Anti-inflammatory Lathyrane Diterpenoids from Euphorbia lathyris

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

Anti-inflammatory Lathyrane Diterpenoids from Euphorbia lathyris Cui-Yun Zhang,†,⊥ Yan-Li Wu,†,⊥ Peng Zhang,† Zhuang-Zhuang Chen,† Hua Li,*,†,‡ and Li-Xia Chen*,† †

Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China

‡

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S Supporting Information *

ABSTRACT: Six new lathyrane diterpenoids (1−6) and 10 known analogues (7−16), were separated from the seeds of Euphorbia lathyris. The absolute configuration of 1 was determined by X-ray crystallography, and the C-2′ configuration of 5 was elucidated by comparing experimental and calculated ECD data. These compounds were studied for their inhibition against nitric oxide (NO) generation induced by lipopolysaccharide in RAW264.7 macrophage cells. Compounds 1−3, 7, 9, 11, 13, 14, and 16 displayed inhibitory effects on NO production, with IC50 values of 2.6−26.0 μM. The new compound 1 (IC50 3.0 ± 1.1 μM), with no obvious cytotoxicity, was selected for further experiments. The production of cytokines such as IL-6 and IL-1β, as well as the protein expression of iNOS, NF-κB, and phosphorylated IκBα, was reduced by 1 dose-dependently. These results suggested that lathyrane diterpenoids may be used as potential antiinflammatory agents and are worth being further researched. physiological mechanisms.12 However, NO and cytokines such as IL-1β and IL-6 are released when the immune system is chronically or overly activated,13 which are proven to be closely related to inflammation. Pharmacological investigations have shown that inflammation is connected with the overproduction of NO. In order to find more potentially anti-inflammatory diterpenoids from E. lathyris, an extract of the seeds of this species was investigated and afforded 16 lathyrane-type diterpenoids including six new (1−6) and 10 known analogues (7−16). Their structures were determined by spectroscopic data, comparison of the experimental and calculated electronic circular dichroism (ECD) spectra, and X-ray crystallography. These compounds were tested for their inhibitory effects against NO production triggered by lipopolysaccharide (LPS) in RAW264.7 macrophage cells, and their structure−activity relationship is discussed. Compound 1 was chosen to study its possible anti-inflammatory mechanism.

Euphorbia (Euphorbiaceae), a large genus including approximately 2160 species, is broadly distributed in both tropical and temperate regions. The genus Euphorbia is also an abundant source of special diterpenoids with various macrocyclic and polycyclic skeletons.1 These characteristic diterpenoids including jatrophane, ningenane, daphnane, tigliane, and lathyrane are characteristic constituents for the Euphorbiaceae and Thymelaeaceae families, which are therefore considered to be important taxonomic markers. Some of these diterpenoids possess various biological activities, such as anticancer, multidrug resistance reversal, antiviral, and anti-inflammatory effects.2 The seeds of Euphorbia lathyris (caper spurge) are utilized as traditional Chinese medicine for treating terminal schistosomiasis, ascities, hydropsy, and snakebites.3 Previous studies on the chemical and biological properties of this plant have resulted in the separation of a series of diterpenoids with a lathyrane skeleton named Euphorbia factors L1−L28,4,5 as powerful multidrug-resistance (MDR) modulators through inhibiting P-glycoprotein.6−8 In recent years, some diterpenoids extracted from this plant, such as Euphorbia factors L1− L11, have also been found to possess anti-inflammatory activity by inhibition of NF-κB signaling activation.9,10 Inflammation is the normal response to infection or injury of the body. It involves the activation of the immune system, including the recruitment of immune cells and antibodies, in order to eliminate invasive pathogens, repair damaged tissue, and accelerate wound healing.11 Nitric oxide (NO) is a pivotal signaling molecule and considered to be the regulator of many © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Studies on constituents of the seeds of E. lathyris yielded six new lathyrane diterpenoids (1−6) and 10 known analogues, Euphorbia factors L3 (7),4 L8 (8),14 L11 (9),15 L2 (10),14 L9 (11),16 L19 (12),4 L22 (13),4 L10 (14),17 and L1 (16),18 and Received: July 23, 2018

A

DOI: 10.1021/acs.jnatprod.8b00600 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds (1−16) from Euphorbia lathyris.

