Identification of potential inflammatory inhibitors from Aster tataricus

Bioorganic Chemistry 92 (2019) 103208

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Identification of potential inflammatory inhibitors from Aster tataricus a,1

Xiang Dong Su a b

, Hyun-Jae Jang

b,1

a

a,⁎

a,⁎

, Hong Xu Li , Young Ho Kim , Seo Young Yang

T

College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, 30 Cheongju, Chungbuk 28116, Republic of Korea

ARTICLE INFO

ABSTRACT

Keywords: Aster tataricus Asteraceae Inflammatory NF-κB MAPKs

Aster tataricus L.f. is a traditional Eastern Asian herbal medicine used for the relief of cough-related illnesses. In this study, 32 known compounds and two novel monoterpene glycosides were isolated from the roots of A. tataricus. With the aid of reported data, elucidation of the root-extract components was carried out using a multitude of spectroscopic techniques. All isolates were investigated for their ability to inhibit nitric oxide (NO) secretion in lipopolysaccharide-activated RAW264.7 cells. Compound 7 remarkably suppressed NO production with an IC50 value of 8.5 µM. In addition, compound 7 exhibited significant inhibitory activity against the production of inflammatory cytokines (prostaglandin E2, interleukin-6, and interleukin-1 beta) and the expression of inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) via inhibition of nuclear factor-kappa B activation. Moreover, compound 7 effectively prevented the downstream activation of the mitogen-activated protein kinase signaling pathway by inhibiting phosphorylation of c-Jun N-terminal kinases, extracellular signal-regulated kinases, and p38. These results outline compound 7 as a potential inhibitor for the broad treatment of inflammatory diseases, such as atopic dermatitis, asthma, and various allergies.

1. Introduction Phlegm and recurrent coughing are two representative clinical symptoms of airway mucus hypersecretion, which are attributed to chronic inflammatory airway illnesses [1]. As an underlying self-defense mechanism, inflammation is a complex physiological process involving both the immune/vascular systems and molecular mediators, all of which are involved in sequestering inflammatory agents such as bacteria, viruses, and damaged cells. Activated macrophages mediate the inflammation process by secreting diverse pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) [2]. NO is a cell-derived signaling molecule produced by inducible nitric oxide synthase (iNOS) [3]. Excessive expression of NO leads to the overproduction of reactive oxygen species, which in turn damage DNA, leading to apoptosis [4]. Nuclear factor-kappa B (NF-κB) is a common transcription factor that expresses inflammatory cytokines and mediator molecules in mammals [2]. Activation of NF-κB is initiated when phosphorylation-induced (carried out by inhibitor of κB kinase) degradation of inhibitor of κB (IκB) occurs, which results in NFκB entering the nucleus and activating pro-inflammatory gene expression [5,6]. Mitogen-activated protein kinases (MAPKs) are a group of

protein kinases involved in a series of cellular responses, including inflammation and apoptosis [7]. The activation of MAPKs (c-Jun Nterminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38) by MAPK kinases (MEKs) has a significant effect on the process of inflammation [5,8]. Aster tataricus L.f. (family Asteraceae) is a perennial plant that possesses small, colorful flowers. It is found in many regions in Eastern Asia, including mainland China, South Korea, and Japan [9]. Its rhizomes and roots have been used as an herbal material to assist in the treatments of cough, asthma, pharyngitis, and dysuria [10]. A variety of chemical constituents have been obtained from A. tataricus, including tetracyclic triterpene ketones, triterpenoid saponins, monoterpenoids, peptides, caffeoylquinic acid derivatives, and flavonoids [10–13]. A study outlining the use of an ethanol extract of A. tataricus for the treatment of diabetic mice showed that anti-inflammatory activity was achieved by suppression of pro-inflammatory cytokines and activation of the NF-κB signaling pathway [14]. In addition, 4-hydroxyphenylacetic acid isolated from A. tataricus showed anti-inflammatory effects by attenuating hypertonic and hypoxia-inducible factor 1-alpha production in seawater aspiration-induced lung injury in rats [15]. The goal of this study was to discover compounds with anti-

Corresponding authors. E-mail addresses: [email protected] (Y.H. Kim), [email protected] (S.Y. Yang). 1 These authors contributed equally to this work. ⁎

https://doi.org/10.1016/j.bioorg.2019.103208 Received 21 February 2019; Received in revised form 25 July 2019; Accepted 15 August 2019 Available online 19 August 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

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inflammatory properties isolated from A. tataricus, which could provide valuable evidence supporting its traditional use in the treatment of coughing and related illnesses. Our phytochemical study led to the isolation of two novel monoterpene glycosides (1 and 2) and 32 known compounds (3–34). Extensive chromatographical techniques were used to isolate the compounds from a crude methanol extract of the roots and rhizomes of A. tataricus. Consequently, all isolates were evaluated for their inhibitory activity against lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells.

