PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1713–1719 (2014) Published online 13 August 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5191
Effect of Kuwanon G Isolated from the Root Bark of Morus alba on Ovalbumin-induced Allergic Response in a Mouse Model of Asthma Hyo Won Jung,1 Seok Yong Kang,2 Jong Seong Kang,3 A Ryun Kim,4 Eun-Rhan Woo4† and Yong-Ki Park1,2*,† 1
Korean Medicine R&D Center, Dongguk University, Gyeongju 740-814, Korea Department of Herbology, College of Korean Medicine, Dongguk University, Gyeongju 740-814, Korea College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea 4 College of Pharmacy, Chosun University, Gwangju 501-759, Korea 2 3
The root bark of Morus alba L. (Mori Cortex Radicis; MCR) is traditionally used in Korean medicine for upper respiratory diseases. In this study, we investigated the antiasthmatic effect of kuwanon G isolated from MCR on ovalbumin (OVA)-induced allergic asthma in mice. Kuwanon G (1 and 10 mg/kg) was administered orally in mice once a day for 7 days during OVA airway challenge. We measured the levels of OVA-specific IgE and Th2 cytokines (IL-4, IL-5, and IL-13) in the sera or bronchoalveolar lavage (BAL) fluids and also counted the immune cells in BAL fluids. Histopathological changes in the lung tissues were analyzed. Kuwanon G significantly decreased the levels of OVA-specific IgE and IL-4, IL-5, and IL-13 in the sera and BAL fluids of asthma mice. Kuwanon G reduced the numbers of inflammatory cells in the BAL fluids of asthma mice. Furthermore, the pathological feature of lungs including infiltration of inflammatory cells, thickened epithelium of bronchioles, mucus, and collagen accumulation was inhibited by kuwanon G. These results indicate that kuwanon G prevents the pathological progression of allergic asthma through the inhibition of lung destruction by inflammation and immune stimulation. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: allergic asthma; kuwanon G; lung inflammation; Morus alba; Mori Cortex Radicis; ovalbumin; Th2 cytokines; immunoglobulin E.
Abbreviations: MCR, Mori Cortex Radicis; OVA, ovalbumin; BAL, bronchoalveolar lavage; H&E, hematoxylin and eosin; PAS, periodic acid–Schiff; Ig, immunoglobulin.
INTRODUCTION Asthma is a common chronic inflammatory disease of the airways characterized by acute symptoms of wheezing, coughing, chest tightness, shortness of breath, reversible airflow obstruction, and bronchospasm (Shepherd et al., 2002). Asthma is caused by a combination of genetic and environmental factors including allergens, air pollution, and other environmental chemicals. It is clinically classified according to the frequency of symptoms, forced expiratory volume in one second, and peak expiratory flow rate, and may also be classified as atopic (extrinsic) or non-atopic (intrinsic) where atopy refers to a predisposition toward type I hypersensitivity reactions (Dietert, 2011). Treatment of acute asthma exacerbation is usually with an inhaled short-acting beta-2 agonist and oral corticosteroids. In chronic asthma, inhaled/intravenous corticosteroids and magnesium sulphate are used in addition to longacting beta agonists, leukotriene receptor antagonists,
* Correspondence to: Yong-Ki Park, Department of Herbology, College of Korean Medicine, Dongguk University, Gyeongju 740-814, Korea. E-mail: [email protected] † Eun-Rhan Woo and Yong-Ki Park contributed equally as co-corresponding authors. Copyright © 2014 John Wiley & Sons, Ltd.
anticholinergic medications (Shepherd et al., 2002), or mast cell stabilizer. However, patients who require frequent or continuous treatment of these medications receive little or no benefit because of the severe side effects when administered over long periods or at high doses (Domingo et al., 2011). Therefore, safer preventive and therapeutic agents for asthma need to be developed. Recent research indicates that the patients with respiratory diseases such as asthma, pulmonary inflammation or embolism, and bacterial pneumonia, particularly those dissatisfied with current treatment, are very likely to seek alternative treatments, and most patients use complementary medicines such as herbal medicines and nutritional supplements (Li and Brown, 2009; Bielory, 2004). This rising interest in alternative medical practices indicates a clear need for more thorough investigation into the safety and efficacy of complementary medicines with multiple therapeutic functions (Chung and Dumont, 2011). Mori Cortex Radicis (MCR) is the dried root bark of Morus alba L. (Sang Bai Pi; Moracae). It is traditionally used for the treatment of cough and upper respiratory system with tropism of the lung channel (Kim et al., 2011). This plant has biological activities such as antihyperlipidemia (Chen et al., 1995), immune modulation (Kim et al., 2000), antibacterial (Nam et al., 2002), antiinflammation (Wang Received 23 December 2013 Revised 28 April 2014 Accepted 27 May 2014
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et al., 2002; El-Beshbishy et al., 2006; Zhang et al., 2009), antiviral (Du et al., 2003), antioxidation (Lee et al., 2011), neuroprotection (Ham et al., 2012; Lee et al., 2012), and antidepressant (Lee et al., 2013). MCR contains many biologically active compounds such as umbelliferone, scopoletin, mucin, tannin, and flavonoids such as morusin, mulberrin, mulberrichromene, cyclomulberin, moracin P, moracin O, mulberrofuran Q, kuwanon E, kuwanon H (Mihara et al, 1995), and 2-arylbenzofurans (Lee et al., 2011), and recently prenylated flavonoids such as licoflavone C, cyclomulberrin, neocyclomorusin, sanggenon I, morusin, kuwanon U, and 2-arylbenzofurans such as moracenin D, moracin P, moracin O, and mulberrofuran Q are recently described (Ham et al, 2012; Lee et al., 2012). We isolated kuwanon G from the methanol extract of MCR and investigated its antiasthmatic effect on ovalbumin (OVA)-induced allergic response in an asthma mice model. Kuwanon G inhibits specific binding of gastrinreleasing peptide (GRP) to GRP-preferring receptors in murine Swiss 3T3 fibroblasts (Mihara et al., 1995) and the growth of cariogenic bacteria such as such as Streptococcus sobrinus and Streptococcus sanguis, and Porpyromonas gingivalis causing periodontitis (Park et al., 2003). However, little is known about the therapeutic effect of kuwanon G on asthma based on experimental evidence although MCR is reported to have antiasthmatic property via enhancement of regulatory T cells and inhibition of Th2 cytokines in a mouse asthma model (Kim et al., 2011). Therefore, to identify the biological activity of kuwanon G, we investigated the antiasthmatic property of kuwanon G on OVA sensitization/ challenge-induced asthma in mice.