correlations from H-5 to C-7, C-4, C-15, C-17, and C-6 and H2-17 to C-5, C-7, and C-6 (Figure 2). The NOESY correlations of H-5 (δH 4.51) with H-3 (5.75), H-2″ (2.12), H-12 (6.40), H-3′ (7.75), H-5′ (7.75), and H-7b (2.70) demonstrated an α-orientation for 5-OH (Figure 3). The absolute configuration of 1 was defined according to the X-ray single-crystal diffraction data (Figure 4). Therefore, the structure of compound 1 was assigned as (2S,3S,4S,5R,9S,11R,15R)-15-acetoxy-3-cinnamoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene. Compound 2 was isolated as a white powder. Its formula, C31H38O8, implying 13 indices of hydrogen deficiency, was determined using HRESIMS (m/z 561.2465 [M + Na]+, calcd for C31H38O8Na+, 561.2464) and 13C NMR data (Table 2). The 1H and 13C NMR spectra of 2 showed the presence of a benzoyl group [δH 8.14 (2H, dd, J = 7.9, 1.3 Hz), 7.62 (1H, m), 7.50 (2H, t, J = 7.9 Hz); δC 166.2, 131.0, 129.9 (×2), 128.6 (×2), 133.2], an exocyclic double bond [δH 5.23 (1H, s), 5.00 (1H, s); δC 144.7, 118.1], an olefinic bond [δH 6.37 (1H, dd, J = 11.4, 0.8 Hz); δC 144.9, 135.0], two acetoxy groups [δH 2.18 (3H, s), 1.96 (3H, s); δC 169.7, 169.8, 22.2, 21.7], and a ketocarbonyl carbon (δC 197.7). The NMR spectra of 2 closely

(−)-(12E,2S,3S,4R,5R,6R,9S,11S,15R)-15-acetoxy-5,6-epoxylathyr-12-en-3-ol-14-one (15)19 (Figure 1). Compound 1, colorless needle crystals, has a formula of C31H38O6 as measured by HRESIMS (m/z 529.2564 [M + Na]+, calcd for C31H38O6Na+, 529.2566) and 13C NMR data, indicating 13 indices of hydrogen deficiency. The 1H and 13C NMR data (Tables 1 and 2) of 1 indicated the presence of a cinnamoyl group [δH 7.54 (2H, m), 7.42 (3H, m), 7.75 (1H, d, J = 15.9 Hz), 6.49 (1H, d, J = 15.9 Hz); δC 167.3, 118.3, 145.4, 134.5, 128.3 (×2), 129.2 (×2), 130.7], an exocyclic double bond [δH 4.89 (1H, br s), 4.67 (1H, br s); δC 148.6, 113.0], an olefinic bond [δH 6.40 (1H, dd, J = 11.4, 1.0 Hz); δC 146.9, 134.3], an acetoxy group [δH 2.12 (3H, s); δC 169.9, 22.3], and a ketocarbonyl carbon (δC 197.3). Its 1H and 13C NMR data were similar to those of the known Euphorbia factor L7a,6 suggesting 1 to be a lathyrane diterpenoid. The main differences in their NMR spectra involved the absence of an acetoxy group at C-17 and a Δ5(6) double bond, whereas an exocyclic olefinic bond [δH 4.89, 4.67 (each 1H, br s, H2-17); δC 148.6 (C-6), 113.0 (C-17)] and an oxygenated methine [δH 4.51 (1H, d, J = 9.8 Hz, H-5); δC 65.7 (C-5)] were present in 1. This observation was corroborated by the HMBC B



Article

Table 1. 1H NMR Data of Compounds 1−6 in CDCl3 (600 MHz, δ in ppm, J in Hz) pos. 1 2 3 4 5 7 8 9 11 12 16 17 18 19 20 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 2″ 2‴

1 1.74 3.52 2.34 5.75 2.70 4.51 2.15 2.70 1.58 2.01 1.17 1.40 6.40 1.01 4.89 4.67 1.17 1.11 1.71 6.49 7.75 7.75 7.42 7.42 7.42 7.54 2.12

dd (14.3, dd (14.3, m t (3.3) m d (9.8) m m m m m dd (11.4, dd (11.4, d (6.7) br s br s m s d (0.9) d (15.9) d (15.9) m m m m m s

2 11.8) 8.4)

8.4) 1.0)

3

4

1.79 3.52 2.38 5.95 2.76 4.80 5.36

dd (14.3, 11.9) dd (14.3, 8.3) m t (3.3) dd (9.4, 3.3) d (9.4) dd (10.9,2.4)