5:1:0.1, 2:1:0.1; 2.0 L for each steps) to obtain 12 sub-fractions (Fr. 2B1–2B12). Fraction 2B2 (670.0 mg) was isolated using YMC RP-18 (1.0 × 60 cm) column chromatography with an MeOH–H2O (1:5, 1:4, 1:2; 900 mL for each steps) elution solvent to give 22 (30.0 mg) and 28 (70.0 mg). Fraction 2B4 (1.3 g) was purified using YMC RP-18 (2.0 × 80 cm) column chromatography with an MeOH–H2O (1:2, 1:1.5; 1.5 L for each steps) elution solvent to give 16 (7.0 mg), 19 (70.0 mg), 29 (6.5 mg), and 30 (40.0 mg). Fraction 2B7 (946.3 mg) was subjected to YMC RP-18 (1.0 × 60 cm) column chromatography with an MeOH–H2O (1:5, 1:2, 1:1; 1L for each steps) elution solvent to obtain 20 (83.0 mg), 21 (10.0 mg), and 32 (14.8 mg). Fraction 2B12 (1.2 g) was separated using YMC RP-18 (2.0 × 60 cm) column chromatography with an MeOH–H2O (1:5, 1:3, 1:1; 1L for each steps) elution solvent to give 1 (10.0 mg), 14 (5.5 mg), 15 (4.0 mg), 18 (1.5 mg), 23 (92.2 mg), and 27 (16.0 mg). Fraction 2C (6.6 g) was isolated using YMC RP-18 (3.0 × 80 cm) column chromatography with an MeOH–H2O (1:5, 1:2, 1:1; 2L for each steps) elution solvent to give 14 sub-fractions (2C1–2C14). Fraction 2C4 (450.1 mg) was subjected to silica gel (1.0 × 60 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (7:1:0, 5:1:0.1; 1.0 L for each steps) to obtain 17 (35.9 mg) and 31 (13.4 mg). Fraction 2C11 (964.7 mg) was separated using silica gel (1.0 × 70 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (5.5:1:0, 4:1:0.1; 1.0 L for each steps) to obtain 25 (10.0 mg) and 26 (79.2 mg). Fraction 2C14 (554.0 mg) was isolated using silica gel (1.0 × 60 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (6:1:0; 1.0 L for each steps) to obtain 33 (287.0 mg) and 34 (55.3 mg). The n-BuOH layer (100.0 g) was subjected to silica gel (5 × 30 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (10:1:0, 5:1:0.1, 2:1:0.1; 6.0 L for each steps) to give 4 fractions (Fr. 3A–3D). Fraction 3B (19.0 g) was separated using silica gel (4 × 15 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (7:1:0.1, 5:1:0.1, 2:1:0.1; 3.0 L for each steps) to give 12 sub-fractions (Fr. 3A1–3D12). Fraction 3B9 (437.0 mg) was isolated using silica gel (1 × 70 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (4:1:0.1; 800 mL for each steps) to give 2 (4.0 mg) and 24 (7.0 mg). Shionoside A1 (1): Colorless needles; [α] –57.7 (c 0.1, MeOH); IR νmax (KBr) 3400, 2910, 1775, 1650, 1375, 1261, 1050 cm−1; 1H NMR (Methanol‑d4, 600 MHz) and 13C NMR data (Methanol‑d4, 150 MHz), see Table 1; HR-ESI-MS m/z 487.2147 [M+Na]+ (calcd for C21H36O11Na, 487.2150). Shionoside A2 (2): Pale yellowish needles; [α] –53.7 (c 0.1, MeOH); IR νmax (KBr) 3385, 2945, 1750, 1675, 1400, 1265, 1045 cm−1; 1H NMR (Methanol‑d4, 600 MHz) and 13C NMR data (Methanol‑d4, 150 MHz), see Table 1; HR-ESI-MS m/z 485.1997 [M+Na]+ (calcd for C21H34O11Na, 485.1993).