MATERIALS AND METHODS Isolation of Kuwanon G. The root bark of M. alba (MCR) was collected on 15 March 2012 in Gyeongju, Gyeongsangbuk-do Province, South Korea (GPS location: 35.748358, 129.235482), and authenticated by Prof. J. H. Lee (Dongguk University, Gyeongju, Korea). A voucher specimen (CSU-1048-17) was deposited in the Herbarium of the College of Pharmacy, Chosun University. The dried root bark of M. alba (12 kg) was extracted three times with MeOH under reflux, and 1511.6 g of residue was produced. The MeOH extract was suspended in water and then partitioned sequentially with equal volumes of dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Each fraction was evaporated in vacuo to yield the residues of CH2Cl2 (318.2 g), EtOAc (192.2 g), n-BuOH (182.4 g), and water (534.3 g) extract. The EtOAc fraction (53 g) was chromatographed over a silica gel column (500 g, 5.0 × 15 cm, twice) using a gradient solvent system of n-hexane-EtOAc (2:1 → 1:8, EtOAc, MeOH) to give five subfractions (E1–E5). Subfraction E2 (8.30 g) was subjected to MCI gel column chromatography (CC, 200 g, 3.5 × 28 cm) eluting with a gradient solvent system of MeOH/H2O (1:1 → 3:1) to yield 16 subfractions (E21–E216). Subfraction E24 (4.03 g) was subjected to silica gel CC (120 g, 2.0 × 40 cm, CHCl3/ MeOH/H2O, 12:1:0.1 → 8:1:0.1) to give six fractions Copyright © 2014 John Wiley & Sons, Ltd.
(E241–E246). Subfraction E245 (2.24 g) was purified by LiChroprep RP 18 CC (MeOH/H2O, 1:1) to yield kuwanon G (1011 mg). IR spectra were recorded on an IMS 85 (Bruker). NMR spectra were recorded on a Varian VNMRS 600 MHz spectrometer (KBSIGwangju center) operating at 600 MHz (1H) and 150 MHz (13C), respectively, with chemical shifts given in ppm (δ). HPLC was performed using a Waters HPLC system equipped with Waters 600 Q-pumps, a 996 photodiode array detector, and an Optimapak C18 column (5 μm, 250 mm × 4.6 mm), solvent [MeOH/H2O 15:85 (0 min), 70:30 (15 min), 100:0 (30 min)], flow rate 1.0 mL/min. TLC was carried out on precoated Kieselgel 60F254 (art. 5715, Merck) and RP-18 F254s (art. 15389, Merck) plates. Column chromatography was performed on silica gel 60 (40–63 and 63–200 μm, Merck) and MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Co.). Low pressure liquid chromatography was carried out over a Merck Lichroprep Lobar®-A RP-18 (240 × 10 mm) column with a FMI QSY-0 pump (ISCO). The physico-chemical characteristics including 1 H-NMR, 13C-NMR, and heteronuclear single quantum coherence of these compounds were identical with those reported in the literature (Oshima et al., 1980; Nomura et al., 1980).
Animals. Six-week-old male BALB/c mice (20 ± 2 g) were purchased from Koatech Co. Ltd. (Gyeonggido, Korea). The animals were housed under controlled environmental conditions at a temperature of 22 ± 3 °C with a relative humidity of 55 ± 5% and 12 h light/dark cycle throughout the study. The care and treatment of animals were in accordance with the guidelines established by the Korean National Institute of Health at the Korean Academy of Medical Sciences for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Dongguk University.
Induction of asthma and drug administration. The mice were divided into five groups and given free access to a standard laboratory diet and water during the experimental period. OVA solution (1 mg/mL in saline) and AlOH3 (20 mg/mL in saline) were mixed in a 1:1 ratio, and the mice were then sensitized with an intraperitoneal injection at a dosage of 0.3 mL/ mice. For the second immunization, 0.1% OVA solution (antigen challenge) was injected peritoneally on days 7 and 14. In addition, a local challenge was performed three times (at 2-day intervals) from day 21 to day 28 by instilling 0.1% OVA solution into the bilateral nasal cavities using a nebulizer. Kuwanon G at a dose of 1 and 10 mg/kg body weight or ketotifen (an antihistamine) as a reference drug at a dose of 10 mg/kg body weight was orally administered to OVA-sensitized animals once daily for seven consecutive days during OVA challenge. Normal and OVA-sensitized/challenged animals were given saline alone on the same schedule. Blood samples were taken from each mouse by cardiac puncture under isoflurane anesthesia 24 h after the oral administration of OVA. Serum was prepared and frozen at 70 °C prior to analysis. The lung tissues were Phytother. Res. 28: 1713–1719 (2014)
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removed from the body, changes were assessed.
and
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histopathological
Serological analysis. The concentrations of OVAspecific IgE (BD sciences, San Diego, CA, USA) and Th2 cytokines IL-4, IL-5 and IL-13 (R&D Systems Inc., MN, USA) in the sera of mice were measured using commercially available enzyme-linked immunosorbant assay (ELISA) or enzyme immunoassay kits according to the manufacturer’s recommendations. The concentration of each substance was calculated from the equations obtained from standard curve plots for the standard solutions in the kits. Inflammatory cell counts in bronchoalveolar lavage fluid. To count the inflammatory cells in bronchoalveolar lavage (BAL) fluids, mice were anesthetized with 50 mg/kg of pentobarbital (Hanlim Pharm., Seoul, Korea), and BAL fluids harvested by lavaging the lungs with saline delivered via a tracheal cannula (Chen et al., 2011). The BAL fluids were deposited onto cytospin slides and stained with Diff-Quik (Dade Behring Inc., Deerfield, IL, USA). Differential cell counts were performed by two independent investigators. Histological analysis of lung tissues. Mice were euthanized with a high dose of pentobarbital and exsanguinated after final OVA challenge. The lungs were obtained from the mid-zone of the left lung of mice and fixed in 4% formalin. The tissues were embedded in paraffin, and serial sections of 5-μm thickness were prepared. After choosing the first section randomly, ten sections in each mouse were stained with hematoxylin and eosin (H&E) for the assessment of the histopathological feature in the lungs, periodic acid–Schiff (PAS) for the assessment of goblet cell menbers, and Masson’s trichrome for the assessment of epithelial and subepithelial collagen thickness.
Figure 1. HPLC analysis of kuwanon G isolated from MRC methanol extract. (A) HPLC chromatogram was analyzed as described in the Materials and Methods Section and (B) the chemical structure of kuwanon G.
Effect of kuwanon G on the production of OVA-specific IgE and Th2 cytokines in OVA-induced asthma mice To investigate the effect of kuwanon G on allergic immune responses in vivo, the levels of allergic mediators OVA-specific IgE and Th2 cytokines were measured in the sera of OVA-sensitized/challenged asthma mice. Initially, OVA-specific IgE serum levels increased in OVA-sensitized/challenged mice. The administration of kuwanon G at doses of 1 and 10 mg/kg in OVA-sensitized/challenged mice for 7 days significantly decreased OVA-specific IgE serum levels compared with that in the OVA-control group (Fig. 2). The administration of ketotifen as a reference drug at a dose of 10 mg/kg also significantly decreased serum IgE levels in OVA-sensitized/challenged mice. Next, we also examined the effect of kuwanon G on the release of Th2 cytokines IL-4, IL-5, and IL-13 in the sera of OVA-sensitized/ challenged mice. The serum levels of IL-4, IL-5, and IL-13 significantly increased in OVA-sensitized/challenged mice compared with those in normal group. The
Statistical analysis. All data were analyzed in GraphPad PRISM 5.0 software (GraphPad Software, Inc., Sandiego, CA, USA). Data are expressed as means ± the standard error of mean (S.E.M.). The significance of treatment effects was determined using one-way analysis of variance followed by Tukey’s post hoc analysis; null hypotheses of no difference were rejected if p-values were less than 0.05.
