Anti-inflammatory Potential of Saponins from Aster

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Anti-inflammatory Potential of Saponins from Aster tataricus via NFκB/MAPK Activation Xiang-Dong Su,†,# Hyun-Jae Jang,§,# Cai-Yi Wang,† Seung Woong Lee,§ Mun-Chual Rho,*,§ Young Ho Kim,*,† and Seo Young Yang*,† †

College of Pharmacy, Chungnam National University, Daejeon 34134, Korea Immunoregulatory Material Research Center, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup, Jeonbuk 56212, Korea

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§

S Supporting Information *

ABSTRACT: Four new aster saponins (1−4) together with five known analogues (5−9) were isolated from Aster tataricus. The chemical structures of 1−4 were elucidated based on spectrometric and spectroscopic analysis and comparison with reported data. The potential anti-inflammatory activities of aster saponins 1−9 were evaluated subsequently by measuring lipopolysaccharide (LPS)-enhanced nitric oxide (NO) formation in murine macrophages. Among these, aster saponin B (6) exhibited the most potent inhibitory activity (IC50: 1.2 μM). Additionally, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels were dose-dependently suppressed by 6 in LPS-activated RAW 264.7 cells. Investigation of the anti-inflammatory mechanism indicated that 6 attenuated the phosphorylation and degradation of the inhibitor of NF-κB (IκB), which led to the blocking of NF-κB p65 translocation to the nucleus. Aster tataricus L. f. is a perennial plant with colorful flowers of the Asteraceae family that is commonly found in mountain meadows, hills, and low-lying marshes.1 This plant is distributed naturally throughout the northern part of mainland China and the eastern parts of Siberia, South Korea, and Japan.2 The rhizomes and roots of A. tataricus have been utilized commonly as an expectorant in Eastern Asian countries for thousands of years and used to eliminate phlegm and treat coughs when compounded with other herbal medicines.3 Previous phytochemical studies have reported a variety of secondary metabolites from A. tataricus, such as tetracyclic triterpene ketones, oleanane-type saponins, monoterpenoids, peptides, and flavonoids.4−7 In the last few decades, a substantial number of biological studies have demonstrated potential therapeutic effects, including antioxidant, antitussive, antibacterial, antidepressant, and antiinflammatory properties, of different extracts and chemical constituents of A. tataricus.6,8,9 Inflammation is an important physiological cellular response activated by the invasion of foreign pathogens and harmful stimuli, and it is recognized as a mechanism of innate immunity that helps to repair injured tissues and remove irritants and damaged cells.10 However, unregulated inflam© XXXX American Chemical Society and American Society of Pharmacognosy

mation causes pathological changes in the human body and contributes to serious illnesses, including allergy, atherosclerosis, cancer, and ischemic heart disease.11 The process of inflammation entails different molecular mediators, the local immune systems, and the vascular system within the injured tissues. Macrophages, a type of leukocyte, play an essential role in stimulating the immune system and initiating inflammatory responses via activating cascades of inflammatory mediators, such as those for tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6), as well as nitric oxide (NO) and prostaglandin E2 (PGE2).12 NO is a gaseous biological mediator that is primarily derived from inducible nitric oxide synthase (iNOS), and this physiologically important molecule exhibits various biological functions, such as vasodilation, which recruits leukocytes to the tissues by relaxing and reducing smooth muscle cells and platelet aggregation.13 However, abnormal production of NO leads to the formation of superoxide anions (ONOO−), DNA damage, and cell death.14 Received: October 15, 2018

A

DOI: 10.1021/acs.jnatprod.8b00856 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) are pivotal signaling pathways that activate inflammatory processes involved in the releases of proinflammatory mediators.13 The NF-κB complex, which consists of RelA (p65), RelB (p50), and an inhibitor of NF-κB (IκB) subunits, is inactive in the cytoplasm; NF-κB released by the degradation of IκB is translocated into the nucleus, and the transcription factor is activated to initiate target gene

expression.15,16 MAPKs are a class of serine or threonine kinases and are involved in the biological cascade regulating embryogenesis, cell differentiation, proliferation, and apoptosis.17 The activation of MAPKs is associated with the phosphorylation of MAPKs, including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 through upstream MAPK kinases (MEKs), such as MEK1/2, MKK4/7, and MKK3/6.18 Therefore, controlling the activaB