1.71 3.49 2.37 5.85 2.92 6.33 4.24

1.86 2.16 1.27 1.43 6.37 1.01 5.23 5.00 1.12 1.19 1.73

m m m dd (11.4, 8.3) dd (11.4, 0.8) d (6.7) br s br s br s br s d (0.9)

2.07 m 1.27 1.43 6.51 0.96 5.30 5.06 1.19 1.20 1.73

m dd (11.4, 8.3) d (11.4) d (6.8) br s br s br s br s s

8.14 7.50 7.62 7.50 8.14

dd (7.9, 1.3) t (7.9) m t (7.9) dd (7.9, 1.3)

8.05 7.46 7.59 7.46 8.05

d (7.7) t (7.7) m t (7.7) d (7.7)

1.96 s 2.18 s

m dd (14.3, 8.3) m t (3.5) dd (9.3, 3.5) d (9.3) t (6.0)

1.78 s 2.22 s

1.50 3.52 2.07 3.98 2.58 5.98 2.07 2.48 1.50 2.27 1.11 1.42 6.53 1.08 4.42 4.09 1.18 1.04 1.84 2.02

dd (14.1, dd (14.1, m t (3.5) dd (11.2, d (11.2) m m m m m dd (11.4, dd (11.4, d (6.8) d (12.2) d (12.2) s s d (0.9) s

2.01 s

resembled those of Euphorbia factor L2,14 a lathyrane diterpenoid from this plant. The absence of a benzoyl group, as well as the chemical shifts of C-3, C-4, C-6, and C-7 in 2, which were deshielded by approximately ΔδC 1.9, 2.0, 2.7, and 1.5 ppm, respectively, suggested that the substituents at C-5 and C-7 might be different in 2 and Euphorbia factor L2. The HMBC correlations from H-7 to C-5, C-17, and the ester carbonyl carbon C-1″ confirmed that an acetoxy group was linked to C-7, and a hydroxy group should correspondingly be connected to C-5. In the NOESY spectrum, H-5 (δH 4.80) correlated to H-2″ (1.96), H-12 (6.37), and H-3 (5.95), and H-7 (δH 5.36) correlated to H-9 (1.27), H-4 (2.76), and H-12 (6.37), demonstrating an α-orientation for 5-OH and a βorientation for the 7-acetoxy group. The absolute configuration of 2 was defined based on biosynthetic considerations6 and comparison with that of 1. Consequently, the structure of compound 2 was established as (2S,3S,4S,5R,7R,9S,11R,15R)7,15-diacetoxy-3-benzoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene. The formula of 3 was determined as C31H38O8 via the HRESIMS ion at m/z 561.2479 [M + Na]+ (calcd for C31H38O8Na+, 561.2464) and 13C NMR data. The 1H and 13C NMR data (Tables 1 and 2) of 3 indicated characteristic signals for a lathyrane diterpenoid skeleton. The highly similar 13 C NMR data of 2 and 3 revealed that their structural differences were restricted to the interchange of substituents at C-5 and C-7. The acetoxy group was attached to C-5 based on the HMBC correlations from H-5 (δH 6.33) to the ester carbonyl carbon C-1″ (δC 170.6), C-6 (δC 146.1), C-17 (δC 118.7), C-15 (δC 92.3), C-7 (δC 78.5), and C-4 (δC 53.3).

5 12.3) 8.2)

3.5)

8.0) 1.0)

6

1.43 3.57 2.23 5.32 2.63 4.58 1.82 1.89 1.01 2.07 1.01 1.33 6.27 1.02 4.22 3.96 1.16 1.00 1.78 5.23

dd (14.1, 12.5) dd (14.1, 8.4) m t (3.5) dd (11.0, 3.5) d (11.0) m m m m m dd (11.6,7.3) d (11.6) d (6.6) d (12.2) d (12.2) s s s br s

7.51 7.43 7.38 7.43 7.51

dd (7.0, 1.4) t (7.6) m t (7.6) dd (7.0, 1.4)

2.00 s 1.99 s

1.68 3.49 2.37 5.92 3.00 6.46 5.57

m dd (14.5, 8.6) m t (3.5) dd (8.6, 3.5) d (8.6) dd (12.5,4.1)

2.37 2.72 1.28 1.48 6.71 0.96 4.08 4.04 1.20 1.36 1.79

m m m dd (11.7, 8.6) dd (11.7, 1.1) d (6.0) d (12.6) d (12.6) s s d (0.9)

