.H., Han, J.K., Choi, C.G., Han,
M.C. (1993). Left paracardiac mass caused by di-
lated pericardiacophrenic vein: report of four cases.
American Journal of Roentgenology, 160,
25-28.
DOI :10.2214/aj r.16 0 .1.8 416638
de Graaf,D.C., Masschelein, G., Vandergeynst, F., De
Brabander, H.F., Jacobs, F.J. (1993).
In Vitro
Germination
of
Nosema apis
(Microspora: Nosematidae) Spores
and its effect on their αα-trehalose/d-glucose ratio.
Journal of Invertebrate Pathology, 62,
220-225. DOI:
http://dx.doi.org/10.1006/jipa.1993.1103
Genersch, E., von der Ohe, W., Kaatz, H., Schroeder,
A., Otten, C., Büchler, R., Berg, S., Ritter, W., Mühlen,
W., Gisder, S., Meixner, M., Liebig, G., Rosenkranz, P.
(2010). The German bee monitoring project: a long
term study to understand periodically high winter
losses of honey bee colonies.
Apidologie, 41
, 332-
352. DOI: https://doi.org/10.1051/apido/2010014
Gisder, S., Hedtke, K., Mockel, N., Frielitz, M.C., Linde,
A., Genersch, E. (2010). Five-year cohort study of
Nosema spp.
in Germany: does climate shape viru-
lence and assertiveness of
Nosema ceranae
?
Ap-
plied and Environmental Microbiology, 76,
3032-
3038. DOI:10.1128/AEM.03097-09
Gisder, S., Mockel, N., Linde, A., & Genersch, E. (2011). A
cell culture model for
Nosema ceranae
and
Nosema
apis
allows new insights into the life cycle of these
important honey bee-pathogenic microsporidia.
En-
vironmental Microbiology, 13,
4 0 4 - 413 . D O I :10 .1111/
j.1462-2920.2010.02346.x
Higes, M., Nozal, M.J., Alvaro, A., Barrios, L., Meana, A.,
Martín-Hernández, R., Bernal, J. L., Bernal, J. (2011).
The stability and effectiveness of fumagillin in
controlling
Nosema ceranae
(Microsporidia) infec-
tion in honey bees (
Apis mellifera
) under labora-
tory and eld conditions.
Apidologie, 42,
36 4 - 37 7.
DOI:10.10 07/s13592-011-0 003-2
Unauthenticated
Download Date | 4/12/18 2:25 PM

lee et AL.
10
Anti-nosemosis activities
Hirasawa, M.,& Takada, K. (2004). Multiple effects
of green tea catechin on the antifungal activity of
antimycotics against Candida albicans.
Journal of
Antimicrobial Chemotherapy
,
53 (2),
225-229.
Huang, W.F., Bocquet, M., Lee, K.C., Sung, I.H., Jiang,
J.H., Chen, Y.W., Wang, C.H. (2008). The comparison
of rDNA spacer regions of
Nosema ceranae
iso-
lates from different hosts and locations.
Journal
of Invertebrate Pathology, 97,
9-13. DO I :10.1016 /j.
j i p . 2 0 0 7. 0 7. 0 0 1
Huang, W.F., Solter, L.F., Yau, P.M., & Imai, B.S. (2013).
Nosema ceranae
escapes fumagillin control in hon-
ey bees.
PLoS Pathog, 9,
e1003185. D OI:10.1371/
jou r nal. ppat .1003185
Kiani, B.H., Suberu, J., & Mirza, B. (2016). Cellular
engineering of
Artemisia annua
 and
Artemisia
dubia
with the
rol ABC
genes for enhanced pro-
duction of potent anti-malarial drug artemisinin.
Ma-
lariya Journal, 15,
252.
Kim, J.H., Park, J.K., & Lee, J.K. (2016). Evaluation of an-
timicrosporidian activity of plant extracts on
Nose-
ma Ceranae
.
Journal of Apicultural Science, 60
(1) ,
167-178. DOI: 10 .1515 / j as - 2016- 0 0 27
Klee, J., Besana, A. M., Genersch, E., Gisder, S., Nan-
etti, A., Tam, D.Q., … Paxton R.J. (2007). Widespread
dispersal of the microsporidian
Nosema ceranae,
an
emergent pathogen of the western honey bee, Apis
mellifera.