Journal of Natural Products Table 1. 1H and

13

Article

C NMR Spectroscopic Data for Compounds 1−4 1 δC, type

2

a,b

pos. aglycone

3-O-inner

3-O-term

28-Oinner

3

δC, type

a,b

δHa,c

1

44.2

CH2

2 3 4 5 6

69.8 90.3 39.0 56.2 18.8

CH CH C CH CH2

7 8 9 10 11 12 13 14 15

33.6 40.2 48.6 37.3 24.2 123.5 144.2 42.6 28.4

CH2 C CH C CH2 CH C C CH2

16

23.7

CH2

17 18

47.5 42.3

C CH

19

46.4

CH2

20 21

31.2 34.3

C CH2

22

33.0

CH2

23 24 25 26 27 28 29 30 Glc-1 2 3 4 5 6

30.2 19.2 16.9 17.9 26.5 176.9 33.5 24.2 106.7 75.6 78.3 72.5 76.9 70.9

CH3 CH3 CH3 CH3 CH3 C CH3 CH3 CH CH CH CH CH CH2

Ara-1 2

105.3 72.9

CH CH

3 4 5

74.5 69.4 66.8

CH CH CH2

Xyl I-1

93.8

2 3 4

77.8 76.9 69.3

1.25, m 2.26, d (13.5) 4.64, br s 3.34, d (3.1) 0.89, 1.50, 1.73, 1.59,

s m m m

1.57, m 1.89, m 5.42, br s

1.28, m 2.04, os 1.74, m 2.05, m 3.16, dd (3.3, 13.7) 1.25, m 1.76, m 1.18, m 1.36, m 1.70, m 2.03, 1.32, 1.34, 1.40, 1.04, 1.21,

m s s s s s

0.93, 1.03, 4.86, 3.99, 4.20, 4.21, 4.01, 4.02, 4.85,

s s d (7.7) os os os os os d (9.2)

δC, type

4

b ,b

δHa,c

44.1

CH2

69.8 90.2 38.9 56.1 18.7

CH CH C CH CH2

33.5 40.3 48.6 37.2 24.0 123.3 144.4 42.6 28.3


23.7

CH2

47.5 42.1

C CH

46.7

CH2

31.0 34.3

C CH2

32.8

CH2

30.1 19.0 16.8 17.7 26.2 176.9 33.3 24.1 106.0 75.5 78.8 72.4 76.8 70.0


1.27, m 2.28, d (13.1) 4.65, br s 3.35, d (3.1) 0.95, 1.59, 1.70, 1.57,

m m m m

1.62, m 1.91, m 5.43, br s

1.36, m 2.12, m 1.95, m 2.12, m 3.20, dd (3.8, 13.6) 1.29, m 1.78, m 1.20, m 1.40, m 1.76, m 2.06, 1.32, 1.37, 1.42, 1.11, 1.26,

m s s s s s

0.92, 0.99, 4.86, 3.99, 4.19, 4.19, 4.02, 4.00, 4.84,


105.3 72.8

CH CH

74.4 69.4 66.8

CH CH CH2

CH

4.78, d (6.8) 4.44, dd (6.8, 8.3) 4.18, os 4.31, os 3.76, d (10.7) 4.29, os 6.58, d (5.5)

95.6

CH

4.77, d (6.7) 4.43, dd (6.7, 8.5) 4.15, os 4.30, os 3.76, d (10.7) 4.28, os 6.26, d (5.4)

CH CH CH

4.33, os 4.52, os 4.31, os

77.7 76.3 70.8

CH CH CH

4.29, os 4.28, os 4.25,d (8.0) C

δC , type

a ,b

δHa,c

44.1

CH2

69.7 90.1 38.8 56.1 18.7

CH CH C CH CH2

33.4 40.2 48.5 37.1 23.8 123.3 144.3 42.6 28.6


23.5

CH2

47.5 42.1

C CH

46.6

CH2

31.0 34.3

C CH2

32.8

CH2

30.1 19.0 16.8 17.7 26.2 176.9 33.3 24.1 108.0 75.6 78.7 72.4 76.9 70.8


105.9 72.9

CH CH

74.8 69.6 67.0

CH CH CH2

95.6 76.4 78.6 71.2

1.27, m 2.27, d (13.3) 4.62, br s 3.34, d (3.1)

δHa,c

44.2

CH2

69.9 90.2 38.9 56.1 18.7

CH CH C CH CH2

33.7 40.4 47.7 37.3 24.2 123.3 144.7 42.5 36.2


74.5

CH

49.5 41.5

C CH

47.4

CH2

31.1 36.2

C CH2

32.5

CH2

30.0 19.0 16.9 17.8 27.4 176.6 33.4 24.8 106.1 75.5 78.8 72.4 76.8 70.1


105.3 72.8

CH CH

74.5 69.4 66.8

CH CH CH2

CH

4.76, d (6.7) 4.42, dd (6.7, 8.4) 4.15, os 4.29, os 3.74, d (10.9) 4.26, os 6.12, d (5.5)

