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A standardized aqueous extract of Anoectochilus formosanus modulated airway hyperresponsiveness in an OVA-inhaled murine model.


Anoectochilus formosanus HAYATA, a Chinese herb, is a valued folk medicine for fever, pain, and diseases of the lung and liver. Allergic asthma is characterized by increased serum IgE level and inflammation of the airways with high levels of interleukin (IL)-4 and IL-5 in bronchoalveolar lavage fluids (BALF). Constriction of airway smooth muscle and development of airway hyperresponsiveness (AHR) are the most important symptoms of allergic asthma. In our previous study, a standardized aqueous extract of A. formosanus (SAEAF) was used to modulate innate immunity of normal mice. In this study, airway inflammatory infiltrations, including T cell differentiation, cytokine modulation, allergic antibodies estimation, pulmonary pathology, and enhanced pause (Penh) of AHR were used to evaluate SAEAF treatment of an ovalbumin (OVA)-inhaled airway allergic murine model. The resulting cytokine profiles demonstrated that SAEAF can significantly reduce Th2 polarization after administration of SAEAF in OVA inhalation. These results also suggest that SAEAF modulates cytokine secretion in allergic asthma. Modulated natural T regulatory cells (CD25 +/CD4 +, Treg) were also shown to increase immunosuppression in the allergic lung inflammation and further down-regulate airway inflammatory infiltration in eosinophils and macrophages. Finally, decreased airway anti-OVA IgE secretion and reduced AHR were observed. Our results indicate that the administration of SAEAF can modulate cytokines and T cell subpopulation by regulating inflammatory cell infiltration and modulating the allergic response.

Keywords: Anoectochilus formosanus Airway hyperresponsiveness Treg Th1/Th2


The family Orchidaceae is one of the most important agricultural resources in Taiwan. The species Anoectochilus formosanus Hayata (1)) is very precious in the Taiwanese folk medicine market because of its diverse pharmacological effects. Previous studies, including ours, have indicated that different kinds of extraction of A. formosanus have anti-hyperglycemia (Shih et al., 2002), anti-osteoporosis (Shih et al., 2001), anti-hyperliposis (Du et al., 2003), anti-fatigue (Ikeuchi et al., 2005), and hepatoprotective (Fang et al., 2008; Wu et al., 2007; Shih et al., 2005) effects. Tseng et al. (2006) demonstrated that hot water extraction of Anoectochilus formosanus could enhance the phagocytosis activity of murine peritoneal macrophage, but its active component and mechanism are still not well known.

Allergic asthma is characterized by increased serum IgE antibody level and inflammation of the airways with high levels of interleukin (IL)-4 and IL-5 in bronchoalveolar lavage fluids (BALF) and airways mucosa (Gilmour and Lavender, 2008; Mattes and Foster, 2003). T regulatory cells (Tregs) have been shown to control the disease phenotype in studies of allergic airway inflammation (Jutel et al., 2006; McGee and Agrawal, 2006; Romagnani, 2006). Constriction of airway smooth muscle and development of airway hyperresponsiveness (AHR) are the most important reactions of bronchial asthma in the narrowing of the airway (Berend et al., 2008; Meurs et al., 2008; Cockcroft and Davis, 2006).

In the present study, a standardized aqueous extract of A. formosanus (SAEAF) was prepared without the ethyl acetate fraction to demonstrate the extract's action in the treatment of bronchial asthma. The in vivo effects of SAEAF in mice were investigated by examining the production of allergen-specific IgE antibody, Th1/Th2 cytokines, Treg modulation, and enhanced pause (Penh) of AHR using an ovalbumin (OVA)-sensitized airway allergic murine model.

