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A constituent of Alpinia katsumadai suppresses allergic airway inflammation
Phytochemistry Letters 22 (2017) 149–153
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
A constituent of Alpinia katsumadai suppresses allergic airway inflammation Chun-Yuan Wang a b
a,1
a,⁎,1
, Zhen-Ming Song
b
, Cheng-Long He , Ling-Yu Zhang
MARK
a
Department of Respiration Medicine, Daqing Oilfield General Hospital, Daqing 163000, China Department of Traditional Chinese Medicine and Clinical Laboratory, The Fifth Hospital Affiliated to Harbin Medical University, Daqing 163316, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Alpinia katsumadai Hayata Asthma Allergic airway inflammation Airway hyperresponsiveness
Two new compounds, named as (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1), and (1R,3R,4S)1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2-benzopyran-5,6,8-triol (2), were isolated from seeds of Alpinia katsumadai Hayata. Structures of compounds 1 and 2 were elucidated and determined on the basis of spectroscopic analysis. Additionally, compound 1 significantly suppressed allergic airway inflammation induced by OVA through reducing airway hyperresponsiveness. Moreover, the inflammation suppression was associated with a marked decrease in the Th2 cytokines and IgE production.
1. Introduction Alpinia katsumadai Hayata (Alpinia katsumadae Hayata), one species of the family Zingiberaceae and synonym of the accepted name Alpinia hainanensis K. Schum., widely distributes in the Southeast Asia (Li et al., 2010; Ngo and Brown, 1998) and previous chemical investigations on this plant have isolated structurally interesting constituents including kayalactones, flavonoids, stilbenes, diarylheptanoids, monoterpenes and sesquiterpenes (Chen et al., 2016; Nam and Seo, 2012), with diversified biological activities, such as anti-oxidant, anti-emetic, antiviral, anti-tumor and cytoprotective activities (Grienke et al., 2010; Jeong et al., 2007; Li et al., 2011; Xu et al., 2013; Yang et al., 1999). A compound isolated from A. katsumadai seeds has been reported to exhibit neuroprotective activities and extract from the same plant could attenuate oxidative stress and asthmatic activity in a mouse model of allergic asthma (Chen et al., 2016; Lee et al., 2010). This paper described the isolation and characterization of two new compounds, (3R)5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1), and (1R,3R,4S)-1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2-benzopyran-5,6,8-triol (2) from the seeds of A. katsumadai. The structures of the type described in this paper have so far mostly be described as of fungal origin. We isolated and identified them from the plant. As the most common chronic inflammatory syndrome, the prevalence of allergic asthma strikingly increased, particularly in children. Asthma is characterized by airway inflammation and hyperresponsiveness leading to recurrent symptoms of breathlessness, coughing and chest tightness (Pedersen et al., 2011). Therapeutic drugs to treat asthma are available as anti-allergic drugs, corticosteroids, and
⁎
1
bronchodilators, unfortunately with severe adverse effects (Kelly, 2011; Tamaoki et al., 2000). The chemical constituents from herbal remedies are believed having better compatibility with the human body. Thereafter, this study was conducted to find new therapeutic agents for the treatment of allergic asthma in an allergic airway inflammation mouse model induced by OVA. 2. Results and discussion Compound 1 was obtained as a white amorphous powder and its molecular formula C13H16O4 was deduced from the HR-ESI–MS and NMR data, with 6° of unsaturation. The presence of phenolic hydroxyl group was determined by its positive reaction with FeCl3-K3[Fe(CN]6] and showing purple in the 10% ethanol sulfate solution, together with the IR spectrum at 3356, 3251 cm−1. The UV spectrum at 225 and 293 nm, together with the IR spectrum at 1715, 1167, 1422, 1316, 838 cm−1, indicated the existence of the conjugated ketone and aromatic ring. The 1H NMR data (Table 1) indicated two methylene signals at δ 2.73 (1H, d, J = 13 Hz, H-4α) and δ 2.79 (1H, d, J = 13 Hz, H-4β), two aryl protons at δ 6.56 (1H, s, H-6), δ 6.92 (1H, s, H-8), one methine signal at δ 1.95 (1H, m, H-9), three methyl signals at δ 0.96 (3H, d, J = 6 Hz, H-10), δ 0.85 (3H, d, J = 6 Hz, H-11), δ 1.21 (3H, s, H-12), and two hydroxyl groups at δ 9.57 (1H, brs, H-5), δ 8.73 (1H, brs, H-7). 13C NMR data (Table 1) exhibited 13 signals, associated with one carbonyl (δ 165.1, C-1); one quaternary carbon connecting with oxygen (δ 84.6, C-3); one methylene (δ 29.2, C-4); six olefinic carbons including two methines (δ 108.1, C-6) and (δ 109.2, C-8), two quaternary carbons (δ 114.7, C-4a; 116.5, C-8a) and two oxygenated quaternary carbons (δ 144.6, C-5; 139.7, C-7); one
Corresponding author. E-mail address: [email protected] (Z.-M. Song). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2017.09.009 Received 11 June 2017; Received in revised form 27 August 2017; Accepted 22 September 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 22 (2017) 149–153
C.-Y. Wang et al.
