Goishi tea consumption inhibits airway hyperresponsiveness in BALB/c mice
Goishi tea consumption inhibits airway hyperresponsiveness in BALB/c mice
Ryoji Hirota 0
Nlandu R Ngatu 0
Mitsuhiko Miyamura 2
Hiroyuki Nakamura 1
Narufumi Suganuma 0
0 Department of Environmental Medicine, Kochi Medical School, Kochi University , Japan
1 Department of Environmental and Preventive Medicine, Kanazawa University Graduate School of Medical Science , Japan
2 Department of Pharmacy, Kochi Medical School Hospital, Kochi University , Japan
Background: Airway hyperresponsiveness (AHR) is one of the important traits that characterize bronchial asthma. Goishi tea is a post-heating fermented tea that has been reported to have higher free radical scavenging activity. In this study, we evaluated the prophylactic effects of Goishi tea on AHR in BALB/c mice. Results: The number of inflammatory cells in BAL fluid was considerably reduced in Goishi tea/Der f and Gallic acid/Der f groups as compared with Tap water/Der f group. Regarding inflammatory cells in BAL, a significant reduction of eosinophils and neutrophils was observed in Goishi tea-treated mice (p < 0.01), as well as in the Gallic acid/Der f group (p < 0.05), as compared with Tap water/Der f group. In asthmatic mice (Tap water/Der f group), the intensity of airway resistance increased simultaneously with the increase in acetylcholine concentration in a dose-dependant way. AHR was significantly inhibited in Goishi tea/Der f and Gallic acid/Der f (p < 0.01) groups as compared with the Tap water/ Der f group. Regarding serum specific-IgG1, significantly lower levels of this antibody were observed in Goishi tea/Der f and Gallic acid/Der f groups as compared with the Tap water/Der f group (p < 0.05). In addition, adiponectin level was significantly higher in the Goishi tea group as compared with the Tap water treated mice (p < 0.01). Conclusions: The results suggest that Goishi tea consumption exerted an inhibitory effect on eosinophilic and neutrophilic infiltration in the lung, attenuated the increase in airway resistance and increased the production of adiponectin; thus reducing Der f induced allergic inflammatory process in mice.
adiponectin; allergen; airway hyperresponsiveness; eosinophil; Goishi tea
Airway hyperresponsiveness (AHR) is one of the
important traits that characterize bronchial asthma, apart
from eosinophilic infiltration, reversible airway
narrowing and chronic inflammation [1,2].
Lately, there has been a growing interest in natural
plants extracts containing flavonoids and polyphenols in
search of new therapies thanks to their bioactive
properties. Epigallocatechin gallate (EGCG) and catechin from
green tea, for example, have been reported to improve
cardiovascular function, increase fat oxidation in mice
and exert free radicals and reactive oxygen species
(ROS) scavenging activity [3-7].
Since more than 100 years ago, a post-heating
fermented tea has been produced in many places in Japan.
Goishi tea, known as goishi-cha in Japanese language,
is one of a post-heating fermented tea which is
produced in Otoyo town, Kochi prefecture, Japan, where
local people referred to its sour taste as the tea gruel.
Nowadays, most fermented tea manufacturers have
already stopped the production because of lower
demand; and only three of them still continue producing
post-heating fermented tea in Japan.
Goishi tea is made from Camelia sinensis leaves, as for
green tea. However, in order to make Goishi tea, two
different fermentation processes are needed that are
performed in two steps: the aerobic fermentation (with
fungi) and the anaerobic fermentation. Green tea is
processed as follows: harvested Camellia sinensis leaves are
heated rubbed and then they are dried. Recently,
traditional tea has become popular especially in Japan because
of its beneficial health effects. Interestingly, Goishi tea is
named tea of legend thanks to its efficacy in diet. Up to
now, there are few publications on Goishi tea and very
little is known about its bioactive properties.
We have demonstrated that Goishi tea manufacturing
process improves the DPPH
(2,2-diphenyl-1-picrylhydrazyl) and superoxide scavenging activity as compared
with green tea . The consumption of green tea has
been reported to increase adiponectin expression; this
chemokine is known to improve airway inflammation
and some cardiovascular diseases [9,10]. Therefore, we
hypothesized that Goishi tea consumption might
attenuate airway inflammation. In this study, we evaluated the
prophylactic effects of Goishi tea consumption in BALB/
c mice induced AHR.