RESULTS Isolation of kuwanon G As shown in Fig. 1, kuwanon G was purified from the methanol extract of MCR. This process yielded a brown powder of kuwanon G, ½α25 D + 466° (c = 0.13, MeOH); UV λmax (MeOH) nm (log ε): 240(3.82), 280(3.75); IR (KBr) νmax: 3350, 1622, 1514, 1446, 1045, 974, 840 cm1; ESI-MS m/z: 691[M H]+; 1H-NMR and 13 C-NMR (Supporting information). The purity of isolated kuwanon G was 96.7% based on the HPLC analysis (Fig. 1A). Copyright © 2014 John Wiley & Sons, Ltd.
Figure 2. Effect of kuwanon G on the production of OVA-specific IgE in the sera of OVA-sensitized/challenged asthma mice. The serum levels of OVA-specific IgE were measured by ELISA. Data are expressed * ** as means ± S.E.M. of ten mice per group. p < 0.05, p < 0.01 and *** p < 0.001 versus normal (a) or OVA-control group (b). Phytother. Res. 28: 1713–1719 (2014)
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administration of kuwanon G in high dose significantly decreased the serum levels of IL-4, IL-5, and IL-13 in OVA-sensitized/challenged mice, compared with those in the OVA-control group (Fig. 3). In contrast, the administration of ketotifen did not decrease the serum levels of these cytokines in OVA-sensitized/challenged mice. These results indicate that kuwanon G may regulate the allergic response in asthma progression. Effect of kuwanon G on the infiltration of inflammatory cells in BAL fluids To investigate the effect of kuwanon G on lung inflammation in OVA-sensitized/challenged asthma mice, inflammatory cells (macrophages, lymphocytes, neutrophils, and eosinophils) were counted in BAL fluids. Total cells, lymphocytes, neutrophils, and eosinophils were significantly higher in BAL fluids of OVA-sensitized/ challenged asthma mice compared with the control group (Fig. 4A). The numbers of cells significantly decreased following the administration of kuwanon G at doses of 1 and 10 mg/kg. Ketotifen also significantly reduced the numbers of these inflammatory cells. There was no difference in the numbers of macrophages in BAL fluids among groups. The OVA-specific IgE levels in BAL fluids of OVA-sensitized/challenged asthma mice were determined. As shown in Fig. 4B, the levels of OVA-specific IgE significantly increased in the BAL fluids of OVA-sensitized/challenged asthma mice. The administration of kuwanon G at dose of 1 and 10 mg/kg significantly decreased the levels of OVA-specific IgE in BAL fluids of asthma mice. Effect of kuwanon G on histopathological changes of lung tissues To investigate the effect of kuwanon G on the histopathological changes of lung tissues by asthma, the lung tissues were stained with H&E, PAS, and trichrome. As shown in the H&E stain (Fig. 5A), the lung tissue of OVA-sensitized/challenged asthma mice displayed the typical pathological features of asthma, including infiltration of numerous inflammatory cells around the bronchiole, peribronchial, vascular region, thickened airway epithelium, and the accumulation of mucus and debris in the lumen of bronchioles in OVA-sensitized/ challenged mice. Kuwanon G at doses of 1 and 10 mg/kg markedly reduced these pathological lung changes in OVA-sensitized and challenged asthma mice, especially at a dose of 10 mg/kg. In addition, kuwanon G at dose of 10 mg/kg inhibited mucin accumulation by goblet cells hyperplasia in the lumen of bronchioles (Fig. 5B) and reduced collagen accumulation around the bronchiole, peribronchial, and vascular region (Fig. 5C) in OVA-sensitized/challenged asthma mice. Ketotifen at a dose of 10 mg/kg also reduced the inflammation in peribronchial and perivascular region.
DISCUSSION Allergic asthma is the most common type of asthma. Inhaling specific substances called allergens (allergy Copyright © 2014 John Wiley & Sons, Ltd.
Figure 3. Effect of kuwanon G on the production of Th2 cytokines in the sera of OVA-sensitized/challenged asthma mice. The serum levels of IL-4 (A), IL-5 (B), and IL-13 (C) were measured by ELISA. Data are expressed as means ± S.E.M. of * ** *** p < 0.01 and p < 0.001 ten mice per group. p < 0.05, versus normal (a) or OVA-control group (b). Phytother. Res. 28: 1713–1719 (2014)
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Figure 4. Effect of kuwanon G on the numbers of inflammatory cells and the production of OVA-specific IgE in the BAL fluid of OVA-sensitized/challenged asthma mice. (A) The numbers of each cell type were counted in the BAL fluids by Giemsa staining. (B) The levels of OVA-specific IgE in the BAL fluids were measured by ELISA. Data are expressed as means ± S.E.M. of ten mice per * ** *** p < 0.001 versus normal (a) group. p < 0.05, p < 0.01 and or OVA-control group (b).
triggers such as pollen, mites, or molds) or non-allergic exposures (smoke, dust, fumes, and sometimes strong smells) brings on asthma exacerbations. Allergic asthma is a chronic inflammatory pulmonary disease characterized by coughing, wheezing, shortness of breath, rapid breathing, and tightening of the chest with an underlying Th2 cell-mediated inflammatory response in the airway (Kudo et al., 2013). The general principles for allergic
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asthma management are avoidance of allergens/triggering factors and symptomatic treatment using a variety of drugs such as short acting beta-agonists, anticholinergics, systemic corticosteroids, and antiinflammatory drugs or, recently, therapies based on immunomodulation such as allergen-specific immunotherapy (Shum et al., 2008). Although pharmacotherapy is required, the current treatment of allergic asthma is not ideal, and most modern drugs transiently improve clinical symptoms but cannot cure the cause. Therefore, it is a challenge to choose the most appropriate treatment for patients from the wide range array of available agents. Herbal medicines have long been used to treat human diseases including respiratory ailments and to maintain good health. The search for appropriate therapeutic agents in immune imbalance-associated diseases such as allergic asthma has focused on medicinal plants because natural products may provide better safety and efficacy than currently used modern drugs. MCR is traditionally used for the treatment of chronic respiratory diseases in Korean clinics, and its biologic activity is well described (Chen et al., 1995; Kim et al., 2000; Nam et al., 2002; Wang et al., 2002; Du et al., 2003; El-Beshbishy et al., 2006; Zhang et al., 2009; Kim et al., 2011; Lee et al., 2011; Lee et al., 2012; Lee et al., 2013). However, aside from kuwanon G, we isolated three other compounds such as kuwanon E, morucin, and sanggenon A from MCR methanol extract. For search immune-modulatory compounds in allergic response, we investigated their effects on the production of IL-4 and IFN-γ in activated mouse spleenocytes. As a result, kuwanon G showed a good inhibitory effect (data not shown). On the basis of these results, we examined the antiasthmatic effect of kuwanon G in vivo and evaluated its therapeutic potential in allergic asthma. On the basis of this perspective, we have confirmed that kuwanon G isolated from the root barks of M. alba (MCR) has an immune-modulatory activity on OVA sensitization/challenge-induced allergic asthma in mice. This mouse model is a widely used experimental animal model of allergic asthma with clinical and pathological
Figure 5. Effect of kuwanon G on the pathological changes of lung tissues in OVA-sensitized/challenged asthma mice. The lung tissues were stained with H&E (A) for histopathological examination, PAS (B) for mucin-released goblet cells, and Masson’s trichrome (C) for collagen-release lung fibrotic cells (×200 original magnification). A representative result of at least three independent experiments is shown. (a) The infiltration of inflammatory cells; (b) thickened epithelium of bronchiole; (c) PAS-stained mucin releasing (violet); and (d) trichrome-stained collagen accumulation (blue). This figure is available in colour online at wileyonlinelibrary.com/journal/ptr. Copyright © 2014 John Wiley & Sons, Ltd.