Table 1 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectral data of compound 1 in DMSO-d6 (δ in ppm, J in Hz).
Table 2 1 H NMR (500 MHz) and 13C NMR (125 MHz) date of compound 2 in DMSO-d6 (δ in ppm, J in Hz).
position
δH
δC
No.
δH
δC
1 3 4
– – 2.73 (1H, d, J = 13) 2.79 (1H, d, J = 13) – – 6.56 (1H, s) – 6.92 (1H, s) – 1.95 (1H, m) 0.96 (3H, d, J = 6) 0.85 (3H, d, J = 6) 1.21 (3H, s) 9.57, 8.73 (brs)
165.1 84.6 29.2
1 3 4 5 6 7 8 9 10 11 12 1′ 2′/6′ 3′/5′ 4′ 7′ 5-OH 6-OH 8-OH
5.62(1H, s) 3.45(1H, m) 2.71(1H, m)
73.8 72.6 37.5 156.2 158.7 99.3 153.5 115.9 139.2 19.8 18.6 135.7 129.3 117.1 113.8 21.6
4a 5 6 7 8 8a 9 10 11 12 5,7-OH
114.7 144.6 108.1 139.7 109.2 116.5 37.1 16.8 18.3 22.7
methine (δ 37.1, C-9); and three methyl carbons (δ 16.8, C-10; 18.3, C-11; 22.7, C-12). The correlations between H-10/H-11 and C-9, H-11 and C-10 in HMBC spectrum, and the correlations between H-9 and H-10/H-11 in 1 H-1H COSY spectrum indicated the presence of an isopropyl group. In the HMBC spectrum (Fig. 1), the correlations between H-6 and C-5/C-7 suggested the positions of hydroxyl groups at C-5 and C-7. The correlations between H-8 and C-8a/C-7/C-1 indicated an ester connecting to the benzene ring. The correlations between H-4 and C-4a/C-8a/C-5 suggested the position of methylene at C-4a. The correlations between H-12 and C-3, as well as H-9/H-10/H-11 and C-3, suggested the position of the methyl and isopropyl groups at C-3. C-3 was confirmed as quaternary carbon connecting to oxygen on the basis of the HMBC spectrum and 13C NMR data. The lactone ring was deduced in compound 1, based on the spectrum together with the unsaturation degree. The correlations between H-4 and C-9, H-12 and C4/C-9 further confirmed the compound 1 structure. The absolute configuration was assigned by comparing the optical rotation of compound 1 ([α]20 D = −57.8) with (3R)-3,4-dihydro-3-methyl-5,6,8trihydroxy-1H-2-benzopyran-1-one ([α]20 D = −61.7) (Krohn et al., 1997), suggesting the absolute configuration of 1 as 3R. Thereafter, compound 1 was elucidated as (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one on the basis of these data. Compound 2 was obtained as a yellow gum and its molecular formula C18H20O4 was deduced from HR-ESI–MS. The IR spectrum at 3312, 2985, 1633, 1462 cm−1, showed the presence of the aromatic ring with hydroxy group. The 1H NMR data (Table 2) exhibited five aryl protons at δ 6.37 (1H, s, H-7), δ 6.86 (1H, d, J = 8 Hz, H-2′/H-6′), δ 6.49 (1H, d, J = 8 Hz, H-3′/H-5′); three methine signals at δ 5.62 (1H, s, H-1), δ 3.45 (1H, m, H-3), δ 2.71 (1H, m, H-4); three methyls at δ 1.29 (3H, d, J = 6.5 Hz, H-11), δ 1.13 (3H, d, J = 6.5 Hz, H-12), δ 1.95 (3H, s, H-7′), and three hydroxyl groups at δ 8.56 (1H, s, H-5), δ 8.94 (1H, s, H-6) and δ 8.81 (1H, s, H-8). The 13C NMR data (Table 2) indicated eighteen carbons, including seven quaternary carbons (δ 156.2, C-5; δ 158.7, C-6; δ 153.5, C-8; δ 115.9, C-9; δ 139.2, C-10; δ 135.7, C-1′; δ 113.8, C-4′), eight methines (δ 73.8, C-1; δ 72.6, C-3; δ 37.5, C-4; δ 99.3, C-7; δ 129.3, C-2′/C-6′; δ 117.1, C-3′/C-5′), and three methyls (δ
6.37(1H, s)
1.29(3H, d, J = 6.5) 1.13(3H, d, J = 6.5) 6.86(1H, d, J = 8) 6.49(1H, d, J = 8) 1.95(3H, 8.56(1H, 8.94(1H, 8.81(1H,
s) s) s) s)
19.8, C-11; δ 18.6, C-12; δ 21.6, C-7′). The plane structure of compound 2 was deduced from HMBC and COSY spectrum (Fig. 2). The correlations of H-2′/6′ with H-3′/5′ in the COSY spectrum, together with the HMBC correlations between H-2′/6′ and C-4′, H-3′/5′ and C-1′/C-4′, H7′ and C-3′/C-4′/C-5′ indicated the presence of a 1,4-substituted benzene ring. The correlations of H-11 with H-3, H-12 with H-4 in the COSY spectrum, as well as the correlations between H-1 and C-3/C-9/C10, H-3 and C-1/C-10, H-4 and C-9/C-10 in HMBC spectrum indicated the presence of a multi-substituted dihydropyrane. The HMBC correlations between H-1 and C-1′/C-2′/C-6′, as well as H-2′/6′ and C-1 indicated the benzene ring linking to the dihydropyrane via C-1 and C-1′. The HMBC correlations between 5-OH and