Thirty BALB/c mice, aged five weeks, were purchased
from Japan SLC (Hamamatsu, Shizuoka, Japan). Animals
were housed under conventional conditions at the
animal facility of Kochi Medical School in filter-topped
macrolon cages with a bedding of wood chips,
temperature of 23C, 50-60% relative humidity, and a 12-h light/
dark cycle. They were divided into six groups of five
mice each: (1) Goishi tea/Dermatophagoides farinaeplus
(Der f); (2) Goishi tea/phosphate buffered saline (PBS),
(3) 1% Gallic acid/Der f, (4) Gallic acid/PBS, (5) Tap
water/Der, and (6) Tap water/PBS groups. They received
standard lab chow ad libitum. Animals were maintained
until they were 7 weeks old (~ 20-24 g body wt) at the
time of sensitization. All research adhered to the animal
facility guidelines of Kochi Medical School (C000144).
Goishi tea extracts preparation
The Goishi tea sample used in this experiment was a
gift from the Otoyo county office staff, Kochi prefecture,
Japan. Goishi tea extract was prepared as previously
described . Briefly, Goishi tea is made following a
traditional process; first of all, the harvested Camellia
sinensis leaves are steamed once in a tank, then
fermented on the flat plate for 7 days. Afterwards, fermented
leaves are moved into another tank and then
re-fermented for 10 days. Finally, the cooled leaves are cut in 2
2 cm pieces, then packed .
Goishi tea solution was made following steps; 20 g of
Goishi tea dried leaves were boiled in 1000 ml distilled
water at 100C for 30 min. The extract was quickly
separated from the leaves by filtration. Thus, we used
the 20 mg/ml solution of Goishi tea throughout the
experiment. On the other hand, 1% Gallic acid was
used as a positive control. In this experiment,
considering the daily intake of water by the mouse strain used
(4 ml), each mouse was receiving 80 mg of Goishi tea
daily for the Goishi tea/Der f and Goishi tea/PBS
groups. Mice received tap water, Goishi and Gallic acid
solutions ad libitum according to mice group from day
1 to day 37.
Allergen and AHR induction
Allergen-exposed mice were actively challenged with an
intratracheal instillation of 4 g of Dermatophagoides
farinaeplus (Der f) plus 62.5 mg Diesel exhaust particles
(DEP) solution on days 13-14-20-21-27-28-34-35, for a
total of eight times as shown on the experimental
protocol (Figure 1). Measurement of AHR to intravenous
acetylcholine (ACh) was performed as previously described
. Briefly, to measure AHR, mice were anesthetized
with sodium pentobarbital (60 mg/kg, i.p.) and the
jugular vein was cannulated for intravenous injection of ACh.
They were injected with pancuronium bromide (0.1 mg/
kg, i.v.) to stop spontaneous respiration and then
ventilated with a rodent ventilator (New England Medical
Instruments, Inc., Medway, MA, USA).
Bronchoconstriction was measured according to the overflow method,
using a bronchospasm transducer (Ugo Basil 7020,
Milan, Italy) connected to the tracheal cannula. Changes
in respiratory overflow volume were measured using an
increasing dose of ACh. The increase in respiratory
overflow volume induced by ACh was represented as a
percentage of the maximal overflow volume (100%) obtained
by clamping the tracheal cannula. This experiment was
perform with two independent experiments and, given
the fact that results were similar, we included only data
from the second experiment in this report.
The area under the curve (AUC) calculated from
dose-response curves for ACh was used to express the
magnitude of AHR. Briefly, AHR chart was saved as
bmp format file; then AUC was selected and calculated
using ImageJ software 1.44p (National Institutes of
Health, USA) with each value of doses converted
logarithmically and represented as arbitrary units.