96.6

CH

1.03, s 1.12, s 4.89, d (7.8) 4.01, os 4.18, os 4.00, os 4.12, os 4.03, os 4.86, dd (1.9, 10.4) 4.79 (6.8) 4.45, dd (6.8, 8.5) 4.18, os 4.31, os 3.76, d (10.6) 4.30, os 6.24 (6.9)

CH CH CH

4.33, os 4.22, os 4.18, os

73.4 78.3 71.1

CH CH CH

4.16, os 4.24, os 4.22, os

0.85, 1.57, 1.73, 1.59,

m m m m

1.62, m 1.91, m 5.42, br s

1.51, m 2.04, m 1.98, m 2.14, m 3.16, dd (3.8, 13.7) 1.27, m 1.79, m 1.18, m 1.37, m 1.80, m 2.00, 1.29, 1.30, 1.39, 1.09, 1.27,

m s s s s s

0.90, 0.92, 4.84, 3.97, 4.19, 4.20, 4.06, 3.99, 4.79,


1.33, m 2.34, d (14.6) 4.69, br s 3.38, d (3.3) 0.95, 1.47, 1.51, 1.61,

m m m m

1.77, os 2.05, m 5.61, br s

1.78, os 2.50, dd (3.1, 14.6) 5.29, br s

3.58, dd (4.1, 14.2) 1.40, m 2.82, d (13.5) 1.33, m 2.43, m 2.25, dt (6.5, 14.6) 2.43, m 1.29, s 1.37, s 1.49, s 1.14, s 1.82, s



Article

Table 1. continued 1 δC, type

2

a,b

pos. 5

65.0

δHa,c

δC, type

CH2

3.99, os

28-O-term

28-O-term

δHa,c

67.1

CH2

28-O-term

Rha I-1

101.7

CH

5.70, s

102.0

CH

2 3 4 5 6 Xyl II-1 2 3 4 5

71.8 82.7 78.6 68.5 19.1 105.6 75.9 79.0 71.6 67.6

CH CH CH CH CH3 CH CH CH CH CH2

71.6 82.5 78.9 68.9 19.3 105.7 75.9 78.9 71.5 67.5


Api-1 2 3 4

112.0 77.8 80.0 74.9

CH CH C CH2

4.76, s 4.40, br s 4.56, os 4.23, os 1.73, d (6.2) 5.33, d (7.7) 3.99, os 4.20, os 4.22, os 3.46, t (10.2) 4.23, dd (2.6, 10.2) 6.01, d (4.6) 4.78, d (4.6)

111.9 78.0 79.9 74.8

CH CH C CH2

5 Rha II-1 2 3 4 5 6

64.6 101.0 72.7 73.1 74.1 70.9 18.8

CH2 CH CH CH CH CH CH3

64.7

CH2

4.17, os 4.32, os 4.06, d (10.3) 5.66, s 4.70, s 4.55, os 4.22, os 4.54, s 1.60, d (6.1)

δC, type

4

b ,b

4.40, os 28-Oinner

3

a,b

δC , type

a ,b

δHa,c

3.85, dd (8.0, 11.4) 4.40, dd (4.7, 11.4) 6.11, s

67.6

CH2

101.8

CH

5.00, br s 4.60, br s 4.55, t (9.3) 4.44, d (6.8) 1.81, d (6.1) 5.36, d (7.7) 3.99, os 4.19, os 4.21, os 3.49, t (10.4) 4.24, dd (4.2, 10.4) 6.05, d (4.5) 4.80, d (4.5)

72.0 72.9 85.8 68.6 18.9 105.7 75.9 78.9 71.5 67.5


3.79, dd (8.7, 11.3) 4.32, dd (7.0, 11.3) 6.35, s

67.9

δHa,c CH2

3.83, dd (4.7, 11.2) 4.38, dd (4.3, 11.6)

4.80, br s 4.66, br s 4.35, d (9.4) 4.46, os 1.81, d (6.0) 5.04, d (7.3) 4.05, os 4.19, os 4.16, os 3.53, t (10.8) 4.25, dd (3.8, 10.8)

4.18, os 4.58, d (10.4) 4.07, d (10.4)

a

Measured in pyridine-d5. b150 MHz. c600 MHz. Coupling constants (in parentheses) are in Hz. Assignments were made based on HSQC, HMBC, COSY, TOCSY, and ROESY experiments; s: singlet, d: doublet, t: triplet, m: multiplet, os: overlapped signals.