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features similar to those of human allergic asthma that are dependent on both humoral and cellular immunity (Epstein, 2006; Kumar, 2008). Recently, with a growing global interest in natural drugs including medicinal plants, their efficacy is often compared with those of antiasthma modern drugs such as the inhaled corticoids, cromones, ketotifen for mild persistent asthma, and inhaled beta-2 agonists and antileukotrienes for prevention of exercise-induced asthma. Ketotifen is an antiallergic drug with antihistaminic activity used for asthma prophylaxis, which protects against bronchoconstriction both immediate and late reactions (Yoshihara et al., 2009). Ketotifen is also known to cause a significant inhibition of total IgE and IL-4 levels and improvement of nasal symptoms through the immunomodulatory function (Devillier, 2001). In the present study, we first identified that kuwanon G effectively prevented the disease progression in OVA sensitization/challenge-induced asthma of mice through reducing the levels of OVA-specific IgE and Th2 cytokines, IL-3, IL-5, and IL-13, and also reducing the infiltration of inflammatory cells in lungs, to a similar degree as the reference drug, ketotifen, or better. In animal models, OVA sensitization/challenge induces a significant increase in the serum IgE and BAL fluid IgE (Hamelmann et al., 1999). IgE is a type of antibody that is present in minute amounts in the body, plays a major role in allergic diseases, binds to allergens, and triggers the release of substances from mast cells as part of the inflammatory process (Martinez and Vercelli, 2013). Our results showed that the serum and BAL fluid concentrations of OVA-specific IgE were significantly reduced in asthma mice after kuwanon G administration. This result suggests that kuwanon G has an effect on allergic asthma in an IgE-dependent manner. Asthma is classically recognized as the typical Th2 disease, with increased IgE levels and eosinophilic inflammation in the airway. Rising Th2 cytokines, particularly IL-4, IL-5, IL-6, IL-9, and IL-13, modulate the asthmatic inflammation, by triggering the activation/recruitment of IgE producing B cells, mast cells, and eosinophils (Kudo et al., 2013). IL-4 is the major factor regulating IgE production by B cells and is required for optimal Th2 differentiation (Deo et al., 2010). IL-5 is a cytokine that is highly specific for eosinophilic inflammation in allergic disease, and antibodies that block IL-5 actions are efficacious in reducing eosinophilic inflammation and airway hyper-responsiveness with severe asthma (Greenfeder et al., 2001; Corren, 2011). IL-13, independent of other Th2 cytokines, is both necessary and sufficient to induce all features of allergic asthma (Wills-Karp, 2004) and increases goblet cell differentiation, activation of fibroblasts, elevation of bronchial hyper-responsiveness, and switching of B cell antibody production from IgM to IgE. IL-13 antagonists are considered an important biological agent for asthma
therapy in patients with poorly controlled asthma (Corren, 2013). In this study, the serum levels of IL-4 and IL-13 were decreased significantly in asthma mice after the administration of kuwanon G at dose of 10 mg/kg. This suggests that kuwanon G has the potential to modulate the Th2 cytokines and could be used as immune-modulatory agent in the treatment of allergic asthma. Research suggests that airway inflammation is central to asthma pathophysiology. As a result of airway inflammation, airway structural remodeling occurs, characterized by epithelial damage, goblet cell hyperplasia, and airway smooth muscle hypertrophy, with profound consequences on the mechanics of airway narrowing in asthma, contributing to the chronicity and progression of the disease (Shikotra and Siddiqui, 2013). Eosinophils are the predominant inflammatory cells in asthmatic lung tissues and contribute to the clinical features of allergic asthma and airway hyper-responsiveness. As expected, in this study, OVA sensitization/ challenge in mice engendered the structural remodeling of lungs with inflammatory alternations characterized by inflammatory cell infiltration in the peribronchial and perivascular areas, mucus overproduction, and goblet cell hyperplasia in the bronchial airways. In contrast, the administration of kuwanon G inhibited increases in total number of cells and eosinophils in BAL fluids and lung tissues and prevented the development of pathological features including mucus hypersecretion and goblet cell hyperplasia in allergic asthma. These findings indicate that the protective effect of kuwanon G on allergic asthma induced by OVA is related to an attenuation of inflammatory cells in the lung tissues and goblet cell hyperplasia in the airways. In summary, our data revealed that the antiallergic effect and immune modulation by kuwanon G in OVAsensitized/challenged asthma mice may be associated with its ability to prevent lung inflammation by decreasing the levels of OVA-specific IgE and Th2 cytokines, IL-3, IL-5, and IL-13, in the sera and BAL fluids. Our findings suggest that kuwanon G is useful in the treatment of respiratory disease such as asthma. Further studies should be conducted to understand the mechanism of kuwanon G in a cellular level using immune cells such as lymphocytes, mast cells, and eosinophils.
Acknowledgement This research was supported by a grant (12172MFDS989) from the Ministry of Food and Drug Safety in 2012.
Conflict of Interest The authors declare that they have no conflict of interest.
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oxide-induced cell death in neuroblastoma SH-SY5Y cells. Arch Pharm Res 35: 163–170. Lee MS, Park WS, Kim YH, et al. 2013. Antidepressant-like effects of Cortex Mori Radicis extract via bidirectional phosphorylation of glucocorticoid receptors in the hippocampus. Behav Brain Res 236: 56–61. Li XM, Brown L. 2009. Efficacy and mechanisms of action of traditional Chinese medicines for treating asthma and allergy. J Allergy Clin Immunol 123: 297–306. Martinez FD, Vercelli D. 2013. Asthma. Lancet 382: 1360–1372. Mihara S, Hara M, Nakamura M, et al. 1995. Non-peptide bombesin receptor antagonists, kuwanon G and H, isolated from mulberry. Biochem Biophys Res Commun 213: 594–599. Nam SY, Yi HK, Lee JC, et al. 2002. Cortex Mori extract induces cancer cell apoptosis through inhibition of microtubule assembly. Arch Pharm Res 25: 191–196. Nomura T, Fukai T, Narita T. 1980. Hypotensive constituent, kuwanon H, a new flavone derivative form the root bark of the cultivated mulberry thee (Morus alba L.) Heterocycles 14: 1943–1951. Oshima Y, Konno C, Hikino H, Matsushita K. 1980. Structure of moracenin B, a hypotensive principle of Morus root barks. Tetrahedron Lett 21: 3381–3384. Park KM, You JS, Lee HY, et al. 2003. Kuwanon G: an antibacterial agent from the root bark of Morus alba against oral pathogens. J Ethnopharmacol 84: 181–185. Shepherd JM, Duddleston DN, Hicks GS, et al. 2002. Asthma: a brief overview. Am J Med Sci 324: 174–179. Shikotra A, Siddiqui S. 2013. The role of tissue eosinophils in asthmatic airway remodelling. Clin Exp Allergy 43: 1302–1306. Shum BO, Rolph MS, Sewell WA. 2008. Mechanisms in allergic airway inflammation – lessons from studies in the mouse. Expert Rev Mol Med 10: e15. Wang R, Lu XC, Wang YW. 2002. Antiinflammatory activity of the extracts of Ramulus mori. Journal of Wuhan Botanical Research 20: 467–446. Wills-Karp M. 2004. Interleukin-13 in asthma pathogenesis. Immunol Rev 202: 175–190. Yoshihara S, Ono M, Yamada Y, et al. 2009. Early intervention with suplatast tosilate for prophylaxis of pediatric atopic asthma: a pilot study. Pediatr Allergy Immunol 20: 486–492. Zhang M, Chen M, Zhang HQ, et al. 2009. In vivo hypoglycemic effects of phenolics from the root bark of Morus alba. Fitoterapia 80: 475–477.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.