C-5/C-6/C-10, H-7 and C-5/ C-6/C-8/C-9, 6-OH and C-5/C-6/C-7, as well as 8-OH and C-7/C-8/C-9 indicated the second benzene ring linking to the dihydropyrane via C-9 and C-10. The relative configuration of 2 was determined based on the correlations of H-1/H-4 with H-11 as well as H-3 with H-12 in NOESY spectrum. The absolute configuration was assigned by comparing the optical rotation of compound 2 ([α]25 D = +42.6) with penicitrinol C ([α]25 D = +33.2) (Chen et al., 2011) and penicitrinol F ([α]25 D = +24.8) (Nong et al., 2013), suggesting the absolute configuration of 2 as 1R, 3R, 4S. Based on these data, the compound 2 was elucidated as (1R, 3R, 4S)-1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2-benzopyran-5,6,8-triol. Asthma is characterized by airway inflammation and airway hyperresponsiveness (AHR), which indicate an exaggerated response of the airway to nonspecific stimuli. AHR results in a temporary airway obstruction and leads to recurrent symptoms of chest tightness, breathlessness and coughing (Jacobsen et al., 2014; Pedersen et al., 2011). Cytokines, such as IL-4, IL-5, and IL-13, are thought to contribute to hyperresponsiveness, as well as orchestrating the recruitment of inflammatory cells (Corry et al., 1996; Grunig et al., 1998). In a Fig. 1. Structure and key HMBC correlations of compound 1.
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Fig. 2. Structure, key 1H-1H COSY and HMBC correlations of compound 2.
IR spectrometer with KBr pellets. The UV spectrum was obtained using a Shimadzu UV-2401-A spectrophotometer with a PhotoMultiplier Tube (Shimadzu, Japan). The NMR data were recorded on a Bruker AV-500 spectrometer, using TMS as an internal standard. The HR-ESI–MS spectra were measured with an API QSTAR Pulsar mass spectrometer (Bruker, Swiss). HPLC was performed by Shimadzu apparatus (LC-6AD pump, SPD-20A UV detector, YMC ODS-A (20 mm × 250 mm, 10 μm), detection at UV 265 nm). Column chromatography was performed with silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Sweden). 4.2. Plant materials A. katsumadai seeds were purchased from Anguo City, Hebei province, China, on August 25th 2016. The plant was authenticated by Prof. Weishen Fen, Henan University of Chinese Medicine. A voucher specimen (No. 20160825) was deposited at Daqing Oilfield General Hospital. 4.3. Extraction and isolation
Fig. 3. Effects of compound 1 on airway AHR in challenged mice. AHR changes were measured in response to increasing doses of aerosolized methacholine (0, 25, 50 and 100 mg/mL) in the normal control, OVA + PBS and OVA + compound 1 groups 24 h after the final challenge. **p < 0.01 vs. normal control group; #p < 0.05, ##p < 0.01 vs. OVA + PBS group, n = 5.
Seeds of A. katsumadai were washed thoroughly and checked without fungal contamination. Air-dried seeds (30.0 kg) were pulverized and extracted with 95% ethanol (40 L × 3 × 5 h) under reflux. The plant residue was further extracted with EtOAc and a residue (A, 1235 g) was afforded after evaporating the solvent. The collected 95% ethanol was evaporated to yield a crude extract (B, 860 g). The extract B was subjected to column chromatography eluted with ethyl acetate, acetone and methanol. The portion of ethyl acetate (85.5 g) was chromatographed over a silica gel with gradient elution of ethyl acetate/ petroleum ether (0:100–100:0) to give 5 fractions (Fr. 1–Fr. 5). Fr. 3 (18.6 g) was further subjected to Sephadex LH-20 column chromatography eluted with methanol to give 4 subfractions (Fr. 3.1–Fr. 3.4). Fr. 3.2 (2.5 g) was purified with HPLC (MeOH/H2O, 60:40, v/v, 1.5 mL/ min) to give compound 1 (27.6 mg, tR = 23.5 min). The residue A was subjected to column chromatography with gradient elution of petroleum ether, CH2Cl2, and MeOH to obtain 6 fractions (Fr. 1–Fr. 6). Fr. 3 (4.1 g) was further purified on column chromatography eluted with petroleum ether, CH2Cl2, and MeOH to give 5 subfractions (Fr.3.1–Fr.3.5). Fr. 3.4 (1.5 g) was purified with Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give 2 (7.3 mg).