Materials and methods

Preparation of SAEAF

A. formosanus plants were purchased from Innorchid Agriculture Biotech Ltd. (Pu-Li, Taiwan) and identified by the Institute of Chinese Pharmaceutical Sciences, China Medical University (plant specimen number: CMCP 1253). Fresh, whole plants of cultured A. formosanus were extracted with water and partitioned with ethyl acetate. The aqueous fraction was further filtered and evaporated under reduced pressure to yield a purple residue, with the yield approximately 2%. A standardized aqueous extract of A. formosanus (SAEAF) was prepared. The kinsenoside content was then determined by high-pressure liquid chromatography (HPLC), under the same conditions as in our previous study (Wu et al., 2007). Conditions used for HPLC were as follows: pump, Shimadzu LC-10ATvp; refractive index detector, Shimadzu RID-10A; column, Mightysil ODS RP-18 GP Aqua column (i.d. 4.6 mm, 250 mm long; 5 [micro]m particle size); guard column, Mightysilk 4.6 mm x 6 mm. The solvent system used was deionized water at a flow rate of 0.5 ml/min. The kinsenoside content in SAEAF was approximately 180 mg/g.


Male BALB/c mice weighing 18 to 22 g each were obtained from the National Laboratory Animal Center (Taiwan). They were 6 to 8 weeks of age at the start of the experiments. The animals were housed in a plastic cage with the Ventilated Micro Isolator System (VMIS) maintained at 21[degrees]C to 24[degrees]C with 12 h of light cycle. The animals received a standard sterile rodent chow diet (TestDiet 5010) and distilled water ad libitum.

Ovalbumin sensitization and challenge

The mice were sensitized with an intraperitoneal injection of 0.1 ml/10 g of mouse with OVA (grade V, Sigma-Aldrich, San Diego, USA) absorbed to aluminum hydroxide gel (AHG; Sigma-Aldrich, San Diego, USA) in saline vehicle (0.5 mg OVA with 2 mg AHG/kg of mice). After 10 days, the mice were subjected to antigen inhalation challenge five times (once each day) by intranasal (i.n.) inoculation of 75 [micro]l OVA (2.0 mg/ml) from day 11 to day 15. After one week of resting, mice were recovered from the airway hyperresponseiveness (from day 16 to day 22). Further one week of SAEAF treatment (0.5 and 1.0 g/kg) and control with distilled water, from day 23 to day 29, the mice were exposed to OVA for the inhalation challenge in day 30. The mice were sacrificed post-OVA challenge to monitor the immunomodulatory effect by lung infiltration. The naive group mice indicated the untreated mice for reference condition.

Noninvasive measurement of airway responsiveness by barometric whole-body plethysmography (WBP)

The airway responsiveness of post-OVA-challenge mice was expressed with the enhanced pause (Penh) as a parameter of altered airway function using the MAX II 1320 Modular Unit (Buxco, Troy, NY, USA). Briefly, the mice were placed in the animal chamber of the plethysmograph, and the pressure differences between this chamber and a reference chamber integral to the main chamber (termed the box pressure signal) were measured and recorded using a differential pressure transducer connected to the amplifier. The box pressure signal is caused by the volume and resultant pressure changes in the main chamber during the respiratory cycle of the mouse. Penh is a dimensionless value that represents a function of the proportion of maximal expiratory to maximal inspiratory box pressure signals and of the timing of expiration. According to the manufacturer's instructions, Penh was calculated as (Te-Tr)/Tr x (PEP/PIP), where Te is expiratory time (seconds); Tr is relaxation time (seconds), defined as the time of pressure decay to 30% of the total expiratory pressure signal (area under the box pressure signal at expiration); PEP is peak expiratory pressure (ml/s); and PIP is peak inspiratory pressure (ml/s). Penh reflects changes in the waveform of the box pressure signal during both inspiration and expiration, and combines these changes with the timing comparison of early and late expiration. Penh was measured for 2.0 min for each experiment.

Bronchoalveolar lavage fluid (BALF) harvest and lung infiltration determinate

After the OVA challenge, the lungs of the mice were anesthetized interperitoneally with pentobarbital (1.5 g/kg), the trachea was cannulated, and 1.0 ml of pyrogen-free normal saline was instilled into the lung. The tracheal cannula was clamped, and the throx was massaged for 15 to 30 sec before the BALF was recovered. One milliliter of saline was then instilled into the lungs, and the procedure was performed three times. BALF (3.0 ml) was harvested and further centrifuged at 300 x g for 10 min at 4[degrees]C. The cell pellets were obtained, and differential cell counts were performed on smears and stained with Liu's stain methods. A minimum of 500 cells per smear were counted using light microscopy and classified into lymphocytes, macrophages, or eosinophils according to standard morphologic criteria. The proportion of each cell population was calculated by total cells.