Collection of blood samples, measurement of total IgE,
allergen-specific IgG1 and adiponectin in the serum
Blood samples were drawn from mice on day 35 in order
to determine the level of serum IgE, allergen-specific
IgG1 and adiponectin. Samples were kept at -80C in the
freezer until analyses were performed with the use of
Figure 1 Experimental protocol. Der f : dermatophoides farinae ;
ACh: acetyl choline; AHR: airway hyperresponsiveness.
specific ELISA kits for mice (mouse IgE kit from
Morinaga & Co. Ltd., Yokohama, Japan; mouse adiponectin
kit from Otsuka Pharmaceutical CO., LTD., Tokyo,
Japan). The mouse IgG1 kit was prepared in our
laboratory (Toxicology laboratory, department of
Environmental Medicine, Kochi Medical School, Kochi, Japan).
Briefly, serum IgG1 was bound with coated Der f antigen,
and then it was detected with horse radish peroxidase
conjugated antibody. Animals were sacrificed using a
high dose of pentobarbital on day 37.
Bronchoalveolar lavage (BAL) and cells count
To perform the bronchoalveolar lavage (BAL) on day 37,
animals were intraperitoneally administered 500 mg/kg
of pentobarbital solution. BAL was performed with the
use of 1.5 ml of saline solution and 80% (1.2 ml) of the
0.9% saline solution were recovered. Number of
inflammatory cells such as eosinophils, neutrophils,
lymphocytes and macrophages in the BAL fluid was recorded.
Histopathological analysis of lung specimens
On day 37, after sacrificing animals, lung specimens
from representative mice were taken and samples were
fixed in 10% formalin, embedded in paraffin, sectioned
at 10 m and stained with hematoxylin and eosin (HE
stain), and periodic acid-schiff (PAS stain). To examine
the specimens, a light microscope (Olympus BX51,
Olympus, Japan) was used at 10 magnification.
Results were represented as the mean standard
deviation. Statistical comparison among the treatment groups
were performed by one-way ANOVA, followed by
nonparametric Tukey test, with the use of SPSS software
package. Results were considered to be statistically
significant when p-value was less than 0.05.
Goishi tea consumption attenuates lung inflammation
On day 37 of the experiment, bronchoalveolar lavage and
cells count were performed for each mice group. The
number of inflammatory cells in BAL fluid, which reflects
airway inflammation intensity, was markedly increased in
the Tap water/Der f group. A significantly lower number
of eosinophils, neutrophils (p < 0.05) and lymphocytes (p
< 0.01) was observed in Goishi tea/Der f mice as
compared with the Tap water/Der f group (Figure 2).
Although the number of macrophages was lower in the
Goishi tea/Der f group than in the asthmatic mice (Tap
water/Der f group), the difference was not significant.
Goishi tea consumption attenuates AHR
Repeated challenges to mice lung with ACh induced
AHR in Tap water/Der f mice in a dose-dependent way.
This process was significantly inhibited in the Goishi
tea/Der f and Gallic acid/Der f groups as compared with
the tap water in the Tap water/Der f group (p < 0.05)
In addition, the AHR inhibitory effect in Gallic acid/
Der f group was more efficient than that of Goishi tea/
Der f group (p < 0.01).
Throughout this experiments, Gallic acid-treated mice
groups and other Der f-non exposed groups showed
reduced AHR as compared to Tap water/PBS group (p
< 0.01), especially for the following ACh doses; 250, 500,
1000 and 2000 g/kg.
On the other hand, Der f non-exposed groups (Goishi
tea/PBS, Gallic acid/PBS, Tap water/PBS) also showed a
significant AHR inhibitory effect as compared to Goishi
tea/Der f group (p < 0.05) (Figure 3).
Effect of Goishi tea consumption on serum levels of
antigen-specific IgG1 and IgE
It is well-known that airways exposure to allergens such
as Der f in sensitive mice species induces an increased
serum level of allergen specific IgG1. In this experiment,
significantly lower levels of Der f specific-IgG1 were
observed in Goishi tea/Der f and Gallic acid/Der f (p <
0.05) groups as compared with the Tap water/Der f
group (Figure 4a). Interestingly, there was no statistically
significant difference in serum IgG1 levels between
Goishi tea/Der f and Gallic acid/Der f groups (p > 0.05).
Similarly, there was no statistically difference in terms of
serum level of Der f specific-IgG1 between Goishi tea/
PBS and Tap water/PBS groups, and also between Gallic
acid/PBS and Tap water/PBS groups (p > 0.05).