8.03 7.46 7.59 7.46 8.03

dd (7.9, 1.1) t (7.9) m t (7.9) dd (7.9, 1.1)

1.69 s 2.24 s

Correspondingly, a hydroxy group should be linked to C-7. The 5-acetoxy and 7-OH groups were inferred to be α- and βoriented, respectively, based on the NOESY correlations of H5 with H-3/H-12/H-2‴/H2-17/H-3′ and of H-7 with H-4/H9/H-12/H-11. Based on biosynthetic considerations,6 the structure of 3 was identified as (2S,3S,4R,5R,7R,9S,11R,15R)5,15-diacetoxy-3-benzoyloxy-7-hydroxy-14-oxolathyra-6(17),12E-diene. The HRESIMS (m/z 441.2270 [M + Na]+, calcd for C24H34O6Na+, 441.2253) and 13C NMR data analyses of compound 4 provided the molecular formula of C24H34O6, suggesting 8 indices of hydrogen deficiency. Its 1H and 13C NMR data (Tables 1 and 2) exhibited the presence of two acetoxy groups [δH 2.02 (3H, s), 2.01 (3H, s); δC 170.7, 169.8, 21.7, 21.0], a ketocarbonyl carbon (δC 194.7), two double bonds [δH 6.53 (1H, dd, J = 11.4, 1.0 Hz), 5.98 (1H, d, J = 11.2 Hz); δC 147.0, 140.2, 132.6, 126.4], and four methyls [δH 1.84 (3H, d, J = 0.9 Hz), 1.18 (3H, s), 1.08 (3H, d, J = 6.8 Hz), 1.04 (3H, s); δC 13.7, 29.3, 16.4, 12.6]. Its NMR spectra were closely related to those of the known acetyl derivative of Euphorbia factor L10 (1b),17 except for the absence of a caproyl group at C-3 in 4 (Figure 2). The key HMBC correlations from H-3 to C-1/C-15, H-16 to C-3, and H-2′ to C-15, as well as the diagnostic NOESY correlations of H-2′ with H-19/H-20/H2-17/H-12/H-8b, corroborated that the hydroxy and acetoxy groups were linked to C-3 and C-15, respectively. The β-orientation of 3-OH was elucidated by the NOESY correlations of H-3 with H-4/H-2, but no correlation with H-2′. The key NOESY correlations of H-9 with H-11, H11 with H-18/H-20/H-9, and H-19 with H-2′/H-18/H-12 C


Journal of Natural Products Table 2.

13

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C NMR Data of Compounds 1−6 in CDCl3 (150 MHz, δ in ppm)

pos.

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 1‴ 2‴

49.1, CH2 37.5, CH 81.8, CH 55.2, CH 65.7, CH 148.6, C 35.7, CH2 22.3, CH2 35.3, CH 25.3, C 28.5, CH 146.9, CH 134.3, C 197.3, C 92.8, C 14.2, CH3 113.0, CH2 29.2, CH3 17.0, CH3 12.7, CH3 167.3, C 118.3, CH 145.4, CH 134.5, C 128.3, CH 129.2, CH 130.7, CH 129.2, CH 128.3, CH 169.9, C 22.3, CH3

49.4, CH2 37.6, CH 81.5, CH 54.9, CH 63.9, CH 144.7, C 80.1, CH 28.8, CH2 30.5, CH 24.4, C 27.7, CH 144.9, CH 135.0, C 197.7, C 92.8, C 14.2, CH3 118.1, CH2 28.9, CH3 16.8, CH3 12.7, CH3 166.2, C 131.0, C 129.9, CH 128.6, CH 133.2, CH 128.6, CH 129.9, CH


44.0, CH2 39.4, CH 80.0, CH 52.1, CH 126.4, CH 140.2, C 32.4, CH2 28.6, CH2 34.2, CH 24.9, C 29.5, CH 147.0, CH 132.6, C 194.7, C 95.4, C 13.7, CH3 64.4, CH2 29.3, CH3 16.4, CH3 12.6, CH3 169.8, C 21.7, CH3

44.8, CH2 38.3, CH 83.5, CH 50.7, CH 124.2, CH 140.7, C 31.9, CH2 28.4, CH2 34.2, CH 25.0, C 29.4, CH 147.3, CH 132.4, C 194.3, C 94.6, C 14.0, CH3 64.0, CH2 29.3, CH3 16.3, CH3 12.4, CH3 173.6, C 73.0, CH 139.4, C 127.3, CH 128.7, CH 128.8, CH 128.7, CH 127.3, CH


169.7, C 21.7, CH3 169.8, C 22.2, CH3

170.6, C 21.6, CH3 169.9, C 22.2, CH3

171.0, C 21.2, CH3

169.6, C 21.6, CH3 170.8, C 21.1, CH3

169.3, C 21.2, CH3 170.0, C 22.4, CH3

Figure 2. Key HMBC correlations of compounds 1, 4, and 6.