1.04 (3H, s), 1.21 (3H, s), 0.93 (3H, s), and 1.03 (3H, s) and an olefinic proton signal at δH 5.42 (1H, br s), together with resonances at δC 123.5, 144.2, and 176.9 in the 13C NMR spectrum, were indicative of an oleanane-type aglycone (Table 1). Moreover, the HSQC, COSY, and HMBC experiments supported two oxymethine groups being located at C-2 and C3 of the aglycone, respectively (Figure 1). In the ROESY spectrum, the correlations at δH 3.34 (H-3)/δH 0.89 (H-5) and δH 3.34 (H-3)/δH 1.32 (H-23) and the correlations at δH 4.64 (H-2)/δH 3.34 (H-3) and δH 4.64 (H-2)/δH 0.89 (H-5) indicated that H-2 and H-3 were α-oriented (Figure 2). Based on detailed analysis of the NMR spectroscopic data, the aglycone of 1 was identified as augustic acid.21 Additionally, the 1H NMR spectrum revealed distinctive peaks for seven anomeric protons at δH 4.86 (1H, d, J = 7.7 Hz), 4.78 (1H, d, J = 6.8 Hz), 6.58 (1H, d, J = 5.5 Hz), 5.70 (1H, s), 5.33 (1H, d, J = 7.7 Hz), 6.01 (1H, d, J = 4.6 Hz), and 5.66 (1H, s), which gave correlations in the HSQC spectrum with seven anomeric carbons at δC 106.7, 105.3, 93.8, 101.7, 105.6, 112.0, and 101.0, respectively. The identification of individual monosaccharides, including a β-glucopyranosyl (Glc), a αarabinopyranosyl (Ara), two β-xylopyranosyl (Xyl I, Xyl II), two α-rhamnopyranosyl (Rha I, Rha II), and a β-apiofuranosyl (Api) was confirmed by evaluation of the key HSQC, COSY, TOCSY, HMBC, and ROESY data (Table 1). The βconfiguration of the glucopyranosyl, xylopyranosyl, and apiofuranosyl units and the α-configuration of the arabinopyr-

tion of NF-κB and MAPKs may be a promising target for the treatment of inflammatory diseases. This work describes the separation and structural characterization of nine aster saponins (1−9) from A. tataricus. Those compounds were investigated for potential anti-inflammatory activity on the lipopolysaccharide (LPS)-activated NF-κB and MAPK signaling pathway in RAW264.7 cells.

■

RESULTS AND DISCUSSION In the current investigation of secondary metabolites with antiinflammatory properties from natural product sources, four new aster saponins (1−4) and five known analogues (5−9) were isolated from the methanol extract of the underground parts of A. tataricus. The known compounds were identified as aster saponin A (5),4 aster saponin B (6),19 aster saponin F (7),4 aster saponin G (8),20 and 3-O-α-L-arabinopyranosyl(1→6)-β-D-trihydroxyolean-12-en-28-oic acid (9),20 by comparison of their physical and spectroscopic data (IR, NMR, and MS) with reported values. These known compounds (5−9) were isolated from A. tataricus in previous studies. Compound 1 was purified as a pale yellowish, amorphous powder, and its molecular formula was deduced to be C68H110O33 based on its deprotonated ion peak [M − H]− at m/z 1453.6857 (calcd for 1453.6857) in the HRESIMS (negative-ion mode). In the 1H NMR spectrum, seven tertiary methyl signals at δH 1.32 (3H, s), 1.34 (3H, s), 1.40 (3H, s), D



Article

Figure 1. Selected key COSY (bold lines) and HMBC (H→C) correlations of new compounds 1−4.

Table 2. Inhibitory Activities of Aster Saponins 1−9 on LPS-Stimulated NO Productiona compound

IC50 (μM)

1 2 3 4 5 6 7 8 9 dexamethasonec

42.1 (32.5−54.5) >50b >50b >50b 2.7 (2.1−3.4) 1.2 (1.1−1.34) 2.1 (1.2−3.8) 13.3 (9.8−18.1) >50b 0.01 (0.01−0.02)

a

Figure 2. Selected key ROESY correlations (red dotted double arrows and blue dotted double arrows) of aglycones of new compounds 1−3.