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Effect of Kuwanon G Isolated from the Root Bark of Morus alba on Ovalbumin-induced Allergic Response in a Mouse Model of Asthma Hyo Won Jung,1 Seok Yong Kang,2 Jong Seong Kang,3 A Ryun Kim,4 Eun-Rhan Woo4† and Yong-Ki Park1,2*,† 1
Korean Medicine R&D Center, Dongguk University, Gyeongju 740-814, Korea Department of Herbology, College of Korean Medicine, Dongguk University, Gyeongju 740-814, Korea College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea 4 College of Pharmacy, Chosun University, Gwangju 501-759, Korea 2 3
The root bark of Morus alba L. (Mori Cortex Radicis; MCR) is traditionally used in Korean medicine for upper respiratory diseases. In this study, we investigated the antiasthmatic effect of kuwanon G isolated from MCR on ovalbumin (OVA)-induced allergic asthma in mice. Kuwanon G (1 and 10 mg/kg) was administered orally in mice once a day for 7 days during OVA airway challenge. We measured the levels of OVA-specific IgE and Th2 cytokines (IL-4, IL-5, and IL-13) in the sera or bronchoalveolar lavage (BAL) fluids and also counted the immune cells in BAL fluids. Histopathological changes in the lung tissues were analyzed. Kuwanon G significantly decreased the levels of OVA-specific IgE and IL-4, IL-5, and IL-13 in the sera and BAL fluids of asthma mice. Kuwanon G reduced the numbers of inflammatory cells in the BAL fluids of asthma mice. Furthermore, the pathological feature of lungs including infiltration of inflammatory cells, thickened epithelium of bronchioles, mucus, and collagen accumulation was inhibited by kuwanon G. These results indicate that kuwanon G prevents the pathological progression of allergic asthma through the inhibition of lung destruction by inflammation and immune stimulation. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: allergic asthma; kuwanon G; lung inflammation; Morus alba; Mori Cortex Radicis; ovalbumin; Th2 cytokines; immunoglobulin E.
Abbreviations: MCR, Mori Cortex Radicis; OVA, ovalbumin; BAL, bronchoalveolar lavage; H&E, hematoxylin and eosin; PAS, periodic acid–Schiff; Ig, immunoglobulin.
INTRODUCTION Asthma is a common chronic inflammatory disease of the airways characterized by acute symptoms of wheezing, coughing, chest tightness, shortness of breath, reversible airflow obstruction, and bronchospasm (Shepherd et al., 2002). Asthma is caused by a combination of genetic and environmental factors including allergens, air pollution, and other environmental chemicals. It is clinically classified according to the frequency of symptoms, forced expiratory volume in one second, and peak expiratory flow rate, and may also be classified as atopic (extrinsic) or non-atopic (intrinsic) where atopy refers to a predisposition toward type I hypersensitivity reactions (Dietert, 2011). Treatment of acute asthma exacerbation is usually with an inhaled short-acting beta-2 agonist and oral corticosteroids. In chronic asthma, inhaled/intravenous corticosteroids and magnesium sulphate are used in addition to longacting beta agonists, leukotriene receptor antagonists,
* Correspondence to: Yong-Ki Park, Department of Herbology, College of Korean Medicine, Dongguk University, Gyeongju 740-814, Korea. E-mail: [email protected] † Eun-Rhan Woo and Yong-Ki Park contributed equally as co-corresponding authors. Copyright © 2014 John Wiley & Sons, Ltd.
anticholinergic medications (Shepherd et al., 2002), or mast cell stabilizer. However, patients who require frequent or continuous treatment of these medications receive little or no benefit because of the severe side effects when administered over long periods or at high doses (Domingo et al., 2011). Therefore, safer preventive and therapeutic agents for asthma need to be developed. Recent research indicates that the patients with respiratory diseases such as asthma, pulmonary inflammation or embolism, and bacterial pneumonia, particularly those dissatisfied with current treatment, are very likely to seek alternative treatments, and most patients use complementary medicines such as herbal medicines and nutritional supplements (Li and Brown, 2009; Bielory, 2004). This rising interest in alternative medical practices indicates a clear need for more thorough investigation into the safety and efficacy of complementary medicines with multiple therapeutic functions (Chung and Dumont, 2011). Mori Cortex Radicis (MCR) is the dried root bark of Morus alba L. (Sang Bai Pi; Moracae). It is traditionally used for the treatment of cough and upper respiratory system with tropism of the lung channel (Kim et al., 2011). This plant has biological activities such as antihyperlipidemia (Chen et al., 1995), immune modulation (Kim et al., 2000), antibacterial (Nam et al., 2002), antiinflammation (Wang Received 23 December 2013 Revised 28 April 2014 Accepted 27 May 2014
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et al., 2002; El-Beshbishy et al., 2006; Zhang et al., 2009), antiviral (Du et al., 2003), antioxidation (Lee et al., 2011), neuroprotection (Ham et al., 2012; Lee et al., 2012), and antidepressant (Lee et al., 2013). MCR contains many biologically active compounds such as umbelliferone, scopoletin, mucin, tannin, and flavonoids such as morusin, mulberrin, mulberrichromene, cyclomulberin, moracin P, moracin O, mulberrofuran Q, kuwanon E, kuwanon H (Mihara et al, 1995), and 2-arylbenzofurans (Lee et al., 2011), and recently prenylated flavonoids such as licoflavone C, cyclomulberrin, neocyclomorusin, sanggenon I, morusin, kuwanon U, and 2-arylbenzofurans such as moracenin D, moracin P, moracin O, and mulberrofuran Q are recently described (Ham et al, 2012; Lee et al., 2012). We isolated kuwanon G from the methanol extract of MCR and investigated its antiasthmatic effect on ovalbumin (OVA)-induced allergic response in an asthma mice model. Kuwanon G inhibits specific binding of gastrinreleasing peptide (GRP) to GRP-preferring receptors in murine Swiss 3T3 fibroblasts (Mihara et al., 1995) and the growth of cariogenic bacteria such as such as Streptococcus sobrinus and Streptococcus sanguis, and Porpyromonas gingivalis causing periodontitis (Park et al., 2003). However, little is known about the therapeutic effect of kuwanon G on asthma based on experimental evidence although MCR is reported to have antiasthmatic property via enhancement of regulatory T cells and inhibition of Th2 cytokines in a mouse asthma model (Kim et al., 2011). Therefore, to identify the biological activity of kuwanon G, we investigated the antiasthmatic property of kuwanon G on OVA sensitization/ challenge-induced asthma in mice.