2. Experimental 2.1. General experimental procedures Optical rotations were determined on a JASCO P-2000 polarimeter (Tokyo, Japan) at 25 °C. Column chromatography (CC) was performed by using silica gel (SiO2; 70–230, 230–400 µm particles size; Fuji Silysia Chemical Ltd., Kasugai, Japan) and YMC RP-18 resins (75 µm, Fuji Silysia Chemical Ltd., Kasugai, Japan). Thin-layer chromatography (TLC) was performed by using pre-coated silica gel 60 F254 and reversed phase (RP)-18 F254S plates (Merck, Darmstadt, Germany). The NMR spectra was recorded by using a JEOL ECA 600 spectrometer (1H, 600 MHz; 13 C, 150 MHz; JEOL Ltd, Tokyo, Japan), δ in ppm relative to Me4Si as an internal standard and J in Hz. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained using an Agilent 6530 accurate-mass quantitation-time of flight-liquid Chromatography/mass spectrometry (Q-TOF LC/MS). The FT-IR spectra were conducted by using Nicolet 380 FT-IR spectrometer; KBr pellets; ṽ in cm−1. 2.2. Plant material Dried rhizomes and roots of A. tataricus were provided by Korean Medicine Application Center, Korea Institute of Oriental Medicine, and taxonomically identified by one of the authors (Prof. Young Ho Kim). A voucher specimen (CNU 18115) was deposited at Herbarium of the College of Pharmacy, Chungnam National University. 2.3. Compounds From the methanolic extract of A. tataricus, thirty-four compounds (1–34) were isolated and structurally elucidated. Compounds dissolved in DMSO were diluted to the final concentration in fresh media before each experiment. To not affect the cell growth, the final DMSO concentration did not exceed 0.5% in all experiments. 2.4. Extraction and isolation

2.5. Acid hydrolysis of compounds 1 and 2

Dried crushed rhizomes and roots (3.3 kg) of A. tataricus were extracted with MeOH (8.0 L × 4 times) under reflux. The MeOH extract (1.6 kg) was suspended in water and partitioned with n-hexane (16.0 g), EtOAc (25.0 g), and n-BuOH (100.0 g) fractions. The n-hexane layer was subjected to silica gel (4.0 × 30 cm) column chromatography with a gradient of n-hexane-acetone (10:1, 5:1, 1:1, 2.0 L for each steps) to give 2 fractions (Fr. 1A and 1B). Fraction 1A (4.3 g) was isolated using YMC RP18 (3.0 × 80 cm) column chromatography with an MeOH–H2O (4:1, 5:1, 10:1; 2.0 L for each steps) elution solvent to obtain compounds 4 (300.0 mg), 5 (140.0 mg), 6 (500.0 mg), 7 (20.0 mg), 9 (20.0 mg), 10 (10.0 mg), 11 (100.0 mg), 12 (8.0 mg), and 13 (2.0 mg). Fraction 1B (1.0 g) was separated using YMC RP-18 (1.5 × 80 cm) column chromatography with an MeOH–H2O (1:1, 2:1; 2.0 L for each steps) elution solvent to obtain compounds 3 (40.0 mg) and 8 (8.0 mg). The EtOAc layer (25.0 g) was subjected to silica gel (4.0 × 15 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (10:1:0, 7:1:0.1, 5:1:0.1, 3:1:0.1, 1:1:0.1; 3.0 L for each steps) to give 6 fractions (Fr. 2A-2F). Fraction 2B (8.0 g) was separated using silica gel (2.0 × 60 cm) column chromatography with a gradient of CHCl3–MeOH–H2O (20:1:0, 10:1:0.1,

Compounds 1 and 2 (3.0 mg) were heated in 3 mL 1 N HCl (dioxane–H2O) at 80 °C for 3 h. The residue was partitioned between EtOAc and H2O to give the yield the sugar fractions. The aqueous layer was evaporated until dry to yield a residue; this was dissolved in anhydrous pyridine (200 µL) and then mixed with a pyridine solution of 0.1 M L-cysteine methyl ester hydrochloride (200 µL). After warming to 60 °C for 1 h, trimethylsilylimidazole solution was added, and the reaction solution was warmed at 60 °C for 1 h. The mixture was evaporated in vacuo to yield a dried product, which was partitioned between n-hexane and H2O. The n-hexane layer was filtered and analyzed by gas chromatography. Retention times of the persilylated monosaccharide derivatives were as follows: D-apiose (tR, 9.29 min) and D-glucose (tR, 14.11 min) were confirmed by comparison with those of authentic standards. 2.6. Measurements of NO production and cell viability The nitric oxide (NO) content secreted from the macrophages was 2

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β-actin; Cell Signaling Technology, Beverly, MA, USA) at 1:1000 or 1:2000 dilution according to a previously described method [16].

Table 1 1 H and 13C NMR spectroscopic data (δ ppm) for 1 and 2. position

Shionoside A1 (1) δC

a

Shionoside A2 (2)

b

δH (J in Hz)

δCa 42.6 57.3

CH CH

2.49, s t-like 2.45, m

39.8 50.6 25.6

C CH CH2

– 1.88, d (102.2) 1.36, dt (4.6, 12.6) 1.74, mc

22.5

CH2

1.42, 2.09, 1.32, 1.72, –

t (6.2) m m mc

1.09, 1.18, 5.42, 3.32, 3.42, 3.35, 3.53, 9.7) 3.61, 3.98, 4.96, 3.93, – 3.76, 3.99, 3.61,

s s d (8.2) mc t (9.7) mc ddd (1.9, 5.7,

1 2

49.9 49.3

CH CH

3 4 5

37.6 50.4 37.9

C CH CH2

6

68.6

CH

2.27, d (2.7) 1.81, ddd (2.7, 7.1, 9.6) – 1.78, d (2.7) 1.13, dt (3.6, 13.4) 2.19, ddd (2.7, 7.1, 13.4) 4.04, dd (3.6, 7.1)