Data are shown as IC50 values and 95% confidence intervals (95% CIs). bNo cytotoxicity was observed at the IC50 concentration. c Dexamethasone was used as the positive control.

anosyl unit were determined by the relatively large coupling constants (J = 4.5−7.9 Hz) of their anomeric protons. The αconfigurations of the rhamnopyranosyl moieties were identified by the multiplicity of their anomeric proton signals. The

HMBC correlation at δH 3.34 (Agly H-3)/δC 106.7 (Glc C-1) and the reverse correlation at δH 4.86 (Glc H-1)/δC 90.3 (Agly C-3) clearly indicated the glucopyranosyl unit to be located at C-3 of the aglycone (Figure 1). Moreover, the linkage of the E



Article

Figure 3. Effects of compound 6 on LPS-stimulated iNOS and COX-2 proteins in RAW 264.7 cells. Cells were preincubated with 6 at 1 and 5 μM for 1 h before induction with LPS (0.1 μg/mL) for 16 h. The expression levels of iNOS, COX-2, and β-actin were analyzed using Western blotting. Dexamethasone (Dexa) was used as a positive control, and the relative intensity was calculated as the ratio of the intensities of the iNOS and COX2 bands to the intensity of the β-actin band using ImageJ software. Data are shown as the means ± SEM of three independent experiments. Asterisks display a significant difference from LPS alone (*p < 0.05).

COSY, HSQC, TOCSY, ROESY, and HMBC) of 2 with those of 1 revealed that the terminal L-Rha (Rha II) moiety of the oligosaccharidic chain linked to C-4 of Xyl I was absent in 2. Accordingly, 2 was elucidated to be 3-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl-2β,3β-dihydroxyolean-12-en-28-oic acid-28-O-β-D-apiofuranosyl-(1→3)-[β-D-xylopyranosyl-(1→ 4)]-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside, and it was named aster saponin A2. Compound 3 displayed two pseudomolecular ion peaks [M − H]− at m/z 1175.5851 (calcd 1175.5855) and [M + HCOO]− at m/z 1221.5902 (calcd 1221.5910) in the HRESIMS (negative-ion mode), consistent with the molecular formula C57H92O25. The 1H NMR spectrum of 3 showed five anomeric proton signals at δH 4.84 (1H, d, J = 7.7 Hz), 4.76 (1H, d, J = 6.7 Hz), 6.12 (1H, d, J = 5.5 Hz), 6.35 (1H, s), and 5.04 (1H, d, J = 7.3 Hz), seven tertiary methyls, and one olefinic proton. Comparison of the NMR data of 3 with those of the known compound 8 revealed that 3 was also an oleanane-type saponin with the same sugar moieties and sugar sequences.20 The only difference was the absence of a hydroxy group at C-16 of the aglycone. The aglycone of 3 was further identified as augustic acid, as in 1 and 2 (Table 1). Thus, the structure of 3 was determined to be 3-O-α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl-2β,3β-dihydroxyolean-12-en-28-oic acid-28-O-β- D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl(1→2)-β-D-xylopyranoside and given the common name aster saponin G2. Compound 4 was obtained as a pale yellowish, amorphous powder with a molecular formula of C46H74O18, as determined from its sodium adduct molecular ion peak [M + Na]+ at m/z 937.4793 (calcd 937.4767) in the HRESIMS (positive-ion mode). Comparison of the NMR data of 4 with those of 3 indicated that 4 contains a hydroxy group at C-16 of the aglycone. In the 1H NMR spectrum, H-16 resonated farther

arabinopyranosyl unit at C-6 of the glucopyranosyl unit was confirmed by the HMBC correlation at δH 4.85 (Glc H-6)/δC 105.3 (Ara C-1) and the reverse correlation at δH 4.78 (Ara H1)/δC 70.9 (Glc C-6) (Figure 1). The linkage of Xyl I at C-28 of the aglycone was revealed by the HMBC correlation between the anomeric proton at δH 6.58 (Xyl I H-1) and Agly (C-28) at δC 176.9 (Figure 1). Furthermore, the sequence of the oligosaccharide chain linked at the C-28 position was elucidated by the HMBC correlations at δH 4.33 (Xyl I H-2)/ δC 101.7 (Rha I C-1) and δH 5.70 (Rha I H-1)/δC 77.8 (Xyl I C-2), δH 4.52 (Xyl I H-3)/δC 101.0 (Rha II C-1) and δH 5.66 (Rha II H-1)/δC 76.9 (Xyl I C-3), δH 4.40 (Rha I H-3)/δC 112.0 (Api C-1) and δH 6.01 (Api H-1)/δC 82.7 (Rha I C-3), and δH 4.56 (Rha I H-4)/δC 105.6 (Xyl II C-1) and δH 5.33 (Xyl II H-1)/δC 78.6 (Rha I C-4) (Figure 1). Acid hydrolysis of 1 with 1 N HCl afforded D-glucose, L-arabinose, D-xylose, Lrhamnose, and D-apiose followed by GC analysis. Consequently, the structure of 1 was determined to be 3-O-α-Larabinopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β-dihydroxyolean-12-en-28-oic acid-28-O-β-D-apiofuranosyl-(1→3)-[β-Dxylopyranosyl-(1→4)]-α- L -rhamnopyranosyl-(1→2)-[α- L rhamnopyranosyl-(1→3)]-β-D-xylopyranoside, and it was named aster saponin C2. The molecular formula of compound 2, C62H100O29, was determined by two ion peaks [M − H]− at m/z 1307.6275 (calcd 1307.6278) and [M + HCOO]− at m/z 1353.6329 (calcd 1353.6327) in the HRESIMS (negative-ion mode). The NMR spectra indicated that 2 possesses an oleanane-type skeleton, as in 1 (Table 1). The key HSQC correlations at δH 4.86 (1H, d, J = 7.9 Hz)/δC 106.0, δH 4.77 (1H, d, J = 6.7 Hz)/δC 105.3, δH 6.26 (1H, d, J = 5.4 Hz)/δC 95.6, δH 6.11 (1H, s)/δC 102.0, δH 5.36 (1H, d, J = 7.7 Hz)/δC 105.7, and δH 6.05 (1H, d, J = 4.5 Hz)/δC 111.9 indicated the presence of six sugar moieties. Comparison of the NMR data (1H, 13C, F