murine model of OVA-induced allergic airway inflammation, compound 1 treatment significantly relieved AHR by decreasing the Penh value, which was consistent to the previous report (Lee et al., 2010). Moreover, the AHR suppression was associated with reducing the levels of IL-4, IL-5 and IL-13 in the lungs, as well as the IgE in the blood (Figs. 3–5). These results warrant further study of compound 1 as the potential agent for the treatment of allergic asthma. 3. Conclusions Two new compounds, (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1), and (1R,3R,4S)-1-(4′-methyl-phenyl)-3,4-dihydro3,4-dimethyl-1H-2-benzopyran-5,6,8-triol (2), were isolated and identified from the seeds of A. katsumadai for the first time. Compound 1 exhibited potent therapeutic effects against allergic asthma. 4. Experimental
4.4. (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1)
4.1. General experimental procedure
[α]20 D = −57.8 (c 0.08, CHCl3). UV (MeOH) λmax (logε): 225 (2.32), 293 (2.23). IR (KBr): νmax 3356, 3251, 2891, 1715, 1167, 1422, 1316, 838 cm−1. 1H and 13C NMR spectroscopic data see Table 1. HRESI–MS: m/z found 235.0928 [M−H]− (calcd. for C13H15O4, 235.0923).
Optical rotations were obtained by a Shenguang SGW-1 digital polarimeter. IR spectra were recorded on a PerkinElmer Spectrum One FT151
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Fig. 4. Effects of compound 1 on the Th2 cytokine secretion. The amount of IL-4 (a), IL-5 (b), and IL-13 (c) were measured by ELISA assay. **p < 0.01 vs. normal control group; ## p < 0.01 vs. OVA + PBS group, n = 5.
4.5. (1R,3R,4S)-1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2benzopyran-5,6,8-triol (2) [α]25 D = +42.6 (c 0.1, MeOH); UV (CH3CN) λmax (log ε): 278 (3.43) (nm). IR (KBr): νmax 3312, 2985, 1633, 1462 cm−1. 1H and 13C NMR spectroscopic data see Table 2. HR-ESI–MS m/z: 299.1265 [M−H]− (calcd. for C18H19O4, 299.1271).
4.6. Experimental animals Female BALB/c mice at six-week-old were purchased from Charles River (Beijing, China) and maintained under pathogen-free conditions, a 12-h light/dark cycle. Mice were free access to food and water during the experimental period. All animal experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals. The experimental protocol was approved by the Ethical Committee on Animal Care and Use, Daqing Oilfield General Hospital, Daqing, China in March 2016.
Fig. 5. Effects of compound 1 on the total IgE production. The total IgE levels of mice were measured by ELISA assay. **p < 0.01 vs. normal control group; ##p < 0.01 vs. OVA + PBS group, n = 5.
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(ANOVA) using the Sigma Stat statistical software (SPSS Inc., Chicago, IL). p < 0.05 was considered as significant difference.
4.7. Experimental procedures Mice were sensitized by intraperitoneal injection of 100 μL PBS with 100 μg OVA emulsified in 20 mg aluminum hydroxide on day 0, and boosted again on day 14. Mice were re-challenged with 1% OVA in PBS intratracheally (i.t.) on days 22–24 and 29–31. The challenged mice were divided into two groups (five per group) and treated with compound 1 (5 μg/kg) or PBS by i.t. injection on D3 twice weekly for three weeks. The normal control (NC) mice were challenged and treated with PBS. On day 32, airway hyperresponsiveness (AHR) of mice was evaluated by non-invasive lung function measurements. On day 33, mice were sacrificed to collect samples for further analysis (Jung et al., 2016).
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4.8. Airway hyperresponsiveness assessment AHR to methacholine was evaluated on day 32 in spontaneouslybreathing mice. Mice were treated with methacholine at different concentrations (0, 25, 50, and 100 mg/mL) generated through an ultrasonic nebulizer for 3 min. The data were expressed as the Penh percentage increase after challenging with methacholine at each concentration, where the Penh value at baseline (challenging after saline) was expressed as 100% (Jung et al., 2016). 4.9. Th2 cytokines measurement Lungs from mice were homogenized in extraction reagent (Thermo Scientific, USA) containing protease inhibitor cocktail. After centrifugation at 8000 × g at 4 °C for 10 min. Th2 cytokine levels were measured by the quantitative ELISA kit according to the manufacturer’s protocol (BD Bioscience, USA). The optical value was measured by a microplate reader at 450 nm. The protein concentration was determined by the Bradford Protein Assay Kit (Bio-Rad, USA). All results were normalized to the total amount of lung tissue protein (Jung et al., 2016). 4.10. Immunoglobulin assay Blood from mice was centrifuged at 1500 rpm for 10 min to collect the serum. The total IgE levels in serum were measured by the enzymelinked immunoassay kit according to the manufacturer’s protocol (BD Bioscience, USA). The optical value was measured by a microplate reader at 450 nm. The protein concentration was determined by the Bradford Protein Assay Kit (Bio-Rad, USA). Data were normalized to the total amount of lung tissue protein (Jung et al., 2016). 4.11. Statistical analysis Values were statistically analyzed by one-way analysis of variance
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Short communication
A constituent of Alpinia katsumadai suppresses allergic airway inflammation Chun-Yuan Wang a b
a,1
a,⁎,1
, Zhen-Ming Song
b
, Cheng-Long He , Ling-Yu Zhang
MARK
a
Department of Respiration Medicine, Daqing Oilfield General Hospital, Daqing 163000, China Department of Traditional Chinese Medicine and Clinical Laboratory, The Fifth Hospital Affiliated to Harbin Medical University, Daqing 163316, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Alpinia katsumadai Hayata Asthma Allergic airway inflammation Airway hyperresponsiveness
Two new compounds, named as (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1), and (1R,3R,4S)1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2-benzopyran-5,6,8-triol (2), were isolated from seeds of Alpinia katsumadai Hayata. Structures of compounds 1 and 2 were elucidated and determined on the basis of spectroscopic analysis. Additionally, compound 1 significantly suppressed allergic airway inflammation induced by OVA through reducing airway hyperresponsiveness. Moreover, the inflammation suppression was associated with a marked decrease in the Th2 cytokines and IgE production.