Ovalbumin -specific IgE in BALF

BALF were added in duplicate onto enzyme linked immuno-sorbent assay (ELISA) plates coated with OVA (20 [micro]g/ml in 0.05 M carbonated buffer, pH 9.5). After incubation overnight at 4[degrees]C, the plates were washed and incubated with HRP-conjugated goat anti-mouse polyclonal IgE (Bethyl Laboratories, Inc. Montgomery, TX, USA) for 1 h, followed by washings and developing with SureBlue Reserve TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Absorbance of the samples was determined at 450 nm. OVA-specific IgE in BALF concentration was calculated with absorbance (A). Units are presented as ELISA Units (EU), calculated as follows:

ELISA units. EU = ([A.sub.sample]-[A.sub.blank])/([A.sub.positive]-[A.sub.blank])

T cell subpopulation evaluation in BALF

BALF cells were determined by the detection of surface markers with specific binding of monoclonal antibodies (mAb) as recommended by the Becton-Dickinson Monoclonal Center (FACScan, BD Biosciences, Mountain View, CA, USA). Briefly, 50 [micro]l of cell suspension (5 x [10.sup.5] cells) were incubated in the presence of saturating concentrations of fluoresein, phycoerythrin or PE-Cy5 conjugated mAb (eBioscience, San Diego, CA USA) on ice for 30 min in the dark. Cells were washed with Dulbecco's modified phosphate buffered saline (DPBS) containing 1% FBS and 0.1% sodium azide. Cytofluorometric analysis was performed on the lymphocyte fraction as determined by side scatter (SCC) and forward scatter (FSC) with laser excitation at 488 nm. The number of leukocyte population was determined in 1 x [10.sup.4] cells. A computer system (CellQuest, BD Biosciences) was used for data acquisition and analysis. List mode data for 1 x [10.sup.4] events of leukocyte were stored. Percentages were calculated on the basis of the number of lymphocytes found in each quadrant. T cells were determined by surface marker CD3 (clone 145-2C11)/CD45 (clone 30-F11), T helper (Th) cells by CD3/CD4 (clone RM4-5), T cytotoxic cells by CD3/CD8 (clone 53-6.7), and natural Treg cells by CD4/CD25 (clone PC61).

Estimation of BALF cytokines

To study the effect of SAEAF on BALF cytokines in OVA-challenged mice, a double-antibodies sandwich ELISA was performed according to the manufacturer's recommendations (eBioscience San Diego, CA, USA). IL-2, IL-4,IL-12, IFN-[gamma], and TNF-[alpha] kits were purchased from eBioscience (ELISA Ready-SET-Go! San Diego, CA USA). Capture antibodies were added to 96-well plates and then incubated overnight at 4 [degrees]C. Each sample well was washed three times, blocked for 60 min at room temperature, and washed again three times. Standards and samples at a dilution of 1:20 were then added to the wells. After 2 h incubation at room temperature, the wells were washed five times, and the detection antibodies were added and then washed seven times. Substrate solution (TMB) was added to each well after incubation in the dark, and then stops solution to terminate the enzyme activity. Absorbance was measured at 450 nm with reference at 570 nm by ELISA reader (Multiskan Spectrum, Thermo Electron Corporation, San Jose. CA, USA).

Histopathological examination

For the histopathological examination of the lungs, the mice were sacrificed and the specimens from the sacrificed mice were embedded in paraffin wax. Sections measuring 5 [micro]m were prepared and bathed in Ehrlich's haematoxylin for 5 min before being rinsed for 5 to 10 min in running water. The sections were subsequently dipped with tap water for 3 to 5 s in 70% alcohol containing 1% hydrochloric acid and then washed again for 5 to 10 min in running water. The slides were dipped in 1% eosin for 5 min and then placed under running water until the nuclei appeared blue. The sections were again dehydrated by the addition of serially concentrated alcohol (70%, 80%, 90%, and 100%). After H&E staining of the slide, lung tissues were prepared from each mouse and sectioned transversely through the mid-portion across the pulmonary bronchioles and bronchiolar branches. Thus, the sections were histologically examined for each mouse.