Article

Figure 4. Effects of compound 6 on the LPS-activated phosphorylation and degradation of IκB and the nuclear translocation of NF-κB in RAW 264.7 cells. Cells were preincubated with compound 6 at 1 and 5 μM for 1 h and then treated with LPS (0.1 μg/mL) for 1.5 h. Cytosolic and nuclear extracts were subjected to Western blotting to analyze the IκB (A) and p65 (B) protein levels, respectively. Dexamethasone (Dexa) was used as a positive control. The protein levels of β-actin and lamin B were used as loading controls, and the band intensities relative to those of the loading controls were quantified using ImageJ software. Data are presented as the means ± SEM of three independent experiments. A significant difference was indicated as asterisks (*p < 0.05) between LPS alone and cotreatment groups.

cancer, and atherosclerosis.23 Thus, the regulation of NO secretion is an important pharmacological strategy for drug development. To investigate the anti-inflammatory activities of the isolated compounds, the inhibitory effects of aster saponins 1−9 were tested. Compounds 1 and 5−8 showed more than 50% inhibition against LPS-induced NO release in RAW264.7 cells at 50 μM (Table 2), and the half-maximal inhibitory concentrations (IC50 values) of these compounds were calculated via nonlinear regression analysis with a sigmoidal dose−response curve (Figure S1, Supporting Information). IC50 values were derived for compounds 1 and 5−8, and the preliminary structure−activity relationship (SAR) between the aster saponins and their NO inhibitory effects was examined to determine the active moieties responsible for the antiinflammatory effects (Figure S2, Supporting Information). The inhibitory activities were found to decrease in the order 6 > 7 > 5 > 8 > 1, and compound 6 (IC50: 1.2 μM), which contains OH-16 and apiofuranosyl- and xylopyranosyl-terminal sugars at the 28-COOH group, showed the most potent antiinflammatory effect. Based on the correlations between the chemical structures of the aster saponins and their inhibitory effects, aster saponin B (6) was shown to exhibit more potent potential anti-

downfield (δH 5.29), which suggested that the hydroxy group might be connected to C-16. This was confirmed by the 1 H−1H COSY correlations between δH 5.29 (H-16) and 1.78 (H-15a)/δH 2.50 (H-15b) and the HMBC correlations between δH 5.29 (H-16) and δC 42.5 (C-14) and between δH 1.78 (H-15a), 3.58 (H-18), 2.25 (H-22a)/2.43 (H-22b) and δC 74.5 (C-16) (Figure 1). The α-orientation of the hydroxy group at C-16 was determined by its multiplicity.19 The 1H NMR spectrum of 4 displayed signals for three anomeric protons at δH 4.89 (1H, d, J = 7.8 Hz), 4.79 (1H, d, J = 6.8 Hz), and 6.24 (1H, d, J = 6.9 Hz) (Table 1). The sugar chain connected to the C-3 of the aglycone was identified as the same one as in 3 by NMR analysis. Moreover, the HMBC correlation at δH 6.24 (Xyl I H-1)/δC 176.6 (C-28) indicated that a xylopyranosyl unit was connected to C-28 of the aglycone (Figure 1). Therefore, 4 was elucidated as 3-O-α-Larabinopyranosyl-(1→6)-β-D-glucopyranosyl-2β,3β,16α-trihydroxyolean-12-en-28-oic acid-28-O-β-D-xylopyranoside, and it was named aster saponin H. NO is a common endogenous molecule that is involved in physiological and pathological events. 22 However, the abnormal overproduction of NO has been implicated in the development of various illnesses, such as circulatory shock, G