7

34.3

CH2

1.58, m

38.6

CH2

8

68.8

CH2

174.3

C

9 (endo) 10 (exo) 1′ 2′ 3′ 4′ 5′

21.0 32.7 104.6 75.2 78.3 72.0 77.1

CH3 CH3 CH CH CH CH CH

23.5 32.7 95.1 74.1 78.5 71.4 77.7

CH3 CH3 CH CH CH CH CH

6′

68.9

CH2

68.5

CH2

1″ 2″ 3″ 4″

111.2 78.2 80.7 75.1

CH CH C CH2

111.2 78.1 80.8 75.2

CH CH C CH2

5″

65.7

CH2

3.52, 3.91, 0.88, 1.07, 4.26, 3.19, 3.36, 3.28, 3.43, 9.3) 3.65, 4.01, 5.06, 3.93, – 3.81, 4.02, 3.61,

65.8

CH2

dd (7.1, 9.6) t (9.6) s s d (7.8) dd (7.8, 9.1) d (9.1) d (9.3) ddd (1.9, 6.3, dd (6.3, 11.3) d (9.7)c d (2.5) d (2.5) d (9.7) d (9.7)c d (2.0)

2.9. Statistical analysis.

δHb (J in Hz)

The results were presented as means ± SEM. One-way ANOVA followed by Dunnett’s test was performed using Prism 5 software (GraphPad software, San Diego, CA, USA) to determine significant differences between the treatment groups and the LPS alone group. Data were considered statistically significant at p < 0.05. 3. Results and discussion 3.1. Identification of compounds 1–34 As part of ongoing research for novel anti-inflammatory agents from medicinal plants, purification of a methanol extract of A. tataricus yielded 34 compounds, including two novel monoterpene glycosides (1 and 2) and 32 known compounds: three monoterpenoids (3–5), five fatty acids (6–10), four triterpenes (11–14), one diterpene (15), one sesquiterpene (16), eleven caffeoylquinic acid derivatives (17–27), three lignans (28–30), and four flavonoids (31–34). Among all isolates, compounds 17, 18, and 20–30 were isolated from A. tataricus for the first time, and compounds 18, 22–25, and 29 were isolated from the Asteraceae family for the first time (Fig. 1). Compound 1 was isolated as colorless needles ([α] = − 57.7, c 0.1, MeOH). High-resolution electro-spray ionization mass spectrometry (HRESIMS) showed a pseudo-molecular ion peak at m/z 487.2147 [M +Na]+ (calcd for 487.2150), which was consistent with a molecular formula of C21H36O11. The infrared (IR) spectrum showed typical absorption bands for hydroxy groups (3400 cm−1) and an ether group (1050 cm−1). The 1H and 13C nuclear magnetic resonance (NMR) spectroscopic data of compound 1, assigned using heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) (Table 1), were indicative of a monoterpene aglycone containing two tertiary methyls [δH 0.88 (3H, s, H-9)/δC 21.0 (C-9) and δH 1.07 (3H, s, H-10)/δC 32.7(C-10)], two methylenes [δH 1.13 (1H, dt, J = 3.6, 13.4 Hz, H-5a) and 2.19 (1H, ddd, J = 2.7, 7.1, 13.4 Hz, H5b)/δC 37.9 (C-5) and δH 1.58 (2H, m, H-7)/δC 34.3 (C-7)], three methines [δH 2.27 (1H, d, J = 2.7 Hz, H-1)/δC 49.9 (C-1), δH 1.81 (1H, ddd, J = 2.7, 7.1, 9.6 Hz, H-2)/δC 49.3 (C-2) and δH 1.78 (1H, d, J = 2.7 Hz, H-4)/δC 50.4 (C-4)], an oxymethylene [δH 3.52 (1H, dd, J = 7.1, 9.6 Hz, H-8a) and 3.91 (1H, t, J = 9.6 Hz, H-8b)/δC 68.8 (C8)], an oxymethine [δH 4.04 (1H, dd, J = 3.6, 7.1 Hz, H-6)/δC 68.6 (C6)], and a quaternary carbon at δC 37.6 (C-3). In the 1H–1H correlated spectroscopy (COSY) spectrum (Fig. 2), a bicyclic monoterpene skeleton of –OCH2(8)–CH(2)–CH(1)–CH (6)–CH2(5)–CH(4)–CH2(7)–CH(1) was established by analysis of the spin system starting at a cross-peak between H-8 (δH 3.52/3.91) and H2 (δH 1.81), followed by a segment from H-1 (δH 2.27) to H-6 (δH 4.04), H-5 (δH 1.13/2.19), and H-4 (δH 1.78), which further correlated with H7 (δH 1.58) and continued to H-1 (δH 2.27). Furthermore, the HMBC correlations (Fig. 2) between H-8 (δH 3.52/3.91) and C-1 (δC 49.9), C-2 (δC 49.3), and C-3 (δC 37.6), with the reversed correlations between H-1 (δH 2.27) and H-2 (δH 1.81) to C-8 (δC 68.8), indicated that the oxymethylene was located at the C-2 position. Correlations from H-9 (δH 0.88) and H-10 (δH 1.07) to C-2 (δC 49.3), C-3 (δC 37.6), and C-4 (δC 50.4), with the reversed correlations from H-2 (δH 1.81) and H-4 (δH 1.78) to C-9 (δC 21.0) and C-10 (δC 32.7), revealed that these two tertiary methyls were located at C-3. Comparison of the NMR data of compound 1 with those of known compound 3 revealed that compound 1 was a derivative of camphanol [11]. The only difference was that H-6 resonated at a lower field (δH 4.04), suggesting that a hydroxy group might be connected to C-6 of the aglycone. This was confirmed by the HMBC correlations (Fig. 2) between H-6 (δH 4.04) and C-2 (δC 49.3), C-4 (δC 50.4), and C-7 (δC 34.3),