(Figure 3) demonstrated an α-orientation for H-9/H-11/CH318 and a β-orientation for CH3-19. Based on biosynthetic considerations,6 the structure of 4 was elucidated as (2S,3S,4R,9S,11R,15R)-15,17-diacetoxy-3-hydroxy-14-oxolathyra-5E,12E-diene. The formula of compound 5 was established as C32H40O8 based on the HRESIMS (m/z 575.2642 [M + Na]+, calcd for C32H40O8Na+, 575.2621) and 13C NMR data. Its NMR data (Tables 1 and 2) demonstrated that 5 was highly similar to Euphorbia factor L18,4 except that the chemical shift of C-2′ was deshielded from δC 42.2 in Euphorbia factor L18 to δC 73.0 in 5, indicating the oxygenation of C-2′. The HMBC correlations from H-2′ (δH 5.23) to C-4′, -8′ (each δC 127.3), C-3′ (δC 139.4), and C-1′ (δC 173.6) confirmed the

above supposition. The absolute configuration of C-2′ was established by comparing its experimental ECD data with the calculated results. The computed ECD spectrum of 5a exhibited positive Cotton effects at ca. 202 and 277 nm and a negative Cotton effect at ca. 239 nm, closely resembling the experimental ECD spectrum of 5 (Figure 5), and defined the (2′R) absolute configuration of 5. The negative Cotton effect at ca. 239 nm was similar to that in the ECD spectrum of D(−)-mandelic acid.20 Based on biosynthetic considerations,6 the structure of 5 was determined to be (2S,3S,4S,9S,11R,15R)-15,17-diacetoxy-3-[(2′R)-hydroxyphenylacetyl]-14-oxolathyra-5E,12E-diene. The formula of 6 was deduced as C31H38O8 via the HRESIMS ion at m/z 561.2474 [M + Na]+ (calcd for D



Article

Figure 3. Key NOESY correlations of compounds 1, 4, and 6.

92.9), C-17 (δC 64.7), and C-4 (δC 52.7) confirmed the linkage of an acetoxy group to C-5, rather than C-15 (Figure 2). The 5-acetoxy group was deduced to be α-oriented based on the NOESY correlations of H-5 (δH 6.46) with H-3 (5.92), H-12 (6.71), H-3′ (8.03), H-17a (4.04), H-8b (2.72), and H-2‴ (2.24) (Figure 3). The β-orientation of 15-OH was determined through the pyridine-induced solvent shifts21−23 for H-1β (δCDCl3 − δpyridine‑d5 = −0.35 ppm), H-2α (−0.13 ppm), H-3α (−0.30 ppm), and H-5β (−0.46 ppm). Based on biosynthetic considerations,6 the structure of 6 was identified as (2S,3S,4R,5R,9S,11R,15R)-5,17-diacetoxy-3-benzoyloxy-15hydroxy-14-oxolathyra-6E,12E-diene. Compounds 1−16 were assayed for their inhibitory activities against NO production triggered by LPS in the mouse macrophage cell line RAW264.7. Cell viability was measured by the CCK8 assay to establish whether the inhibition of NO generation was caused by cytotoxicity of these compounds. All compounds showed low cytotoxicity (IC50 > 50 μM, Table S2, Supporting Information). Dexamethasone was employed as the positive control, with an IC50 value of 7.9 ± 1.3 μM (Table 3).

Figure 4. X-ray ORTEP drawing of compound 1.

Table 3. Effects of Compounds 1−16 on Macrophage NO Production Triggered by LPS compound

IC50 (mean ± SD, μM)

1 2 3 4 5 6 7 8 9

3.0 ± 1.1 4.0 ± 1.3 5.0 ± 1.2 >50 >50 >100 8.3 ± 1.4 ≈100 15.2 ± 1.1

a

compound

IC50 (mean ± SD, μM)

10 11 12 13 14 15 16 dexamethasonea

>100 2.6 ± 1.1 >100 26.0 ± 1.0 9.3 ± 1.2 >100 9.9 ± 1.4 7.9 ± 1.3

Positive control.