1. Introduction Alpinia katsumadai Hayata (Alpinia katsumadae Hayata), one species of the family Zingiberaceae and synonym of the accepted name Alpinia hainanensis K. Schum., widely distributes in the Southeast Asia (Li et al., 2010; Ngo and Brown, 1998) and previous chemical investigations on this plant have isolated structurally interesting constituents including kayalactones, flavonoids, stilbenes, diarylheptanoids, monoterpenes and sesquiterpenes (Chen et al., 2016; Nam and Seo, 2012), with diversified biological activities, such as anti-oxidant, anti-emetic, antiviral, anti-tumor and cytoprotective activities (Grienke et al., 2010; Jeong et al., 2007; Li et al., 2011; Xu et al., 2013; Yang et al., 1999). A compound isolated from A. katsumadai seeds has been reported to exhibit neuroprotective activities and extract from the same plant could attenuate oxidative stress and asthmatic activity in a mouse model of allergic asthma (Chen et al., 2016; Lee et al., 2010). This paper described the isolation and characterization of two new compounds, (3R)5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1), and (1R,3R,4S)-1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2-benzopyran-5,6,8-triol (2) from the seeds of A. katsumadai. The structures of the type described in this paper have so far mostly be described as of fungal origin. We isolated and identified them from the plant. As the most common chronic inflammatory syndrome, the prevalence of allergic asthma strikingly increased, particularly in children. Asthma is characterized by airway inflammation and hyperresponsiveness leading to recurrent symptoms of breathlessness, coughing and chest tightness (Pedersen et al., 2011). Therapeutic drugs to treat asthma are available as anti-allergic drugs, corticosteroids, and
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bronchodilators, unfortunately with severe adverse effects (Kelly, 2011; Tamaoki et al., 2000). The chemical constituents from herbal remedies are believed having better compatibility with the human body. Thereafter, this study was conducted to find new therapeutic agents for the treatment of allergic asthma in an allergic airway inflammation mouse model induced by OVA. 2. Results and discussion Compound 1 was obtained as a white amorphous powder and its molecular formula C13H16O4 was deduced from the HR-ESI–MS and NMR data, with 6° of unsaturation. The presence of phenolic hydroxyl group was determined by its positive reaction with FeCl3-K3[Fe(CN]6] and showing purple in the 10% ethanol sulfate solution, together with the IR spectrum at 3356, 3251 cm−1. The UV spectrum at 225 and 293 nm, together with the IR spectrum at 1715, 1167, 1422, 1316, 838 cm−1, indicated the existence of the conjugated ketone and aromatic ring. The 1H NMR data (Table 1) indicated two methylene signals at δ 2.73 (1H, d, J = 13 Hz, H-4α) and δ 2.79 (1H, d, J = 13 Hz, H-4β), two aryl protons at δ 6.56 (1H, s, H-6), δ 6.92 (1H, s, H-8), one methine signal at δ 1.95 (1H, m, H-9), three methyl signals at δ 0.96 (3H, d, J = 6 Hz, H-10), δ 0.85 (3H, d, J = 6 Hz, H-11), δ 1.21 (3H, s, H-12), and two hydroxyl groups at δ 9.57 (1H, brs, H-5), δ 8.73 (1H, brs, H-7). 13C NMR data (Table 1) exhibited 13 signals, associated with one carbonyl (δ 165.1, C-1); one quaternary carbon connecting with oxygen (δ 84.6, C-3); one methylene (δ 29.2, C-4); six olefinic carbons including two methines (δ 108.1, C-6) and (δ 109.2, C-8), two quaternary carbons (δ 114.7, C-4a; 116.5, C-8a) and two oxygenated quaternary carbons (δ 144.6, C-5; 139.7, C-7); one
Corresponding author. E-mail address: [email protected] (Z.-M. Song). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2017.09.009 Received 11 June 2017; Received in revised form 27 August 2017; Accepted 22 September 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Table 1 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectral data of compound 1 in DMSO-d6 (δ in ppm, J in Hz).