dd (5.7, 11.2) dd (2.3, 11.2) d (2.5) d (2.5) d (9.7) d (9.7) d (2.0)

Assignments were done by HSQC, HMBC, COSY and ROESY experiments. a Spectra were measured in methanol‑d4 at 600. b 150 MHz. c Overlapped signals. Coupling constants (in parentheses) are in Hz.

determined using a previously described method [16,17]. Briefly, RAW264.7 cells (TIB-71, ATCC, Manassas, VA, USA) were seeded at a density of 1 × 105 cells/well in 96-well plates. After pretreatment with compounds for 1 h, the cells were treated with LPS (0.1 µg/mL) for 18 h. Mixtures of the cell media and Griess reagent (each 100 μL) were measured at 550 nm using a spectrophotometer (Varioskan LUX, Thermo Fisher Scientific Inc., Waltham, MA, USA). The MTT (3-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was performed to determine cell viability after treatment with the test samples for 24 h [17]. 2.7. Determination of PGE2, IL-1β, IL-6, and TNF-α RAW264.7 cells (2 × 106 cells/well) were cultured in a 6-well plate for 24 h, and cells were treated by LPS (0.1 µg/mL) for 12 h after being pretreated with the samples for 1 h. The PGE2, IL-1β, IL-6, and TNF-α in the supernatants were quantified using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to manufacturer’s protocol. 2.8. Western blot analysis RAW264.7 cells (2 × 106 cells/mL) were pretreated with compound 7 at 10 and 30 μM for 1 h before being treated with LPS (0.1 μg/mL) for 16 h, 1.5 h, and 0.5 h. Whole cell, cytosolic, and nuclear lysates were extracted using a Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA, USA) and an NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer’s instruction. Western blot analysis was carried out with the primary and appropriate secondary antibody (iNOS, COX-2, pIκB, IκB, p65, p-JNK, JNK, p-ERK1/2, ERK1/2, p-p38, p38, Lamin B, or 3

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Fig. 1. Chemical Structures of compounds 1–34.

and the reversed correlations between H-2 (δH 1.81), H-4 (δH 1.78), H-7 (δH 1.58), and C-6 (δC 68.6). In the rotating frame Overhause effect spectroscopy (ROESY) spectrum (Fig. 3), spatial proximities were observed between H-2/H-4 and H-7/H-10 (exo), indicating an α-orientation of H-2. Consequently, ROE correlations (Fig. 3) were observed between H-6 with H-8/H-9 (endo), but no correlations were observed between H-4 (δH 1.78) and H-7 (δH 1.58), revealing a β-orientation of H-6. Additionally, the 1H NMR spectrum (Table 1) of compound 1 showed signals for two anomeric protons at δH 4.26 (1H, d, J = 7.8 Hz, H-1′) and 5.06 (1H, d, J = 2.5 Hz, H-1″), which gave HSQC correlations with two anomeric carbon signals at δC 104.6 and 111.2. The analysis of chemical shifts and interpretation of the 1H–1H COSY, HSQC, and HMBC data revealed the existence of one β-glucopyranosyl and one βapiofuranosyl moiety. The β-configurations of glucopyranosyl and apiofuranosyl units were indicated by the relatively large coupling constants of the anomeric protons. Acid hydrolysis of compound 1 with 1 N HCl afforded D-glucose and D-apiose. The HMBC correlations (Fig. 2) between H-8 (δH 3.52/3.91) and C-1′ (δC 104.6), with the reversed correlation between H-1′ (δH 4.26) and C-8 (δC 68.8), confirmed