MATERIALS AND METHODS Isolation of Kuwanon G. The root bark of M. alba (MCR) was collected on 15 March 2012 in Gyeongju, Gyeongsangbuk-do Province, South Korea (GPS location: 35.748358, 129.235482), and authenticated by Prof. J. H. Lee (Dongguk University, Gyeongju, Korea). A voucher specimen (CSU-1048-17) was deposited in the Herbarium of the College of Pharmacy, Chosun University. The dried root bark of M. alba (12 kg) was extracted three times with MeOH under reflux, and 1511.6 g of residue was produced. The MeOH extract was suspended in water and then partitioned sequentially with equal volumes of dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Each fraction was evaporated in vacuo to yield the residues of CH2Cl2 (318.2 g), EtOAc (192.2 g), n-BuOH (182.4 g), and water (534.3 g) extract. The EtOAc fraction (53 g) was chromatographed over a silica gel column (500 g, 5.0 × 15 cm, twice) using a gradient solvent system of n-hexane-EtOAc (2:1 → 1:8, EtOAc, MeOH) to give five subfractions (E1–E5). Subfraction E2 (8.30 g) was subjected to MCI gel column chromatography (CC, 200 g, 3.5 × 28 cm) eluting with a gradient solvent system of MeOH/H2O (1:1 → 3:1) to yield 16 subfractions (E21–E216). Subfraction E24 (4.03 g) was subjected to silica gel CC (120 g, 2.0 × 40 cm, CHCl3/ MeOH/H2O, 12:1:0.1 → 8:1:0.1) to give six fractions Copyright © 2014 John Wiley & Sons, Ltd.
(E241–E246). Subfraction E245 (2.24 g) was purified by LiChroprep RP 18 CC (MeOH/H2O, 1:1) to yield kuwanon G (1011 mg). IR spectra were recorded on an IMS 85 (Bruker). NMR spectra were recorded on a Varian VNMRS 600 MHz spectrometer (KBSIGwangju center) operating at 600 MHz (1H) and 150 MHz (13C), respectively, with chemical shifts given in ppm (δ). HPLC was performed using a Waters HPLC system equipped with Waters 600 Q-pumps, a 996 photodiode array detector, and an Optimapak C18 column (5 μm, 250 mm × 4.6 mm), solvent [MeOH/H2O 15:85 (0 min), 70:30 (15 min), 100:0 (30 min)], flow rate 1.0 mL/min. TLC was carried out on precoated Kieselgel 60F254 (art. 5715, Merck) and RP-18 F254s (art. 15389, Merck) plates. Column chromatography was performed on silica gel 60 (40–63 and 63–200 μm, Merck) and MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Co.). Low pressure liquid chromatography was carried out over a Merck Lichroprep Lobar®-A RP-18 (240 × 10 mm) column with a FMI QSY-0 pump (ISCO). The physico-chemical characteristics including 1 H-NMR, 13C-NMR, and heteronuclear single quantum coherence of these compounds were identical with those reported in the literature (Oshima et al., 1980; Nomura et al., 1980).
Animals. Six-week-old male BALB/c mice (20 ± 2 g) were purchased from Koatech Co. Ltd. (Gyeonggido, Korea). The animals were housed under controlled environmental conditions at a temperature of 22 ± 3 °C with a relative humidity of 55 ± 5% and 12 h light/dark cycle throughout the study. The care and treatment of animals were in accordance with the guidelines established by the Korean National Institute of Health at the Korean Academy of Medical Sciences for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Dongguk University.
Induction of asthma and drug administration. The mice were divided into five groups and given free access to a standard laboratory diet and water during the experimental period. OVA solution (1 mg/mL in saline) and AlOH3 (20 mg/mL in saline) were mixed in a 1:1 ratio, and the mice were then sensitized with an intraperitoneal injection at a dosage of 0.3 mL/ mice. For the second immunization, 0.1% OVA solution (antigen challenge) was injected peritoneally on days 7 and 14. In addition, a local challenge was performed three times (at 2-day intervals) from day 21 to day 28 by instilling 0.1% OVA solution into the bilateral nasal cavities using a nebulizer. Kuwanon G at a dose of 1 and 10 mg/kg body weight or ketotifen (an antihistamine) as a reference drug at a dose of 10 mg/kg body weight was orally administered to OVA-sensitized animals once daily for seven consecutive days during OVA challenge. Normal and OVA-sensitized/challenged animals were given saline alone on the same schedule. Blood samples were taken from each mouse by cardiac puncture under isoflurane anesthesia 24 h after the oral administration of OVA. Serum was prepared and frozen at 70 °C prior to analysis. The lung tissues were Phytother. Res. 28: 1713–1719 (2014)
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removed from the body, changes were assessed.
and
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histopathological
Serological analysis. The concentrations of OVAspecific IgE (BD sciences, San Diego, CA, USA) and Th2 cytokines IL-4, IL-5 and IL-13 (R&D Systems Inc., MN, USA) in the sera of mice were measured using commercially available enzyme-linked immunosorbant assay (ELISA) or enzyme immunoassay kits according to the manufacturer’s recommendations. The concentration of each substance was calculated from the equations obtained from standard curve plots for the standard solutions in the kits. Inflammatory cell counts in bronchoalveolar lavage fluid. To count the inflammatory cells in bronchoalveolar lavage (BAL) fluids, mice were anesthetized with 50 mg/kg of pentobarbital (Hanlim Pharm., Seoul, Korea), and BAL fluids harvested by lavaging the lungs with saline delivered via a tracheal cannula (Chen et al., 2011). The BAL fluids were deposited onto cytospin slides and stained with Diff-Quik (Dade Behring Inc., Deerfield, IL, USA). Differential cell counts were performed by two independent investigators. Histological analysis of lung tissues. Mice were euthanized with a high dose of pentobarbital and exsanguinated after final OVA challenge. The lungs were obtained from the mid-zone of the left lung of mice and fixed in 4% formalin. The tissues were embedded in paraffin, and serial sections of 5-μm thickness were prepared. After choosing the first section randomly, ten sections in each mouse were stained with hematoxylin and eosin (H&E) for the assessment of the histopathological feature in the lungs, periodic acid–Schiff (PAS) for the assessment of goblet cell menbers, and Masson’s trichrome for the assessment of epithelial and subepithelial collagen thickness.
Figure 1. HPLC analysis of kuwanon G isolated from MRC methanol extract. (A) HPLC chromatogram was analyzed as described in the Materials and Methods Section and (B) the chemical structure of kuwanon G.