Compounds 1−3, 7, and 11 exhibited notable inhibition on NO production (IC50 = 2.0−8.3 μM), showing that lathyrane diterpenoids with an exocyclic Δ6(17) double bond have more significant inhibitory effects than those with a 5α,6β-epoxy (e.g., 15) or Δ5(6) double bond (e.g., 4, 5, and 12) or a Δ6(7) double bond (e.g., 6). Although the presence of the exocyclic Δ6(17) double bond in 8 and 10 is similar to 11, both compounds showed no inhibitory effects, indicating that an aromatic moiety at C-3 and a nitrogen-containing aromatic

Figure 5. Experimental and calculated ECD spectra of compound 5.

C31H38O8Na+, 561.2464) and 13C NMR data. Its NMR data (Tables 1 and 2) are highly similar to those of the known Euphorbia factor L204 except for the absence of an acetoxy group, indicating that C-5 or C-15 was substituted by a hydroxy group. The HMBC correlations from H-5 (δH 6.46) to C-1″ (δC 169.3), C-6 (δC 135.3), C-7 (δC 132.3), C-15 (δC E



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Figure 6. Compound 1 inhibited the production of NO, IL-1β, and IL-6. (A) NO inhibition curve of compound 1, with an IC50 of 3.0 ± 1.1 μM. (B) Survival rate of RAW264.7 cells treated with various concentrations of compound 1 for 24 h. No obvious cytotoxicity at concentrations up to 50 μM was observed. ***, p < 0.001 compared with control (treated with 0 μM compound 1). (C, D) Productions of IL-1β and IL-6 in RAW264.7 cell culture medium after treating with compound 1 for 3 h and LPS for 24 h. ###, p < 0.01, compared with the control group, for which neither compound 1 nor LPS was given. **, p < 0.01 and ***, p < 0.001, compared with that only stimulated with LPS.

inflammatory cytokine responses. In inflammation, overproduction of NO is mainly induced by aberrant expression of iNOS, which could be up-regulated in macrophages by LPS stimulation.24,25 Compound 1 dramatically reduced the expression of iNOS dose-dependently (Figure 7A). NF-κB is a transcription factor and plays key roles in the regulation of inflammation, immune responses, cell survival, and proliferation.26 Therefore, the effect of compound 1 on LPS-induced activation of NF-κB was assessed. After exposure to LPS, the expression of NF-κB/P65 was increased in RAW264.7 cells, while it was strikingly reduced by compound 1 at 5 μM (Figure 7A). Phosphorylation of IκBα is the key step in the process of NF-κB activation.26 As expected, phosphorylated IκBα (pIκBα) was elevated after being induced by LPS, and pretreatment with 10 μM compound 1 could effectively inhibit the phosphorylation of IκBα (Figure 7B). In addition, compound 1 eliminates LPS-induced p65 nuclear translocation (Figure 7C). Overall, these results suggest that compound 1 exerts its anti-inflammatory roles by interfering with the phosphorylation of IκBα, thereby blocking the expression and nuclear translocation of NF-κB, and it also exhibits antiinflammatory effects by reducing the expression of iNOS. In summary, six new lathyrane diterpenoids (1−6) and 10 known analogues (7−16) were separated from the seeds of E. lathyris. Compounds 1−3, 7, 9, 11, 13, 14, and 16 displayed inhibition on NO production induced by LPS in RAW264.7 macrophage cells. Further, compound 1 decreased the production of inflammatory factors, such as IL-1β and IL-6, and also reduced the expression of iNOS and NF-κB and the phosphorylation of IκBα. These findings reveal that lathyrane