that the sugar chain was linked to C-8 of the aglycone. This was further confirmed by ROE correlations between H-1′ (δH 4.26) and H-8 (δH 3.52/3.91). Furthermore, the linkage of the β-apiofuranosyl unit at C-6′ of the β-glucopyranosyl was supported by a HMBC correlation (Fig. 2) between H-1″ (δH 5.06) and C-6′ (δC 68.9), and a reversed correlation between H-6′ (δH 3.65/4.02) and δC 111.2 (C-1″). This was further strengthened by the observed downfield shift by ~5 ppm of C-6′ (δC 68.9) and the ROESY correlations between H-6′ (δH 3.65/4.02) and H1″ (δH 5.06). Therefore, the structure of compound 1 was characterized as (2-endo, 6-exo)-6-hydroxy-3,3-dimethylbicyclo[2.2.1]hept-2-yl)methyl-8-O-β-D-apiofuranosyl-(1 → 6)-β-D-glucopyranoside, namely shionoside A1. Compound 2 was obtained as pale yellowish needles ([α] = − 53.7, c 0.1, MeOH), and its molecular formula was C21H34O11, determined by HRESIMS with a sodium adduct molecular ion peak at m/z 485.1997 [M +Na]+ (calcd for 485.1993). Its IR spectrum showed characteristic absorption bands for hydroxy groups (3385 cm−1), a carbonyl group (1750 cm−1), and an ether group (1400 cm−1). The 1H NMR spectrum (Table 1) of compound 2 showed two tertiary methyls at δH 1.09 (3H, s, H-9) and δH 1.18 (3H, s, H-10), and two anomeric protons at δH 5.42 (1H, 4

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OH

HO

O

HO

OH

HO

O

O OH

O HO

O

O

HO

OH

O

O O

OH

OH OH

OH

COSY Fig. 2. Selected COSY (bold line) and HMBC (H → C) correlations of 1 and 2.

Fig. 3. Selected ROESY correlations for the aglycones of 1 and 2.

d, J = 8.2 Hz, H-1′) and δH 4.96 (1H, d, J = 2.5 Hz, H-1″). Acid hydrolysis of compound 2 with 1 N HCl afforded D-glucose and D-apiose. Comparison of the NMR spectra (1H, 13C, 1H–1H COSY, HSQC, ROESY, and HMBC) of compound 2 with those of compound 1 indicated that compound 2 was also a monoterpenoid with identical sugars present in the same sequence as found in compound 1. Moreover, the 1H–1H COSY correlations (Fig. 2) between H-2 (δH 2.45) and H-1 (δH 2.49), together with the spin system from H-6 (δH 2.09/1.42) through the methylene group H-5 (δH 1.36/1.74) to H-4 (δH 1.88) and continuing to H-7 (δH 1.72/1.32) and H-1 (δH 2.49), established a segment of –CH(2)–CH (1)–CH2(6)–CH2(5)–CH(4)–CH2(7)–CH(1), which confirmed a bicyclic monoterpene skeleton. In the 13C NMR spectrum (Table 1), an additional signal was observed at δC 174.3, indicating the presence of a carbonyl group at the C-2 position of the aglycone. This was confirmed by the HMBC spectrum in which long-range correlation between H-1 (δH 2.49)/ H-2 (δH 2.45) and C-8 (δC 174.3) was observed (Fig. 2). The ROE correlations (Fig. 3) from H-2 (δH 2.45) to H-1 (δH 2.49), H-7 (δH 1.72), and H-10 (δH 1.18, exo) suggested a β-orientation of H-2. Thus, the aglycone was identified as (1R,2R,3S)-3,3-dimethyl-bicyclo[2.2.1]heptane-2-carboxylic acid, namely endo-camphenanic acid [18]. Furthermore, using the HMBC spectrum, the substitution position of the sugar chain was confirmed to be C-8 of the aglycone by correlations with the anomeric proton signal of H-1′ (δH 5.42) and C-8 (δC 174.3). Therefore, the structure of compound 2 was elucidated as 2-endo-(3,3-dimethyl-bicyclo [2.2.1]hept-2-yl)carboxylate-8-O-β-D-apiofuranosyl-(1 → 6)-β-D-glucopyranoside, namely shionoside A2.