Statistical analysis

Results are expressed as mean [+ or -] SD. One-way ANOVA was used for multiple group comparisons and was used in the Duncan's test for post hoc examination. Differences with P < 0.05 were considered significant.


Quantitative measurement of kinsenoside using HPLC

The linear relationship and AUC of kinsenoside standard solutions are calculated and shown in Fig. 1A. Kinsenoside is identified using HPLC with RI detector at a retention time of 12.2 min (Fig. 1A). The results from our research indicate that the amounts of the three analytes of SAEAF are 180 mg/g (Fig. 1B). The structure of kinsenoside is presented in Fig. 1C.


Effect of SAEAF administration on cellular distribution in BALE of OVA-inhaled allergic mice

No observable abnormal clinical signs were attributable to the SAEAF dosing, and there was neither loss of body weight nor gross abnormality. The changes of cellular distribution in the BALF of OVA-inhaled allergic mice at 1, 6, 12, 24, 48 and 72 h after treatment with various doses of SAEAF are shown in Table 1. Intranasal challenge of 75 [micro]l OVA (2.0 mg/ml) of sensitized mice without SAEAF treatment (controls) induced a marked increase in the number of infiltrated cells and eosinophils in BALF (Table 1) by 1 h after the OVA challenge. The levels of the infiltrated cells peaked at 12 h and then gradually returned 72 h after the OVA challenge. For evaluation of the prophylactic effect of SAEAF on allergen-induced airway inflammation, SAEAF treatment reduced eosinophil infiltration by 24 h (SAEAF, 0.5 g/kg) and 12 h (SAEAF, 1.0 g/kg) after OVA challenge, respectively. Moreover, lymphocytes showed significant increase after 24 h post-OVA challenge.
Table 1

Changes of infiltrated cell counts in BALF.

                               OVA-challenged allergic BALF
                               infiltrated cells, x 10 (6)

            Naive               Control

Lymphocyte  0.13 [+ or -]0.03  1.64 [+ or -]0.10 #

Macrophage  1.29 [+ or -]0.22  4.13 [+ or -]0.89 #

Eosinophil  0.01 [+ or -]0.01  1.80 [+ or -]0.15 #

                 OVA-challenged allergic BALF
                 infiltrated cells, x 10 (6)

            SAEAF, 0.5 g/kg       SAEAF, 1.0 g/kg

Lymphocyte  2.74 [+ or -]0.06 *  2.27 [+ or -]0.50

Macrophage  3.41 [+ or -]1.29    1.24 [+ or -]0.32 *

Eosinophil  0.73 [+ or -]0.23 *  0.65 [+ or -]0.06 *

# P <0.05, represents significant difference when compared with naive
group which without any treatment.
* P<0.05, represents significant difference when compared with control
group which is administered water. Data shown as mean [+ or -] SD.

Flow cytometry analysis of T-cell population in the BALE of sensitized mice

The effect of SAEAF on the percentage change of T-cell subsets was determined by flow cytometry with immunofluorescence of monoclonal antibodies to direct staining of CD3, CD4, CD8, CD25 and CD45 molecules on infiltrated lymphocytes in the BALF of challenged mice (Table 2). In the control group mice, the significant decrease in natural Tregs in the 48 h post-challenge indicate the immuno-hyperreactive status in regulating late asthmatic response (LAR). We found that the increased percentage subset of natural Tregs (CD4+/CD25+) after 48 h post-challenge indicated immunosuppression in an asthmatic response.
Table 2

Changes of infiltrated T cell subset in BALF.