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Figure 5. Effects of compound 6 on LPS-stimulated MAP kinase activation in RAW 264.7 cells. Cells were pretreated with compound 6 and a positive control, dexamethasone, for 1 h and incubated with LPS (0.1 μg/mL) for 0.5 h. Western blotting was performed to measure phosphorylated JNK, ERK, and p38 in whole cell lysates. The loading proteins were normalized to the total JNK, ERK, and p38 protein levels. Three independent experiments were quantitatively evaluated using ImageJ software. The results are shown as the means ± SEM of three independent experiments. A significant difference was indicated as asterisks (*p < 0.05) between LPS alone and cotreatment groups.

examined using Western blot analysis. The group treated with 6 showed significant suppression of the phosphorylation and degradation of IκB in the cytoplasm (Figure 4A) and that the releases of p65 into the nucleus was blocked (Figure 4B) compared with the findings in the group treated with LPS alone. Therefore, the anti-inflammatory activity of 6 was exerted by inhibiting NF-κB activity in LPS-treated macrophages. In addition to the NF-κB signaling pathway, the MAPK mechanism, including JNK, ERK, and p38, contributes to the inflammatory response in macrophages activated by LPS.27 Thus, Western blot analysis was used to assess whether MAPKs are associated with the anti-inflammatory effect of 6. As shown in Figure 5, the enhanced phosphorylation of JNK, ERK, and p38 MAPKs caused by LPS was attenuated by 6 in RAW264.7 cells with no change in the total JNK, ERK, and p38 MAPK protein levels. In particular, 6 most significantly inhibited the phosphorylation of JNK at 1 μM, indicating its potential as a JNK inhibitor. JNK and p38 have been reported to be involved in the development of the inflammatory response by upregulating the expression of inflammatory mediators, such as iNOS and COX-2.28,29 Therefore, these results suggest that the MAPK signaling pathway may be an important target in the anti-inflammatory activity of 6.

inflammatory activity than the other compounds isolated from A. tataricus. Accordingly, the mechanism of the activity of aster saponin B on LPS-activated inflammatory molecules in RAW264.7 macrophages was examined. The inflammatory response is mainly caused by the secretion of inflammatory mediators, including NO, iNOS, COX-2, PGE2, IL-6, and TNF-α,24 which are closely related to acute or chronic inflammatory diseases.25 Thus, 6 was investigated by Western blotting and ELISA analysis to determine whether it inhibited iNOS, COX-2, IL-6, and TNF-α production in LPS-treated RAW264.7 cells. As shown in Figure 3, 6 suppressed LPS-enhanced iNOS and COX-2 enzyme expression in a dose-dependent manner, resulting in the inhibition of pro-inflammatory mediators such as NO and PGE2. Additionally, the LPS-activated pro-inflammatory cytokine IL-6 was suppressed by 6, but no effect on TNF-α release was observed (Figure S3, Supporting Information). NF-κB is a key transcription factor for the regulation of proinflammatory mediators, and NF-κB subunits p50 and p65 are bound to IκB in the inactive form.13,14 The phosphorylation and degradation of IκB results in the release and translocation of NF-κB into the nucleus to initiate pro-inflammatory gene expression, including the expression of iNOS and COX-2.26 To investigate whether 6 affects the NF-κB signaling cascades, the activation of IκB and p65 in cytosolic and nuclear extracts was H