Comparison with reported data confirmed the chemical structures of compounds 3–34 as the following known compounds: shionoside A (3) [11], shionoside B (4) [11], (3,3-dimethylbicyclo[2.2.1]hept-2-yl)methyl-O-β-D-glucopyranoside (5) [11], lachnophyllol (6) [19], lachnophyllol acetate (7) [20], cetylic acid (8) [21], α-linolenic acid (9) [22], α-linoleic acid (10) [23], bungeolic acid (11) [24], (+)-isobauerenol (12) [25], epifriedelinol (13) [26], shionon (14) [27], (4α)-17-(acetyloxy)kauran-18-oic acid (15) [28], (+)-spathulenol (16) [29], benzylO-β-D-glucopyranoside (17) [30], jaboticabin acid (18) [31], caffeic acid (19) [32], methyl chlorogenate (20) [33], methyl 4-caffeoylquinate (21) [33], 4-O-feruloylquinic acid methyl ester (22) [34], 3,5-Odicaffeoyl-1-O-methylquinic acid methyl ester (23) [35], arillanin B (24) [36], parispolyside F (25) [37], helonioside A (26) [37], helonioside B (27) [37], lariciresinol 9-O-β-D-glucopyranoside (28) [38], pinoresinol O-β-D-glucopyranoside (29) [39], isolariciresinol 9-O-β-Dglucopyranoside (30) [40], kaempferol (31) [41], astragaline (32) [42], quercetin (33) [43], and isoquercetin (34) [43]. The overproduction of NO in cells is associated with inflammatory diseases and carcinogenesis. Various reports have monitored NO levels within cells when subjected to the anti-inflammatory properties of phytochemicals derived from natural products [44–46]. Thus, the antiinflammatory inhibitory effects of the A. tataricus isolates (1–34) on NO production were evaluated in LPS-activated RAW264.7 macrophages. Ten compounds, fatty acids (6–10), terpenoids (11–15), and flavonoids (31 and 33), displayed potent inhibitory effects, with half-maximal inhibitory concentration (IC50) values of 6.9–47.2 µM (Table 2). Of 5

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Table 2 Inhibitory effects of compounds 1–34 against LPS-induced NO secretion.a compounds

IC50 (µM)

compounds

IC50 (µM)

compounds

IC50 (µM)

1 2 3 4 5 6 7 8 9 10 11 12

> 50 > 50 > 50 > 50 > 50 26.34 ± 1.92 8.45 ± 1.32 > 50 17.98 ± 0.95 40.98 ± 2.40 22.31 ± 0.59 28.65 ± 1.44

13 14 15 16 17 18 19 20 21 22 23 24

6.90 ± 0.72 > 50 47.22 ± 0.99 > 50 > 50 > 50 > 50 > 50 > 50 > 50 > 50 > 50

25 26 27 28 29 30 31 32 33 34 dexamethasone

> 50 > 50 > 50 > 50 > 50 > 50 30.00 ± 2.07 > 50 39.22 ± 2.83 > 50 0.010 ± 0.005

Results are expressed as the IC50 values (mean ± SEM) of three independent experiments (n = 3). a Macrophages cytotoxicity was not observed at the IC50.

Fig. 4. Effect of compound 7 on LPS-induced pro-inflammatory cytokine production in RAW264.7 cells. PGE2, TNF-α, IL-6, and IL-1β levels were measured using enzyme-linked immunosorbent assay, with dexamethasone (Dexa) as a positive control. Results are presented as the means ± SEM of duplicate experiments. *p < 0.05 compared to the LPS-alone treatment group.

these bioactive isolates, lachnophyllol acetate (7, IC50: 8.5 µM), exhibited a higher inhibitory activity than lachnophyllol (6, IC50: 26.3 µM), which may be due to the presence of an acetyl group at the acetylenic alcohol in compound 7. Inflammation symptoms such as edema and pain can be attributed to PGE2 stimulation in response to inflammatory stimuli, such as tissue injury and exogenous microorganisms; it is well known that nonsteroidal anti-inflammatory drugs suppress PGE2 synthesis [47]. Pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, are key mediators of inflammation processes that lead to the pathogenesis of acute or chronic inflammatory diseases; thus, their blockade has been studied for the development of anti-inflammatory drugs [48]. To investigate whether these pro-inflammatory mediators were regulated by compound 7, we assessed the PGE2, TNF-

α, IL-6, and IL-1β levels from supernatants in LPS-treated RAW264.7 cells. Compound 7 showed significant inhibitory activity against the release of PGE2 and pro-inflammatory cytokines IL-6 and IL-1β, but not TNF-α, suggesting that anti-inflammatory activity of compound 7 was due to the suppression of PGE2, IL-6, and IL-1β, as well as NO (Fig. 4). Chronic expression of NO and PGE2, catalyzed and synthesized by iNOS and cyclooxygenase-2 (COX-2), respectively, contributes to the mediation of inflammatory processes [49]. To evaluate the inhibitory effects of compound 7 on NO and PGE2 levels, as well as iNOS and COX-2 expression, we performed Western blot analysis (Fig. 5). Protein expression of iNOS and COX-2 were reduced by compound 7 in a dosedependent manner. Therefore, inhibitory activity of compound 7 on NO and PGE2 levels was significantly correlated with a decrease in both 6

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Fig. 5. Effect of compound 7 on LPS-stimulated iNOS and COX-2 expression in RAW264.7 cells. iNOS and COX-2 protein levels were measured by Western blot analysis with β-actin serving as a loading control and dexamethasone (Dexa) as a positive control. The band intensity of proteins was quantified using ImageJ software. Data are expressed as the means ± SEM. *p < 0.05 compared to the LPS-alone treatment group.