                                 OVA-challenged allergic BALF
                                 infiltrated T cells, %

             Naive                Control

CD25+, CD4+  17.93 [+ or -]2.35  4.06 [+ or -]2.38 #

CD4+, CD3+   26.38 [+ or -]5.82  6.57 [+ or -]5.13 #

CD8+, CD3+    8.93 [+ or -]3.23  2.38 [+ or -]1.41 #

CD3+, CD45+  47.22 [+ or -]6.73  8.52 [+ or -]8.94 #

                   OVA-challenged allergic BALF
                      infiltrated T cells, %

             SAEAF, 0.5 g/kg        SAEAF, 1.0 g/kg

CD25+, CD4+  11.22 [+ or -]2.43 *  10.52 [+ or -]3.25 *

CD4+, CD3+   27.67 [+ or -]6.95 *  22.05 [+ or -]3.83 *

CD8+, CD3+    6.92 [+ or -]1.21 *   8.60 [+ or -]5.69 *

CD3+, CD45+  41.47 [+ or -]4.33 *  38.93 [+ or -]6.67 *

# P<0.05, represents significant difference when compared with naive
group which without any treatment.
* P<0.05, represents significant difference when compared with control
group which is administered water. Data shown as mean [+ or -] SD.

Effect of SAEAE on the OVA-challenged mice for BALF cytokine and IgE infiltration

BALE cytokine infiltrations showed decreased IL-4 expression after SAEAF administration and an increase in IFN-[gamma] and IL-12 at higher doses of SAEAF (1.0 g/kg) administration. The Th l polarization environment and further reduction of the inflammatory TNF-[alpha] expression in SAEAF administration post-OVA challenge were likewise indicated (Table 3). Anti-OVA IgE was also significantly reduced in SAEAF administration (P <0.05 in 0.5 g/kg and P <0.01 in 1.0g/kg; Fig. 2)

Table 3

Effect of SAEAF on the production of cytokines in BALE.

                                   OVA-challenged allergic
                                   BALF, pg/ml

              Naive                Control

IL-2          11.7 [+ or -] 1.1    13.3 [+ or -] 2.7

IL-4           5.0 [+ or -] 3.6    38.3 [+ or -] 15.6 #

IL-12         31.6 [+ or -] 5.2    40.0 [+ or -] 7.3

IFN-[gamma]  112.2 [+ or -] 48.2  184.9 [+ or -] 60.3

TNF-[alpha]   40.8 [+ or -] 6.3    41.2 [+ or -] 8.4

               OVA-challenged allergic BALF, pg/ml

              SAEAF, 0.5 g/kg       SAEAF, 1.0 g/kg

IL-2          14.3 [+ or -] 3.0     13.1 [+ or -] 2.4

IL-4          17.1 [+ or -] 6.2 *   11.5 [+ or -] 6.6 *

IL-12         47.7 [+ or -] 8.8     59.0 [+ or -] 7.2 *

IFN-[gamma]  159.2 [+ or -] 41.3   137.8 [+ or -] 35.2

TNF-[alpha]   27.2 [+ or -] 4.7 *   20.8 [+ or -] 4.6 *

# P<0.05, represents significant difference when compared with naive
group which was without any treatment.
* P<0.05, represents significant difference when compared with control
group which was administered water. Data shown as mean [+ or -] SD.

Effect of SAEAF on OVA-induced respiratory resistance

Enhanced pause (Penh) was monitored at 1, 6, and 24 h after aerosol inhalation of OVA (Fig. 3). OVA-challenged mice induced a pronounced immediate bronchoconstriction in the IAR at 1 to 6 h post-OVA challenge. After the immediate response, a late bronchoconstriction (LAR) at around 24 h post-OVA challenge was observed. Further bronchoconstriction can be reduced after SAEAF (0.5 g/kg) administration in 1, 6 and 24 h (P <0.01) post-OVA challenge (Fig. 3).


Histological examination of the effect of SAEAF on inflammatory cell infiltration in lung

Examination of lung sections under light microscopy revealed an infiltration of inflammatory cells (macrophage and eosinophil) into the airway, around the bronchi, bronchioles, and alveoli of post-OVA-challenged mice in the untreated group. After the OVA challenge, the inflammatory cells of treated mice were observed beneath the airway smooth muscles, and were tracked through the muscle toward the epithelium (Fig. 4B). In SAEAF administration, reduced airway inflammatory infiltration (Figs. 4C and D) dominated, affecting the peribronchial and peribronchiolar smooth muscles, mucosal epithelium, and alveoli after OVA challenge.