group at C-7 are probably critical for the inhibition of NO production. Deacetylation at C-15 in 10 led to an elevated inhibitory effect, as observed for 9. Compared to 4, 5, and 12, compounds 13 and 14 exhibited stronger activity, suggesting that deacetylation at C-17 may increase the activity in the compounds possessing a Δ5(6) double bond. However, the type and size of the C-3 substituents are also crucial for the suppression of NO production. Compound 6, with a Δ6(7) double bond, showed no inhibitory activity, while compound 16 showed inhibitory activity with an IC50 value of 9.9 ± 1.4 μM, indicating that the 6,17-epoxy moiety could increase inhibitory effects against NO generation. Among the new compounds (1−6), compound 1 displayed the strongest inhibitory activity, with an IC50 value of 3.0 ± 1.1 μM (Table 3, Figure 6A), and was selected for further antiinflammatory studies. Before the inhibitory activity of compound 1 on NO production was examined, its cytotoxic effect was measured to exclude its cytotoxicity on RAW264.7 cells. Cell viability was not significantly affected by compound 1 up to 50 μM for 24 h, but it dropped at 100 μM (Figure 6B). Thus, the concentration of 1 was limited to 50 μM in subsequent experiments. As shown in Figure 6C and D, the production of inflammatory cytokines, including IL-1β and IL-6, was significantly elevated in the cell supernatant of LPS-induced RAW264.7 cells, as compared with that of untreated cells. The up-regulation of these cytokines was significantly decreased by treating with compound 1 in a dose-dependent manner. The anti-inflammatory activities of compound 1 prompted an investigation of the possible mechanism of modulating proF



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Extraction and Isolation. The powder of dried seeds of E. lathyris (10.0 kg) was extracted with petroleum ether (9.5 L) overnight at room temperature to get rid of the fatty oils. The residue was extracted with 95% EtOH (3 × 30 L× 3 h) to give a total extract after removing solvent in vacuo. The extract (1.02 kg) was suspended in water (1.5 L) and extracted with EtOAc (4 × 1.5 L). The EtOAc extract (715.8 g) was subjected to silica gel CC (10 × 160 cm) eluted with petroleum ether−EtOAc (100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 2:1, 1:1, and 0:1, v/v) to afford eight fractions (E1−E8). Fraction E3 (36.4 g) was recrystallized with petroleum ether−MeOH to afford compound 7 (25.6 g). Fraction E4 (75.8 g) was recrystallized with petroleum ether−MeOH to yield compound 10 (10.95 g) and another impure white powder (0.95 g), which was purified by silica gel CC (1.5 × 70 cm) eluted with petroleum ether−EtOAc (100:0 to 0:1) to afford compound 9 (168.0 mg). Fraction E5 (36.4 g) was recrystallized with petroleum ether−MeOH to give compound 16 (14.9 g). The mother liquor (5.72 g) was isolated by silica gel CC (2.5 × 80 cm) with an increasing acetone percentage in petroleum ether from 30% to 100% to give eight subfractions (E51−E58). Subfraction E57 (2.62 g) was subjected to a silica gel CC (2 × 70 cm) using petroleum ether−acetone (100:1 to 1:1) as eluent to afford nine fractions (E571−E579). Fraction E578 (267.6 mg) was separated by HPLC (MeOH−H2O, 6:1) to obtain compound 1 (19.7 mg). Fraction E6 (9.49 g) was crystallized with petroleum ether−MeOH to afford compound 8 (1.87 g), and the rest were applied to a silica gel column (3.5 × 80 cm) using petroleum ether−acetone (from 100:0 to 0:1) as eluent to yield nine fractions (E61−E69). Subfraction E66 (1.4 g) was separated on a silica gel column (2 × 70 cm) eluted with petroleum ether−acetone (100:1 to 0:1) to obtain E662 (362.4 mg). Separation of E662 through preparative HPLC (MeOH−H2O, 4:1) yielded 11 subfractions (E662-1 to E662-11). Separation of subfractions E662-1 (6.1 mg) and E662-2 (12.5 mg) through preparative HPLC (MeOH−H2O, 7:3) gave compounds 4 (1.9 mg, tR = 25.9 min) and 15 (3.2 mg, tR = 27.0 min). Compound 2 (16.1 mg, tR = 24.2 min) was separated from subfraction E662-4 (47.0 mg) by preparative HPLC (MeOH−H2O, 3:1). Subfractions E662-7 (24.8 mg) and E662-8 (94.7 mg) were subjected to preparative HPLC (MeOH−H2O, 4:1) to yield compounds 14 (12.7 mg, tR = 28.0 min) and 11 (68.7 mg, tR = 29.5 min), successively. Fraction E67 (2.75 g) was separated by using silica gel CC (3 × 80 cm) eluted with petroleum ether−acetone (100:1 to 0:1) to obtain E674 (1.38 g), which was separated by preparative HPLC (MeOH−H2O, 4:1) to afford 10 subfractions, E674-1 to E674-10. Compound 13 (26.4 mg, tR = 182.3 min) was purified from subfraction E674-8 (67.4 mg) by HPLC (MeOH−H2O, 7:3). E674-2 (17.0 mg) and E674-4 (23.1 mg) were purified by preparative HPLC (MeOH−H2O, 7:3) to yield compounds 5 (2.6 mg, tR = 58.0 min) and 12 (4.3 mg, tR = 42 min). Subfraction E674-3 (118.6 mg) was separated by preparative HPLC (MeOH−H2O, 7:3) to afford compounds 3 (11.5 mg, tR = 59.3 min) and 6 (21.9 mg, tR = 65.7 min). (2S,3S,4S,5R,9S,11R,15R)-15-Acetoxy-3-cinnamoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene (1): colorless needles (CH2Cl2); mp 94−96 °C; [α]D25 +124 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 273.4 (4.2) nm; IR (KBr) νmax 3436, 2986, 2925, 2873, 1713, 1629, 1450, 1383, 1274, 1174, 1009, 906 cm−1; 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data (Tables 1 and 2); HRESIMS m/z 529.2564 [M + Na]+ (calcd for C31H38O6Na+, 529.2566). (2S,3S,4S,5R,7R,9S,11R,15R)-7,15-Diacetoxy-3-benzoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene (2): white powder (MeOH); [α]25 D +72 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 256.6 (4.6) nm; IR (KBr) νmax 3450, 2957, 2931, 1743, 1631, 1452, 1276, 1116, 1011, 908, 712 cm−1; 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data (Tables 1 and 2); HRESIMS m/z 561.2465 [M + Na]+ (calcd for C31H38O8Na+, 561.2464). (2S,3S,4R,5R,7R,9S,11R,15R)-5,15-Diacetoxy-3-benzoyloxy-7-hydroxy-14-oxolathyra-6(17),12E-diene (3): white powder (MeOH); [α]25 D −13 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 254.8 (4.5) nm; IR (KBr) νmax 3437, 2928, 1745, 1629, 1453, 1277, 1115, 1008, 912, 713 cm−1; 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3)