Journal of Invertebrate Pathology, 96,
1-10.
DOI:10.1016 /j. j ip.2 0 07.02.014
Kulić, M., Aleksić, N., Stanimirović, Z., Ristić, S., Meden-
ica, S. (2009). Examination of genotoxic effects of
fumagillin in vivo.
Genetika, 41
, 329-338.
Kwon, H.C., Jung, C.M., Shin, C.G., Lee, J.K., Choi, S.U.,
Kim, S.Y., Lee, K.R. (2000). A new caffeoyl quinic acid
from aster scaber and its inhibitory activity against
human immunodeciency virus-1 (HIV-1) integrase.
Chemical and Pharmaceutical Bulletin, 48,
1796 -
1798 .
Li, Z.J.,Liu, M.,Dawuti, G., Dou, Q.,Ma, Y.,Liu, H.
G., Aibai, S. (2017).Antifungal activity of gallic acid
in vitro and in vivo.
Phytotherapy Research,
31( 7) ,
1039-10 45.
Lopez, M.I., Pettis, J.S., Smith, I.B., & Chu, P.S. (2008).
Multiclass determination and conrmation of an-
tibiotic residues in honey using LC-MS/MS.
Journal
of Agricultural and Food Chemistry, 56
, 155 3 -1559 .
DOI:10.1021/jf 073236w
Mahilranjan, S., Nandakumar, J., Kailayalingam,
R.,Manoharan, N.A.,SriVijeindran, S. (2014). Screen-
ing the antifungal activity of essential oils against
decay fungi from palmyrah leaf handicrafts.
Biologi-
cal Research, 47(1),
35.
Molina, J.M., Goguel, J., Sarfati, C., Michiels, J.F., De-
sportes-Livage, I., Balkan, S., … Decazes J.M. (2000).
Trial of oral fumagillin for the treatment of intesti-
nal microsporidiosis in patients with HIV infection.
ANRS 054 Study Group. A
gence Nationale de Re-
cherche sur le SIDA. AIDS, 14,
1341-13 48 .
Molina, J.M., Tourneur, M., Sarfati, C., Chevret, S., de
Gouvello, A., Gobert, J.G., Balkan, S., Derouin, F. (2002).
Fumagillin treatment of intestinal microsporidiosis.
The New England Journal of Medicine, 346
, 19 63 -
1969. D O I:10.105 6/NEJ Moa0129 24
Montagner, C.,de Souza, S.M.,Groposoa, C.,Delle
Monache, F., Smânia, E.F., Smânia, A.(2008). Anti-
fungal activity of coumarins.
Zeitschrift für Natur-
forschung C,63(1-2),
21- 8 .
Pajuelo, A.G., Torres, C., & Bermejo, F.J.O. (2008). Col-
ony losses: a double blind trial on the inuence of
supplementary protein nutrition and preventative
treatment with fumagillin against
Nosema
ceranae.
Journal of Apicultural Research, 47,
84-86.
Pohorecka, K. (2004). Laboratory studies on the ef-
fect of standardized
Artemisia absinthium
L. extract
on
Nosema apis
infection in the worker
Apis mellif-
era
.
Journal of Apicultural Science, 48,
131-136 .
Porrini, M.P., Fernández, N.J., Garrido, P.M., Gende, L.B.,
Medici, S.K., Eguaras, M.J. (2011). In vivo evaluation of
antiparasitic activity of plant extracts on
Nosema
ceranae
(Microsporidia).
Apidologie, 42,
700-707.
DOI:10.10 07/s13592-011-0 076-y
Unauthenticated
Download Date | 4/12/18 2:25 PM

J. APIC. SCI. Vol. 62 No. 1 2018
11
Ptaszynska, A.A., Borsuk, G., Anusiewicz, M., & Mu-
lenko, W. (2012). Location of Nosema spp. spores
within the body of the honey bee.
Medycyna we-
terynaryjna,
68 (10),
618 - 621.
Ptaszyńska, A.A., Paleolog, J., & Borsuk, G. (2016)
Nosema ceranae infection promotes prolifera-
tion of yeasts in honey bee intestines.