Effect of kuwanon G on the production of OVA-specific IgE and Th2 cytokines in OVA-induced asthma mice To investigate the effect of kuwanon G on allergic immune responses in vivo, the levels of allergic mediators OVA-specific IgE and Th2 cytokines were measured in the sera of OVA-sensitized/challenged asthma mice. Initially, OVA-specific IgE serum levels increased in OVA-sensitized/challenged mice. The administration of kuwanon G at doses of 1 and 10 mg/kg in OVA-sensitized/challenged mice for 7 days significantly decreased OVA-specific IgE serum levels compared with that in the OVA-control group (Fig. 2). The administration of ketotifen as a reference drug at a dose of 10 mg/kg also significantly decreased serum IgE levels in OVA-sensitized/challenged mice. Next, we also examined the effect of kuwanon G on the release of Th2 cytokines IL-4, IL-5, and IL-13 in the sera of OVA-sensitized/ challenged mice. The serum levels of IL-4, IL-5, and IL-13 significantly increased in OVA-sensitized/challenged mice compared with those in normal group. The
Statistical analysis. All data were analyzed in GraphPad PRISM 5.0 software (GraphPad Software, Inc., Sandiego, CA, USA). Data are expressed as means ± the standard error of mean (S.E.M.). The significance of treatment effects was determined using one-way analysis of variance followed by Tukey’s post hoc analysis; null hypotheses of no difference were rejected if p-values were less than 0.05.
RESULTS Isolation of kuwanon G As shown in Fig. 1, kuwanon G was purified from the methanol extract of MCR. This process yielded a brown powder of kuwanon G, ½α25 D + 466° (c = 0.13, MeOH); UV λmax (MeOH) nm (log ε): 240(3.82), 280(3.75); IR (KBr) νmax: 3350, 1622, 1514, 1446, 1045, 974, 840 cm1; ESI-MS m/z: 691[M H]+; 1H-NMR and 13 C-NMR (Supporting information). The purity of isolated kuwanon G was 96.7% based on the HPLC analysis (Fig. 1A). Copyright © 2014 John Wiley & Sons, Ltd.
Figure 2. Effect of kuwanon G on the production of OVA-specific IgE in the sera of OVA-sensitized/challenged asthma mice. The serum levels of OVA-specific IgE were measured by ELISA. Data are expressed * ** as means ± S.E.M. of ten mice per group. p < 0.05, p < 0.01 and *** p < 0.001 versus normal (a) or OVA-control group (b). Phytother. Res. 28: 1713–1719 (2014)
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administration of kuwanon G in high dose significantly decreased the serum levels of IL-4, IL-5, and IL-13 in OVA-sensitized/challenged mice, compared with those in the OVA-control group (Fig. 3). In contrast, the administration of ketotifen did not decrease the serum levels of these cytokines in OVA-sensitized/challenged mice. These results indicate that kuwanon G may regulate the allergic response in asthma progression. Effect of kuwanon G on the infiltration of inflammatory cells in BAL fluids To investigate the effect of kuwanon G on lung inflammation in OVA-sensitized/challenged asthma mice, inflammatory cells (macrophages, lymphocytes, neutrophils, and eosinophils) were counted in BAL fluids. Total cells, lymphocytes, neutrophils, and eosinophils were significantly higher in BAL fluids of OVA-sensitized/ challenged asthma mice compared with the control group (Fig. 4A). The numbers of cells significantly decreased following the administration of kuwanon G at doses of 1 and 10 mg/kg. Ketotifen also significantly reduced the numbers of these inflammatory cells. There was no difference in the numbers of macrophages in BAL fluids among groups. The OVA-specific IgE levels in BAL fluids of OVA-sensitized/challenged asthma mice were determined. As shown in Fig. 4B, the levels of OVA-specific IgE significantly increased in the BAL fluids of OVA-sensitized/challenged asthma mice. The administration of kuwanon G at dose of 1 and 10 mg/kg significantly decreased the levels of OVA-specific IgE in BAL fluids of asthma mice. Effect of kuwanon G on histopathological changes of lung tissues To investigate the effect of kuwanon G on the histopathological changes of lung tissues by asthma, the lung tissues were stained with H&E, PAS, and trichrome. As shown in the H&E stain (Fig. 5A), the lung tissue of OVA-sensitized/challenged asthma mice displayed the typical pathological features of asthma, including infiltration of numerous inflammatory cells around the bronchiole, peribronchial, vascular region, thickened airway epithelium, and the accumulation of mucus and debris in the lumen of bronchioles in OVA-sensitized/ challenged mice. Kuwanon G at doses of 1 and 10 mg/kg markedly reduced these pathological lung changes in OVA-sensitized and challenged asthma mice, especially at a dose of 10 mg/kg. In addition, kuwanon G at dose of 10 mg/kg inhibited mucin accumulation by goblet cells hyperplasia in the lumen of bronchioles (Fig. 5B) and reduced collagen accumulation around the bronchiole, peribronchial, and vascular region (Fig. 5C) in OVA-sensitized/challenged asthma mice. Ketotifen at a dose of 10 mg/kg also reduced the inflammation in peribronchial and perivascular region.
DISCUSSION Allergic asthma is the most common type of asthma. Inhaling specific substances called allergens (allergy Copyright © 2014 John Wiley & Sons, Ltd.
Figure 3. Effect of kuwanon G on the production of Th2 cytokines in the sera of OVA-sensitized/challenged asthma mice. The serum levels of IL-4 (A), IL-5 (B), and IL-13 (C) were measured by ELISA. Data are expressed as means ± S.E.M. of * ** *** p < 0.01 and p < 0.001 ten mice per group. p < 0.05, versus normal (a) or OVA-control group (b). Phytother. Res. 28: 1713–1719 (2014)
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Figure 4. Effect of kuwanon G on the numbers of inflammatory cells and the production of OVA-specific IgE in the BAL fluid of OVA-sensitized/challenged asthma mice. (A) The numbers of each cell type were counted in the BAL fluids by Giemsa staining. (B) The levels of OVA-specific IgE in the BAL fluids were measured by ELISA. Data are expressed as means ± S.E.M. of ten mice per * ** *** p < 0.001 versus normal (a) group. p < 0.05, p < 0.01 and or OVA-control group (b).