Table 2 1 H NMR (500 MHz) and 13C NMR (125 MHz) date of compound 2 in DMSO-d6 (δ in ppm, J in Hz).
position
δH
δC
No.
δH
δC
1 3 4
– – 2.73 (1H, d, J = 13) 2.79 (1H, d, J = 13) – – 6.56 (1H, s) – 6.92 (1H, s) – 1.95 (1H, m) 0.96 (3H, d, J = 6) 0.85 (3H, d, J = 6) 1.21 (3H, s) 9.57, 8.73 (brs)
165.1 84.6 29.2
1 3 4 5 6 7 8 9 10 11 12 1′ 2′/6′ 3′/5′ 4′ 7′ 5-OH 6-OH 8-OH
5.62(1H, s) 3.45(1H, m) 2.71(1H, m)
73.8 72.6 37.5 156.2 158.7 99.3 153.5 115.9 139.2 19.8 18.6 135.7 129.3 117.1 113.8 21.6
4a 5 6 7 8 8a 9 10 11 12 5,7-OH
114.7 144.6 108.1 139.7 109.2 116.5 37.1 16.8 18.3 22.7
methine (δ 37.1, C-9); and three methyl carbons (δ 16.8, C-10; 18.3, C-11; 22.7, C-12). The correlations between H-10/H-11 and C-9, H-11 and C-10 in HMBC spectrum, and the correlations between H-9 and H-10/H-11 in 1 H-1H COSY spectrum indicated the presence of an isopropyl group. In the HMBC spectrum (Fig. 1), the correlations between H-6 and C-5/C-7 suggested the positions of hydroxyl groups at C-5 and C-7. The correlations between H-8 and C-8a/C-7/C-1 indicated an ester connecting to the benzene ring. The correlations between H-4 and C-4a/C-8a/C-5 suggested the position of methylene at C-4a. The correlations between H-12 and C-3, as well as H-9/H-10/H-11 and C-3, suggested the position of the methyl and isopropyl groups at C-3. C-3 was confirmed as quaternary carbon connecting to oxygen on the basis of the HMBC spectrum and 13C NMR data. The lactone ring was deduced in compound 1, based on the spectrum together with the unsaturation degree. The correlations between H-4 and C-9, H-12 and C4/C-9 further confirmed the compound 1 structure. The absolute configuration was assigned by comparing the optical rotation of compound 1 ([α]20 D = −57.8) with (3R)-3,4-dihydro-3-methyl-5,6,8trihydroxy-1H-2-benzopyran-1-one ([α]20 D = −61.7) (Krohn et al., 1997), suggesting the absolute configuration of 1 as 3R. Thereafter, compound 1 was elucidated as (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one on the basis of these data. Compound 2 was obtained as a yellow gum and its molecular formula C18H20O4 was deduced from HR-ESI–MS. The IR spectrum at 3312, 2985, 1633, 1462 cm−1, showed the presence of the aromatic ring with hydroxy group. The 1H NMR data (Table 2) exhibited five aryl protons at δ 6.37 (1H, s, H-7), δ 6.86 (1H, d, J = 8 Hz, H-2′/H-6′), δ 6.49 (1H, d, J = 8 Hz, H-3′/H-5′); three methine signals at δ 5.62 (1H, s, H-1), δ 3.45 (1H, m, H-3), δ 2.71 (1H, m, H-4); three methyls at δ 1.29 (3H, d, J = 6.5 Hz, H-11), δ 1.13 (3H, d, J = 6.5 Hz, H-12), δ 1.95 (3H, s, H-7′), and three hydroxyl groups at δ 8.56 (1H, s, H-5), δ 8.94 (1H, s, H-6) and δ 8.81 (1H, s, H-8). The 13C NMR data (Table 2) indicated eighteen carbons, including seven quaternary carbons (δ 156.2, C-5; δ 158.7, C-6; δ 153.5, C-8; δ 115.9, C-9; δ 139.2, C-10; δ 135.7, C-1′; δ 113.8, C-4′), eight methines (δ 73.8, C-1; δ 72.6, C-3; δ 37.5, C-4; δ 99.3, C-7; δ 129.3, C-2′/C-6′; δ 117.1, C-3′/C-5′), and three methyls (δ
6.37(1H, s)
1.29(3H, d, J = 6.5) 1.13(3H, d, J = 6.5) 6.86(1H, d, J = 8) 6.49(1H, d, J = 8) 1.95(3H, 8.56(1H, 8.94(1H, 8.81(1H,
s) s) s) s)
19.8, C-11; δ 18.6, C-12; δ 21.6, C-7′). The plane structure of compound 2 was deduced from HMBC and COSY spectrum (Fig. 2). The correlations of H-2′/6′ with H-3′/5′ in the COSY spectrum, together with the HMBC correlations between H-2′/6′ and C-4′, H-3′/5′ and C-1′/C-4′, H7′ and C-3′/C-4′/C-5′ indicated the presence of a 1,4-substituted benzene ring. The correlations of H-11 with H-3, H-12 with H-4 in the COSY spectrum, as well as the correlations between H-1 and C-3/C-9/C10, H-3 and C-1/C-10, H-4 and C-9/C-10 in HMBC spectrum indicated the presence of a multi-substituted dihydropyrane. The HMBC correlations between H-1 and C-1′/C-2′/C-6′, as well as H-2′/6′ and C-1 indicated the benzene ring linking to the dihydropyrane via C-1 and C-1′. The HMBC correlations between 5-OH and C-5/C-6/C-10, H-7 and C-5/ C-6/C-8/C-9, 6-OH and C-5/C-6/C-7, as