Regarding the serum level of serum total IgE,
significantly lower titers were noted in the Goishi tea/Der f
and the Gallic acid/Der f groups (p < 0.01) as compared
with the Tap water/Der f group (Figure 4b). Although
lower levels of serum total IgE were also observed in
Goishi tea/PBS and Gallic acid/PBS groups when
compared with the Tap water/PBS group, the difference was
not significant (p > 0.05). Taken together, the data
suggest that Goishi tea consumption, as well as Gallic acid,
exerted an immunomodulatory activity that could
inhibit airway inflammation in mice.
Effect of Goishi tea consumption on adiponectin
Goishi tea consumption significantly increased
adiponectin expression as compared with tap water-treated mice
(vs. Tap water/Der f group; p < 0.05) (Figure 5). That was
also true for gallic acid treated mice (vs. Tap water/Der f
group; p < 0.01) as shown in Figure 5. The Goishi tea/
PBS group had a relatively higher level of adiponectin as
compared with the Goishi tea/Der f group; however, the
difference was not significant (p > 0.05).
Figure 2 Goishi tea consumption attenuates lung inflammation. #: p < 0.05;##: p < 0.01(vs. Tap water/Der f group). *: p < 0.05 (vs. Tap water/
PBS group). p: p-value by one-way ANOVA test; BAL: bronchoalveolar lavage; Der f: Dermatophagoides farinae. The figure shows that the number
of eosinophils (a), neutrophils(b) and lymphocytes (c) in BAL fluid were significantly lower in the Goishi tea/Der f and the Gallic acid/Der f groups
(vs. Tap water/Der f; p < 0.01 and p < 0.05, respectively). Mice were intraperitoneally administered 500 mg/kg of pentobarbital solution in 1.5 ml
of 0.9% saline solution, and 80% (1.2 ml) of the solution were recovered. The number of inflammatory cells was recorded. Results are mean SD
of data from 5 mice in each group.
As for the trend of body weight, tap water treated
mice that were exposed to the allergen had a relatively
lower body weight than allergen non-exposed mice (Tap
water/PBS), whereas Goishi tea consumption did not
induce a significant change in the body weight as
compared with the Goishi tea/PBS group (p > 0.05) (data
Histological evaluation of lung specimens from Goishi
tea-treated mouse and controls
After sacrificing mice using high dose of pentobarbital,
full lung specimens were taken from animals for
histological analysis. The hematoxylin and eosin staining of
lung specimens showed a marked goblet cells
hyperplasia and eosinophilic infiltration in allergen-challenged
control mice (Tap water/Der f group), while the number
of those cells was reduced in Goishi tea and Gallic acid
treated mice (Goishi tea/Der f and Gallic acid/Der f)
(Figure 6). Lung specimens in Goishi tea/PBS and Gallic
acid/PBS showed similar to Tap water/PBS (data not
Asthma is an escalating public health problem in
children and adults; patients have an exaggerated immune
response to allergens leading to lung inflammation and
AHR , and antioxidants are thought to play a
significant role in mediating the pathogenesis of asthma
[13,14]. The intake of antioxidant foods could be
beneficial in preventing some episodes of asthma and a
Figure 3 Goishi tea consumption attenuates AHR.#: p < 0.05 (vs. Tap water/Der f group);##: p < 0.01 (vs. Tap water/Der f group); *: p < 0.05
(vs. Goishi tea/Der f group). **: p < 0.01 (vs. Goishi tea/Der f group). AHR: airway hyper responsiveness; Der f: Dermatophagoides farinae. The
figure shows that the increase in AHR, following ACh challenges, was significantly inhibited in Goishi tea/Der f and Gallic acid/Der f as compared
with the Tap water/Der f group (p < 0.01). In addition, the AHR inhibitory effect in Gallic acid/Der f group was more efficient than that of Goishi
tea/Der f group (p < 0.01). On the other hand, all Der f non-exposed groups showed significantly reduced airway reactivity to ACh throughout
the experiment (p < 0.01, vs Tap water/Der f). To measure AHR to ACh, changes in respiratory overflow volume were measured using an
increasing dose of ACh. The area under the curve (AUC) calculated from dose-response curves for ACh was used to express the magnitude of
AHR. Briefly, each dose was converted logarithmically; AUC was calculated and represented as arbitrary units.
number of epidemiologic studies have reported a
positive association between dietary antioxidant status and
lung function, and the protective effect of dietary
antioxidant supplementation on asthma [15,16].