In the treatment of asthma, the emphasis in recent decades has been on the suppression of inflammatory responses and [beta]2-bronchodilator agents to alleviate the occurrence of asthma and airway smooth muscle caused by the contraction symptoms (Papi et al., 2009; Barnes and Adcock, 2009). Steroids and [beta]2-bronchodilators for the control of asthma symptoms and lung function improvement are most effective, but because of potential side effects, these drugs are still a cause for concern. In recent years, Th2 cells have been known as the dominant modulators in regulated downstream cell differentiation and proliferation including B cells class switch to secreting IgE, IgG1 and eosinophils, and attract a large number of inflammatory cell infiltrations in the lungs (Larche et al., 2003). Bronchoalveolar lavage fluid of asthmatic murine model can be found in a large number of inflammatory cells.

In the present study, SAEAF was found to significantly modulate the pulmonary environment of cytokines, significantly enhancing IL-12 and reducing IL-4 (Table 3). It can provide a type I environment to promote Th1 cell differentiation. The resulting decrease in anti-OVA IgE (Fig. 2) and down-regulation of inflammatory cell infiltration, including macrophages and eosinophils (Table 1, Fig. 4), can reduce the symptoms of airway hyperresponsiveness (Figs. 3 and 4). Kinsenoside is the active ingredient of A. formosanus (Du et al., 2000). In our research, the high yield of kinsenoside of culture A. formosanus was developed as a hepatoprotective product (Wu et al., 2007). The structure of kinsenoside is similar to that of lacto-N-biose (Galbetal-3GlcNAc), a prebiotic in enhanced bifidobacterial growth (Kiyohara et al., 2009). It may be an active prebiotic in regulating the microbiota population in the gastrointestinal tract. Probiotics, which include Lactobacillus rhamnosus GG or Bifidobacterium lactis (Bb-12), can modulate intestine immune response and reduce the asthmatic lung infiltration in macrophage and eosinophil (Feleszko et al., 2007), as the results reveal (Table 1).

Natural T regulatory cells (Tregs) were known as T lymphocytes present CD4 and CD25 surface markers. Tregs play an immuno-suppression role in regulating the immune response that inhibits the functional cell differentiation, including Th1 and Th2 cell proliferation, which can inhibit the secretion of Th1 and Th2 cell cytokines (Xu et al., 2003; Suto et al., 2001). In this present study, lFN-[gamma] and IL-4 (P <0.05) cytokines in BALF were reduced (Table 3). Infiltrated Tregs may play a major role in the immuno-suppression of allergic hyperreactivity (Table 2). However, IFN-[gamma] not reaching significant difference might be another ingredient activity of SAEAF that can increase IFN-[gamma] expression to counterpart the Treg immunosupression. Another ingredient of SAEAF is water-soluble polysaccharide. Polysaccharide harvest from Ganoderma can stimulate Th1 immune response (Lin, 2005; Lin et al., 2006). SAEAF contains lower water-soluble polysaccharide (4.5%) might be the Th1 modulator and we will isolate and study this ingredient in further T helper cell differentiation.

In conclusion, SAEAF is responsible for the suppression and regression of the pulmonary allergy through reduced IL-4 by Tregs for immuno-suppression and enhanced IL-12 and IFN-[gamma] by Th1 differentiation. This results in a down-regulation of anti-OVA IgE and inflammatory infiltration, and finally to reduced AHR. Our findings may shed light on the novo effect fraction of folk medicine in the regulation of herbal medicine for multiple modulated pathways.


This study was supported by grants from the National Sciences Council of the Republic of China (NSC96-2317-B-039-003. NSC97-2317-B-039-005).


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C.-C. Hsieh (a), H.-B. Hsiao (b), W.-C. Lin (c), *

(a) Department of Animal Science and Biotechnology, Tunghai University, Taichung, Taiwan, ROC

(b) Department of Life Science, National Chung Hsing University, Taichung, Taiwan, ROC

(c) Department of Pharmacology, School of Medicine, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan, ROC

* Corresponding author. Tel.: +886 4 22053366x2229; fax: +886 4 22053764. E-mail address: (W.-C. Lin).

(1) This orchid species is not under international protection and conservation.

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doi: 10.1016/j.phymed.2009.12.012
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Author:Hsieh, C.-C.; Hsiao, H.-B.; Lin, W.-C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jul 1, 2010
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