PLoS ONE,
11( 10 ) ,
e0164477. https://doi.org/10.1371/journal.
pone.0164477
Rhimi, W., Salem, I.B., Immediato, D., Saidi, M., Boulila,
A., Cafarchia, C. (2017). Chemical Composition, Anti-
bacterial and Antifungal Activities of Crude Dittri-
chia viscosa (L.) Greuter Leaf Extracts.
Molecules,
22
, 942.
Sardi, J.C.,Gullo, F.P.,F reires, I.A .,Pitangui, N.S.,Segal-
la, M.P.,Fusco-Almeida, A.M.,... , Mendes-Giannini,
M.J.(2016). Synthesis, antifungal activity of caffeic
acid derivative esters, and their synergism with u-
conazole and nystatin against Candida spp.
Diag-
nostic Microbiology and Infectious Disease,
86 (4),
387-391.
Sprague, V., & Becnel, J.J. (1998). Note on the name-
author-date combination for the taxon “Microspori-
dies” Balbiani, 1882, when ranked as a phylum.
J
our-
nal of Invertebrate Pathology,
71
, 91-94.
Sprague, V., & Becnel, J.J. (1999). Appendix: checklist
of available generic names for Microsporidia with-
type species and type hosts, Wittner M., Weiss L. M.
(eds.): Microsporidia and Microsporidiosis. ASM Press,
Washington, D.C. 517-530.
Stanimirović, Z., Aleksić, N., Kulić, M., & Maletić, M.
(2010). Fumagillininduced chromosome aberrations
in mouse bone-marrow cells.
Archives of Biological
Sciences, 62
, 47-55.
Strachecka, A., Krauze, M., Olszewski, K., Borsuk,
G., Paleolog, J., Merska, M., …, Grzywnowicz, K.
(2014a). Unexpectedly strong effect of caffeine
on the vitality of western honeybees (
Apis mellif-
era
).
Biochemistry (Moscow), 79(11),
119 2 -12 0 1.
Strachecka, A., Olszewski, K., Paleolog, J., Borsuk, G.,
Bajda, M. (2014b). Coenzyme Q10 treatments inu-
ence the lifespan and key biochemical resistance
systems in the honeybee,
Apis mellifera
.
Archives of
Insect Biochemistry and Physiology,
86(3),
165-17 9 .
DOI: 10.1002/ arch.21159
Strachecka, A., Olszewski, K., & Paleolog, J. (2015).
Curcurmin stimulates biochemical mechanisms of
Apis Mellifera resistance and extends the apian life-
span.
Journal of Apiculture Science, 59(1),
129 -141.
DOI: http://doi.org/10.1515/jas-2015-0014
Tariq, K.A., Chishti, M.Z., Ahmad, F., & Shawl, A.S.
(2009). Anthelmintic activity of extracts of
Arte-
misia absinthium
against ovine nematodes.
Vet-
erinary Parasitology, 160,
83-8 8 . DOI :10 .1016/j .vet-
par.2008.10.084
Whittington, R., & Winston, M.L. (2003). Effects of
Nosema bombi
and its treatment fumagillin on bum-
ble bee (
Bombus occidentalis
) colonies.
Journal of In-
vertebrate Pathology, 84
, 54-58.
Williams, G.R., Sampson, M.A., Shutler, D., & Rogers,
R.E. (2008). Does fumagillin control the recently
detected invasive parasite
Nosema ceranae
in
western honey bees (
Apis mellifera
)?
Journal of In-
vertebrate Pathology, 99,
342-3 44. D OI:10.1016/ j.
jip.2008.04.005
Williams, G.R., Shutler, D., Burgher-MacLellan, K.L., &
Rogers, R.E. (2014). Infra-population and -communi-
ty dynamics of the parasites
Nosema apis
and
Nose-
ma ceranae
, and consequences for honey bee (
Apis
mellifera
) hosts.
PloS One 9
, e99465. DOI:10.1371/
Journal.pone.0099465
Yanase, T., Tamura, M., Fujita, K., Kodama, S., Tanaka,
K. (1993). Inhibitory effect of angiogenesis inhibitor
TNP-470 on tumor growth and metastasis of hu-
man cell lines in vitro and in vivo.
Cancer Research,
53
, 2566-2570.
Unauthenticated
Download Date | 4/12/18 2:25 PM

lee et AL.
12
Anti-nosemosis activities
Unauthenticated
Download Date | 4/12/18 2:25 PM