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Statistical Analysis. The IC50 values and 95% confidence intervals were calculated using nonlinear regression analysis using Prism 5 software (GraphPad software, San Diego, CA, USA). Oneway ANOVA followed by Dunnett’s test was used to show significant differences between the treatment groups and the LPS alone group. The results were considered statistically significant at p < 0.05.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a JASCO P-2000 polarimeter. The FT-IR spectra were collected on a Nicolet 380 FT-IR spectrometer using KBr pellets. 1D and 2D NMR spectra were recorded using a JEOL ECA 600 spectrometer (JEOL, Japan); Me4Si was used as an internal standard. HRESIMS was performed using an Agilent 6530 accuratemass quadrupole time-of-flight liquid chromatography/mass spectrometer (Q-TOF LC/MS). Column chromatography (CC) separations were performed using silica gel (SiO2; 70−230, 230− 400 μm particle size; Fuji Silysia Chemical Ltd., Japan). Thin-layer chromatography (TLC) separations were performed using precoated silica gel 60 F254 and reverse-phase (RP)-18 F254S plates (Merck, Darmstadt, Germany). Biological Material. The rhizomes and roots of Aster tataricus (3.3 kg) were collected on December 9, 2016, in Daejeon, Korea, and the samples were authenticated by one of the authors (Y.H.K.). The age of the rhizomes and roots was one year old. A deposition of the plant’s voucher specimen (CNU 18115) was conserved in the Laboratory of Natural Products Research, College of Pharmacy, Chungnam National University. Extraction and Isolation. The crushed samples (3.3 kg) of A. tataricus were extracted with methanol (8.0 L × 4 times) by refluxing. The methanol extract (1.6 kg) was suspended in H2O (1.2 L) and partitioned with n-BuOH (1.2 L × 4 times) to generate the n-BuOH (100.0 g) fraction. The n-BuOH layer (100.0 g) was separated using silica gel (5 × 30 cm) CC with a gradient solvent system of CHCl3− MeOH−H2O (10:1:0, 5:1:0.1, and 2:1:0.1) to produce four fractions (Fr. 3A−3D). Fraction 3B (19.0 g) yielded 12 subfractions (Fr. 3B1− 3B12) by silica gel (4 × 15 cm) CC using CHCl3−MeOH−H2O (7:1:0.1, 5:1:0.1, and 2:1:0.1) as mobile phase. Fraction 3B5 (1.7 g) was chromatographed by silica gel (2 × 80 cm) CC using a gradient mixture of CHCl3−MeOH−H2O−formic acid (1.5:1:0.1:0.1) to obtain 4 (9.0 mg), 5 (70.0 mg), and 8 (80.0 mg). Separation of fraction 3B7 (445.9 mg) led to the isolation of 3 (6.0 mg), 6 (70.0 mg), 7 (100.0 mg), and 9 (13.0 mg) by silica gel (1 × 80 cm) CC using a gradient solvent system of CHCl3−MeOH−H2O−formic acid (1.7:1:0.1:0.1; 2.0). The purification of fraction 3B12 (543.6 mg) gave 1 (50.0 mg) and 2 (14.0 mg) using silica gel (1 × 80 cm) CC with CHCl3−MeOH−H2O−formic acid (3.5:1:0.1:0.1; 1.0 L) as mobile phase. Aster saponin C2 (1): pale yellowish, amorphous powder; [α]25 D −46.6 (c 0.1, MeOH); IR (KBr) νmax 3390, 2900, 1750, 1650, 1350, 1250, 1050 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 1453.6857 [M − H]− (calcd for C68H109O33, 1453.6857). Aster saponin A2 (2): pale yellowish, amorphous powder; [α]25 D −24.6 (c 0.1, MeOH); IR (KBr) νmax 3400, 2950, 1760, 1640, 1350, 1250, 1050 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 1307.6275 [M − H]− (calcd for C62H99O29, 1307.6278) and 1353.6329 [M + HCOO]− (calcd for C63H101O31, 1353.6327). Aster saponin G2 (3): white, amorphous powder; [α]25 D −20.4 (c 0.1, MeOH); IR (KBr) νmax 3380, 2900, 1750, 1650, 1350, 1265, 1050 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 1175.5851 [M − H]− (calcd for C57H91O25, 1175.5855) and 1221.5902 [M + HCOO]− (calcd for C58H93O27, 1221.5910). Aster saponin H (4): pale yellowish, amorphous powder; [α]25 D −12.6 (c 0.1, MeOH); IR (KBr) νmax 3425, 2950, 1765, 1665, 1400, 1225, 1045 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 937.4793 [M + Na]+ (calcd for C46H74NaO18, 937.4767). Acid Hydrolysis. Acid hydrolysis and identification of the monosaccharides were based on the same method as described in a previous paper from this laboratory.32 Determination of Secreted NO, PGE2, IL-6, and TNF-α Levels and Cell Viability. The bioassay experiments were performed using a previously described method.30,31 Western Blot Analysis. Western blot analysis was performed according to a previously described method.30

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00856. 1D and 2D NMR, HRESIMS, and IR data for compounds 1−4, inhibition percentage curves figure, graphical depiction of structure−activity relationships, and PGE2, TNF-α, and IL-6 production figure (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M. C. Rho). Tel: +82-63-5705230. Fax: +82-63-570-5239. *E-mail: [email protected] (Y. H. Kim). Tel: +82-42-821-5933. Fax: +82-42-823-6566. *E-mail: [email protected] (S. Y. Yang). Tel: +82-42-8217321. Fax: +82-42-823-6566. ORCID

Hyun-Jae Jang: 0000-0002-4383-4465 Seung Woong Lee: 0000-0003-1025-7363 Young Ho Kim: 0000-0002-5212-7543 Seo Young Yang: 0000-0002-5248-1374 Author Contributions #

X. D. Su and H. J. Jang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2018R1A6A3A11047338) and by a grant from the KRIBB Research Initiative Program (KGM5241911).

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REFERENCES

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Anti-inflammatory Potential of Saponins from Aster

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