well as 8-OH and C-7/C-8/C-9 indicated the second benzene ring linking to the dihydropyrane via C-9 and C-10. The relative configuration of 2 was determined based on the correlations of H-1/H-4 with H-11 as well as H-3 with H-12 in NOESY spectrum. The absolute configuration was assigned by comparing the optical rotation of compound 2 ([α]25 D = +42.6) with penicitrinol C ([α]25 D = +33.2) (Chen et al., 2011) and penicitrinol F ([α]25 D = +24.8) (Nong et al., 2013), suggesting the absolute configuration of 2 as 1R, 3R, 4S. Based on these data, the compound 2 was elucidated as (1R, 3R, 4S)-1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2-benzopyran-5,6,8-triol. Asthma is characterized by airway inflammation and airway hyperresponsiveness (AHR), which indicate an exaggerated response of the airway to nonspecific stimuli. AHR results in a temporary airway obstruction and leads to recurrent symptoms of chest tightness, breathlessness and coughing (Jacobsen et al., 2014; Pedersen et al., 2011). Cytokines, such as IL-4, IL-5, and IL-13, are thought to contribute to hyperresponsiveness, as well as orchestrating the recruitment of inflammatory cells (Corry et al., 1996; Grunig et al., 1998). In a Fig. 1. Structure and key HMBC correlations of compound 1.
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Fig. 2. Structure, key 1H-1H COSY and HMBC correlations of compound 2.
IR spectrometer with KBr pellets. The UV spectrum was obtained using a Shimadzu UV-2401-A spectrophotometer with a PhotoMultiplier Tube (Shimadzu, Japan). The NMR data were recorded on a Bruker AV-500 spectrometer, using TMS as an internal standard. The HR-ESI–MS spectra were measured with an API QSTAR Pulsar mass spectrometer (Bruker, Swiss). HPLC was performed by Shimadzu apparatus (LC-6AD pump, SPD-20A UV detector, YMC ODS-A (20 mm × 250 mm, 10 μm), detection at UV 265 nm). Column chromatography was performed with silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Sweden). 4.2. Plant materials A. katsumadai seeds were purchased from Anguo City, Hebei province, China, on August 25th 2016. The plant was authenticated by Prof. Weishen Fen, Henan University of Chinese Medicine. A voucher specimen (No. 20160825) was deposited at Daqing Oilfield General Hospital. 4.3. Extraction and isolation
Fig. 3. Effects of compound 1 on airway AHR in challenged mice. AHR changes were measured in response to increasing doses of aerosolized methacholine (0, 25, 50 and 100 mg/mL) in the normal control, OVA + PBS and OVA + compound 1 groups 24 h after the final challenge. **p < 0.01 vs. normal control group; #p < 0.05, ##p < 0.01 vs. OVA + PBS group, n = 5.
Seeds of A. katsumadai were washed thoroughly and checked without fungal contamination. Air-dried seeds (30.0 kg) were pulverized and extracted with 95% ethanol (40 L × 3 × 5 h) under reflux. The plant residue was further extracted with EtOAc and a residue (A, 1235 g) was afforded after evaporating the solvent. The collected 95% ethanol was evaporated to yield a crude extract (B, 860 g). The extract B was subjected to column chromatography eluted with ethyl acetate, acetone and methanol. The portion of ethyl acetate (85.5 g) was chromatographed over a silica gel with gradient elution of ethyl acetate/ petroleum ether (0:100–100:0) to give 5 fractions (Fr. 1–Fr. 5). Fr. 3 (18.6 g) was further subjected to Sephadex LH-20 column chromatography eluted with methanol to give 4 subfractions (Fr. 3.1–Fr. 3.4). Fr. 3.2 (2.5 g) was purified with HPLC (MeOH/H2O, 60:40, v/v, 1.5 mL/ min) to give compound 1 (27.6 mg, tR = 23.5 min). The residue A was subjected to column chromatography with gradient elution of petroleum ether, CH2Cl2, and MeOH to obtain 6 fractions (Fr. 1–Fr. 6). Fr. 3 (4.1 g) was further purified on column chromatography eluted with petroleum ether, CH2Cl2, and MeOH to give 5 subfractions (Fr.3.1–Fr.3.5). Fr. 3.4 (1.5 g) was purified with Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give 2 (7.3 mg).
