Goishi tea consumption inhibits airway hyperresponsiveness in BALB/c mice
© Hirota et al; licensee BioMed Central Ltd. 2011
Received: 1 February 2011
Accepted: 11 August 2011
Published: 11 August 2011
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.
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).
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.
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 23°C, 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 farinae plus (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 100°C 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
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 -80°C in the freezer until analyses were performed with the use of 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 μm 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
Goishi tea consumption attenuates AHR
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 μg/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
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 expression
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 not shown).
Histological evaluation of lung specimens from Goishi tea-treated mouse and controls
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 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 expression .
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 present results.
The reduction of AHR and airway inflammation observed in Goishi tea-treated mice might result from the combination of the upregulation of adiponectin expression that it induces, its antioxidant activity and the inhibition of specific-IgG1 expression which were observed in this study.
Goishi tea extract inhibits airway inflammation and remodeling and has potentially beneficial effects in asthma.
The authors thank Dr. ME, Dr. MBA, Dr. AS, Dr. DN for their wonderful support during the implementation of this study. This study was supported by a grant from the Japanese Ministry of Economy.
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