Fig. 6. Effect of compound 7 on LPS-induced IκB and NF-κB activation in RAW264.7 cells. For measurement of IκB and NF-κB activity, Western blot analysis was performed with cytosolic and nuclear extracts using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific). β-Actin and Lamin B served as loading controls to normalize levels of IκB and NF-κB respectively, with dexamethasone (Dexa) used as a positive control. Band intensities of the IκB/β-actin and NFκB/Lamin B ratios were quantitated using ImageJ software. Data are expressed as the means ± SEM. *p < 0.05 compared to the LPS-alone treatment group.

7

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Fig. 7. Effects of compound 7 on LPS-induced JNK, ERK, and p38 MAPK activation in RAW264.7 cells. The phosphorylation levels of JNK, ERK, and p38 were examined by Western blot analysis with total-JNK, -ERK, and -p38 levels used to normalize for loading controls, respectively, with dexamethasone (Dexa) serving as a positive control. The band intensities of the p-JNK/JNK, p-ERK/ERK, and p-p38/p38 ratios were quantitated using ImageJ software. Data are expressed as the means ± SEM. *p < 0.05 compared to the LPS-alone treatment group.

iNOS and COX-2 expression. The expression of pro-inflammatory mediators or genes, such as iNOS and COX-2, are mainly regulated by transcription factor NF-κB when activated by LPS or other inflammatory cytokines [50]. Phosphorylation and subsequent degradation of IκB activates the inflammatory process via translocation of the liberated NF-κB to the nucleus [49]. Thus, we examined whether compound 7 inhibited the phosphorylation and degradation of IκB and translocation of NF-κB to the nucleus. Compound 7 significantly suppressed the phosphorylation and degradation of IκB in the cytosol, and decreased translocation of p65, a subunit of the heterodimer of NF-κB, into the nucleus (Fig. 6). These data suggest that the inhibitory activity of compound 7 on LPSstimulated pro-inflammatory mediators may be involved in reduction of NF-κB activity via IκB phosphorylation and degradation. Three major MAPKs, JNK, ERK, and p38 MAPK, regulate physiological homeostasis in response to a variety of extracellular stress signals, including mitosis, osmosis, ultraviolet radiation, and heat shock [51]. The MAPK signal cascade is involved in inflammation processes and includes phosphorylation of JNK, ERK, and p38 MAPK by MEKs [52]; therefore, the MAPK signaling pathway is desirable as a target for the treatment of inflammatory disorders. To examine whether phosphorylation of MAPKs was suppressed by compound 7, we evaluated its inhibitory activity on LPS-stimulated p-JNK, p-ERK, and p-p38 MAPK via Western blot analysis. Phosphorylation of JNK, ERK, and p38 MAPK was significantly inhibited by compound 7 (Fig. 7). Interestingly, when comparing the band intensity ratios of p-JNK/JNK, p-ERK/ERK, and pp38/p38, compound 7 had a greater impact on phosphorylation levels of p38 MAPK than JNK and ERK (Fig. 7). Several studies have reported that iNOS expression is regulated by activation of p38 MAPK and the

NF-κB signaling pathway in LPS-stimulated macrophages [52,53]. Thus, these data suggest that the anti-inflammatory activity of compound 7 is due to inhibition of the p38 MAPK signaling cascades. 4. Conclusion Previous studies have reported the anti-inflammatory activity of extracts from A. tataricus [54,55]. However, the specific chemical component(s) responsible for the observed inhibitory activity have not been elucidated. In the present study, two new monoterpenoid glycosides (1 and 2), along with known derivatives 3–34, were isolated from A. tataricus, and their chemical structures identified by HRESIMS and NMR spectroscopic analyses. All isolates (1–34) were evaluated for their inhibitory effects on LPS-stimulated NO release in RAW264.7 cells. Based on their IC50 values, lachnophyllol acetate (7) exhibited the most potent anti-inflammatory activity, possibly due in part to the presence of an acetyl group at the terminal lachnophyllol residue. Furthermore, compound 7 inhibited iNOS and COX-2 protein expression, as well as the inflammatory mediators NO, PGE2, IL-1β, and IL-6. Its anti-inflammatory activity may be a result of inhibition of NF-κB and MAPK activation. Therefore, our findings suggest that compound 7 could be a candidate drug for the treatment of inflammatory diseases mediated via the NF-κB and MAPK signaling pathways. Declaration of Competing Interest The authors declare that there are no conflicts of interest. 8

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Acknowledgments

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