murine model of OVA-induced allergic airway inflammation, compound 1 treatment significantly relieved AHR by decreasing the Penh value, which was consistent to the previous report (Lee et al., 2010). Moreover, the AHR suppression was associated with reducing the levels of IL-4, IL-5 and IL-13 in the lungs, as well as the IgE in the blood (Figs. 3–5). These results warrant further study of compound 1 as the potential agent for the treatment of allergic asthma. 3. Conclusions Two new compounds, (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1), and (1R,3R,4S)-1-(4′-methyl-phenyl)-3,4-dihydro3,4-dimethyl-1H-2-benzopyran-5,6,8-triol (2), were isolated and identified from the seeds of A. katsumadai for the first time. Compound 1 exhibited potent therapeutic effects against allergic asthma. 4. Experimental
4.4. (3R)-5,7-dihydroxy-3-isopropyl-3-methylisochroman-1-one (1)
4.1. General experimental procedure
[α]20 D = −57.8 (c 0.08, CHCl3). UV (MeOH) λmax (logε): 225 (2.32), 293 (2.23). IR (KBr): νmax 3356, 3251, 2891, 1715, 1167, 1422, 1316, 838 cm−1. 1H and 13C NMR spectroscopic data see Table 1. HRESI–MS: m/z found 235.0928 [M−H]− (calcd. for C13H15O4, 235.0923).
Optical rotations were obtained by a Shenguang SGW-1 digital polarimeter. IR spectra were recorded on a PerkinElmer Spectrum One FT151
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Fig. 4. Effects of compound 1 on the Th2 cytokine secretion. The amount of IL-4 (a), IL-5 (b), and IL-13 (c) were measured by ELISA assay. **p < 0.01 vs. normal control group; ## p < 0.01 vs. OVA + PBS group, n = 5.
4.5. (1R,3R,4S)-1-(4′-methyl-phenyl)-3,4-dihydro-3,4-dimethyl-1H-2benzopyran-5,6,8-triol (2) [α]25 D = +42.6 (c 0.1, MeOH); UV (CH3CN) λmax (log ε): 278 (3.43) (nm). IR (KBr): νmax 3312, 2985, 1633, 1462 cm−1. 1H and 13C NMR spectroscopic data see Table 2. HR-ESI–MS m/z: 299.1265 [M−H]− (calcd. for C18H19O4, 299.1271).
4.6. Experimental animals Female BALB/c mice at six-week-old were purchased from Charles River (Beijing, China) and maintained under pathogen-free conditions, a 12-h light/dark cycle. Mice were free access to food and water during the experimental period. All animal experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals. The experimental protocol was approved by the Ethical Committee on Animal Care and Use, Daqing Oilfield General Hospital, Daqing, China in March 2016.
Fig. 5. Effects of compound 1 on the total IgE production. The total IgE levels of mice were measured by ELISA assay. **p < 0.01 vs. normal control group; ##p < 0.01 vs. OVA + PBS group, n = 5.
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(ANOVA) using the Sigma Stat statistical software (SPSS Inc., Chicago, IL). p < 0.05 was considered as significant difference.
4.7. Experimental procedures Mice were sensitized by intraperitoneal injection of 100 μL PBS with 100 μg OVA emulsified in 20 mg aluminum hydroxide on day 0, and boosted again on day 14. Mice were re-challenged with 1% OVA in PBS intratracheally (i.t.) on days 22–24 and 29–31. The challenged mice were divided into two groups (five per group) and treated with compound 1 (5 μg/kg) or PBS by i.t. injection on D3 twice weekly for three weeks. The normal control (NC) mice were challenged and treated with PBS. On day 32, airway hyperresponsiveness (AHR) of mice was evaluated by non-invasive lung function measurements. On day 33, mice were sacrificed to collect samples for further analysis (Jung et al., 2016).
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4.8. Airway hyperresponsiveness assessment AHR to methacholine was evaluated on day 32 in spontaneouslybreathing mice. Mice were treated with methacholine at different concentrations (0, 25, 50, and 100 mg/mL) generated through an ultrasonic nebulizer for 3 min. The data were expressed as the Penh percentage increase after challenging with methacholine at each concentration, where the Penh value at baseline (challenging after saline) was expressed as 100% (Jung et al., 2016). 4.9. Th2 cytokines measurement Lungs from mice were homogenized in extraction reagent (Thermo Scientific, USA) containing protease inhibitor cocktail. After centrifugation at 8000 × g at 4 °C for 10 min. Th2 cytokine levels were measured by the quantitative ELISA kit according to the manufacturer’s protocol (BD Bioscience, USA). The optical value was measured by a microplate reader at 450 nm. The protein concentration was determined by the Bradford Protein Assay Kit (Bio-Rad, USA). All results were normalized to the total amount of lung tissue protein (Jung et al., 2016). 4.10. Immunoglobulin assay Blood from mice was centrifuged at 1500 rpm for 10 min to collect the serum. The total IgE levels in serum were measured by the enzymelinked immunoassay kit according to the manufacturer’s protocol (BD Bioscience, USA). The optical value was measured by a microplate reader at 450 nm. The protein concentration was determined by the Bradford Protein Assay Kit (Bio-Rad, USA). Data were normalized to the total amount of lung tissue protein (Jung et al., 2016). 4.11. Statistical analysis Values were statistically analyzed by one-way analysis of variance
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