We hypothesized that Goishi tea consumption could
possibly attenuate airway inflammation as it shares
similar chemical components with green tea such as
polyphenols, that reduce inflammatory process in injured
lungs . In addition, as mentioned earlier, Goishi tea
contains gallic acid which inhibits pro-inflammatory
cytokines release by mast cells and upregulates IL-10
In this study, while an increased cellularity was
observed in the allergen exposed control group (Tap
water/Der f), there was a reduction of inflammatory
cells both in lung specimens and BAL fluid in the
Goishi tea/Der f group. In particular, Goishi tea
consumption inhibited eosinophilic infiltration and goblet
cells hyperplasia in mice lungs, a characteristic
histological feature of airway allergic inflammation. This
experiment also showed that Goishi tea consumption, as well
as that of Gallic acid, significantly inhibited the
expression of specific IgG1 and IgE in mice as compared with
asthmatic mice group (Tap water/Der f). This suggests
that Goishi tea consumption has attenuated airway
inflammation in Goishi tea/Der f mice.
There are some foods that may increase adiponectin
production  which has been reported to inhibit
allergen-induced AHR, according to previous experimental
studies [18-20]. In our experiment, we performed the
measurement of this hormone in mice sera. Goishi tea
has markedly increased adiponectin expression; this fact
may have possibly contributed to the attenuation of
AHR that was observed in the Goishi tea-treated mice
groups (Goishi tea/Der f, Goishi tea/PBS).
This study has some limitation. The results are from
animals kept in pathogen free condition and might not
reflect exactly what can be found in humans who are
subjects to different airway inflammation triggers.
Further research in humans is needed to confirm the
The reduction of AHR and airway inflammation
observed in Goishi tea-treated mice might result from
the combination of the upregulation of adiponectin
Figure 4 Effect of Goishi tea consumption on serum levels of antigen-specific IgG1 and total IgE. *: p < 0.05 (vs. Tap water/PBS group);#:
p < 0.05 (vs. Tap water/Der f group); IgG1: immunoglobulin G1, Der f: Dermatophagoides farinae. The figure 4a shows that the serum level of Der
f specific-IgG1 was significantly lower in Goishi tea/Der f and Gallic acid/Der f groups (p < 0.05) as compared with the Tap water/Der f group. The
serum level of Der f specific-IgG1 was significantly higher in the Tap water/Der f group (p < 0.05) groups as compared with the Tap water/PBS
group. The figure 4b shows that the serum level of total IgE was significantly lower in Goishi tea/Der f and Gallic acid/Der f groups (p < 0.05) as
compared with the Tap water/Der f group. The serum level of total IgE was significantly higher in the Tap water/Der f group (p < 0.05) as
compared with the Tap water/PBS group.
expression that it induces, its antioxidant activity and
the inhibition of specific-IgG1 expression which were
observed in this study.
Figure 5 Effect of Goishi tea consumption on adiponectin expression. *: p < 0.05 (vs. Tap water/PBS);##: p < 0.01 (vs. Tap water/Der f) Der f:
Dermatophagoides farinae. The figure shows that Goishi tea (p < 0.05) as well as Gallic acid (p < 0.01) consumption significantly increased
adiponectin production as compared with the Tap water treated mice (p < 0.05).
Figure 6 Histological evaluation of lung specimens from Goishi tea-treated mouse and controls. Der f: Dermatophagoides farinae; H&E:
hematoxylin and eosin staining; PAS: periodic acid-schiff, al, alveolus; br, bronchiole; v, blood vessel. Scale bars = 100 m. The figure shows a
marked goblet cells hyperplasia (G), eosinophilic infiltration (E) and smooth muscle mass enlargement (S) in lung specimens from Der
fchallenged control mice (Tap water/Der f), while these processes were reduced in Goishi tea and Gallic acid-treated mice groups.
RH conceived of the study, participated in its design and coordination and
wrote the manuscript, and carried out the biological studies. NN carried out
the animal experiments and wrote in drafting the manuscript. MM carried
out the cellular studies. HN evaluated histological data. NS performed the
statistical analysis. All authors read and approved the final manuscript.
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