triggers such as pollen, mites, or molds) or non-allergic exposures (smoke, dust, fumes, and sometimes strong smells) brings on asthma exacerbations. Allergic asthma is a chronic inflammatory pulmonary disease characterized by coughing, wheezing, shortness of breath, rapid breathing, and tightening of the chest with an underlying Th2 cell-mediated inflammatory response in the airway (Kudo et al., 2013). The general principles for allergic
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asthma management are avoidance of allergens/triggering factors and symptomatic treatment using a variety of drugs such as short acting beta-agonists, anticholinergics, systemic corticosteroids, and antiinflammatory drugs or, recently, therapies based on immunomodulation such as allergen-specific immunotherapy (Shum et al., 2008). Although pharmacotherapy is required, the current treatment of allergic asthma is not ideal, and most modern drugs transiently improve clinical symptoms but cannot cure the cause. Therefore, it is a challenge to choose the most appropriate treatment for patients from the wide range array of available agents. Herbal medicines have long been used to treat human diseases including respiratory ailments and to maintain good health. The search for appropriate therapeutic agents in immune imbalance-associated diseases such as allergic asthma has focused on medicinal plants because natural products may provide better safety and efficacy than currently used modern drugs. MCR is traditionally used for the treatment of chronic respiratory diseases in Korean clinics, and its biologic activity is well described (Chen et al., 1995; Kim et al., 2000; Nam et al., 2002; Wang et al., 2002; Du et al., 2003; El-Beshbishy et al., 2006; Zhang et al., 2009; Kim et al., 2011; Lee et al., 2011; Lee et al., 2012; Lee et al., 2013). However, aside from kuwanon G, we isolated three other compounds such as kuwanon E, morucin, and sanggenon A from MCR methanol extract. For search immune-modulatory compounds in allergic response, we investigated their effects on the production of IL-4 and IFN-γ in activated mouse spleenocytes. As a result, kuwanon G showed a good inhibitory effect (data not shown). On the basis of these results, we examined the antiasthmatic effect of kuwanon G in vivo and evaluated its therapeutic potential in allergic asthma. On the basis of this perspective, we have confirmed that kuwanon G isolated from the root barks of M. alba (MCR) has an immune-modulatory activity on OVA sensitization/challenge-induced allergic asthma in mice. This mouse model is a widely used experimental animal model of allergic asthma with clinical and pathological
Figure 5. Effect of kuwanon G on the pathological changes of lung tissues in OVA-sensitized/challenged asthma mice. The lung tissues were stained with H&E (A) for histopathological examination, PAS (B) for mucin-released goblet cells, and Masson’s trichrome (C) for collagen-release lung fibrotic cells (×200 original magnification). A representative result of at least three independent experiments is shown. (a) The infiltration of inflammatory cells; (b) thickened epithelium of bronchiole; (c) PAS-stained mucin releasing (violet); and (d) trichrome-stained collagen accumulation (blue). This figure is available in colour online at wileyonlinelibrary.com/journal/ptr. Copyright © 2014 John Wiley & Sons, Ltd.
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features similar to those of human allergic asthma that are dependent on both humoral and cellular immunity (Epstein, 2006; Kumar, 2008). Recently, with a growing global interest in natural drugs including medicinal plants, their efficacy is often compared with those of antiasthma modern drugs such as the inhaled corticoids, cromones, ketotifen for mild persistent asthma, and inhaled beta-2 agonists and antileukotrienes for prevention of exercise-induced asthma. Ketotifen is an antiallergic drug with antihistaminic activity used for asthma prophylaxis, which protects against bronchoconstriction both immediate and late reactions (Yoshihara et al., 2009). Ketotifen is also known to cause a significant inhibition of total IgE and IL-4 levels and improvement of nasal symptoms through the immunomodulatory function (Devillier, 2001). In the present study, we first identified that kuwanon G effectively prevented the disease progression in OVA sensitization/challenge-induced asthma of mice through reducing the levels of OVA-specific IgE and Th2 cytokines, IL-3, IL-5, and IL-13, and also reducing the infiltration of inflammatory cells in lungs, to a similar degree as the reference drug, ketotifen, or better. In animal models, OVA sensitization/challenge induces a significant increase in the serum IgE and BAL fluid IgE (Hamelmann et al., 1999). IgE is a type of antibody that is present in minute amounts in the body, plays a major role in allergic diseases, binds to allergens, and triggers the release of substances from mast cells as part of the inflammatory process (Martinez and Vercelli, 2013). Our results showed that the serum and BAL fluid concentrations of OVA-specific IgE were significantly reduced in asthma mice after kuwanon G administration. This result suggests that kuwanon G has an effect on allergic asthma in an IgE-dependent manner. Asthma is classically recognized as the typical Th2 disease, with increased IgE levels and eosinophilic inflammation in the airway. Rising Th2 cytokines, particularly IL-4, IL-5, IL-6, IL-9, and IL-13, modulate the asthmatic inflammation, by triggering the activation/recruitment of IgE producing B cells, mast cells, and eosinophils (Kudo et al., 2013). IL-4 is the major factor regulating IgE production by B cells and is required for optimal Th2 differentiation (Deo et al., 2010). IL-5 is a cytokine that is highly specific for eosinophilic inflammation in allergic disease, and antibodies that block IL-5 actions are efficacious in reducing eosinophilic inflammation and airway hyper-responsiveness with severe asthma (Greenfeder et al., 2001; Corren, 2011). IL-13, independent of other Th2 cytokines, is both necessary and sufficient to induce all features of allergic asthma (Wills-Karp, 2004) and increases goblet cell differentiation, activation of fibroblasts, elevation of bronchial hyper-responsiveness, and switching of B cell antibody production from IgM to IgE. IL-13 antagonists are considered an important biological agent for asthma
therapy in patients with poorly controlled asthma (Corren, 2013). In this study, the serum levels of IL-4 and IL-13 were decreased significantly in asthma mice after the administration of kuwanon G at dose of 10 mg/kg. This suggests that kuwanon G has the potential to modulate the Th2 cytokines and could be used as immune-modulatory agent in the treatment of allergic asthma. Research suggests that airway inflammation is central to asthma pathophysiology. As a result of airway inflammation, airway structural remodeling occurs, characterized by epithelial damage, goblet cell hyperplasia, and airway smooth muscle hypertrophy, with profound consequences on the mechanics of airway narrowing in asthma, contributing to the chronicity and progression of the disease (Shikotra and Siddiqui, 2013). Eosinophils are the predominant inflammatory cells in asthmatic lung tissues and contribute to the clinical features of allergic asthma and airway hyper-responsiveness. As expected, in this study, OVA sensitization/ challenge in mice engendered the structural remodeling of lungs with inflammatory alternations characterized by inflammatory cell infiltration in the peribronchial and perivascular areas, mucus overproduction, and goblet cell hyperplasia in the bronchial airways. In contrast, the administration of kuwanon G inhibited increases in total number of cells and eosinophils in BAL fluids and lung tissues and prevented the development of pathological features including mucus hypersecretion and goblet cell hyperplasia in allergic asthma. These findings indicate that the protective effect of kuwanon G on allergic asthma induced by OVA is related to an attenuation of inflammatory cells in the lung tissues and goblet cell hyperplasia in the airways. In summary, our data revealed that the antiallergic effect and immune modulation by kuwanon G in OVAsensitized/challenged asthma mice may be associated with its ability to prevent lung inflammation by decreasing the levels of OVA-specific IgE and Th2 cytokines, IL-3, IL-5, and IL-13, in the sera and BAL fluids. Our findings suggest that kuwanon G is useful in the treatment of respiratory disease such as asthma. Further studies should be conducted to understand the mechanism of kuwanon G in a cellular level using immune cells such as lymphocytes, mast cells, and eosinophils.
Acknowledgement This research was supported by a grant (12172MFDS989) from the Ministry of Food and Drug Safety in 2012.
Conflict of Interest The authors declare that they have no conflict of interest.
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Phytother. Res. 28: 1713–1719 (2014)