Figure 7. Compound 1 reduced the expression of iNOS and NF-κB and the phosphorylation of IκBα and blocked the LPS-induced nuclear translocation of NF-κB. (A) Compound 1 significantly reduced the expression level of iNOS and NF-κB in a dose-dependent manner. RAW264.7 cells were pretreated with compound 1 for 3 h before LPS was added. The expression of iNOS and NF-κB was detected by Western blotting. (B) Compound 1 reduced the phosphorylation of IκBα. (C) Compound 1 eliminates LPS-induced nuclear translocation of NF-κB. RAW264.7 cells stained for NF-κB (green) and nuclei (DAPI, blue).

diterpenoids can be promising anti-inflammatory agents for chronic inflammation or autoimmune disease.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 hot stage microscope melting point apparatus (uncorrected). Optical rotations were recorded on a PerkinElmer 241 polarimeter. UV spectroscopic data were acquired on a Shimadzu UV 2201 spectrophotometer. ECD spectra were recorded on a Bio-Logic Science MOS-450 spectrometer. NMR spectra were measured on an AV-600 spectrometer. Chemical shift values are depicted in δ (ppm) by using the resonances of the solvent CDCl3 (δH 7.27 and δC 77.23) or pyridine-d5 (δH 7.58 and δC 135.9) as references, and the coupling constants are expressed as J in Hz. HRESIMS data were collected on an Agilent 6210 TOF mass spectrometer. X-ray crystallographic data were obtained from a SuperNova, Dual, AtlasS2 diffractometer with Cu Kα radiation (λ = 1.541 84 Å). Silica gel (200−300 mesh, Qingdao Marine Chemical Factory) and octadecyl silica gel (Merck Chemical Company Ltd., Germany) were applied for column chromatography (CC). Silica gel GF254 for TLC was purchased from Qingdao Marine Chemical Factory (Qingdao, China). RPHPLC was performed on an LC-6AD liquid chromatography system equipped with an ODS column (C18, 250 × 20 mm, 120 Å, 5 μm, YMC Co. Ltd.) and SPD-20A UV detector (Shimadzu, Kyoto, Japan). Plant Material. The seeds of Euphorbia lathyris were collected from Suqian, Jiangsu Province, China, in March 2017, and were authenticated by Dr. Jin-Cai Lu, Shenyang Pharmaceutical University. A specimen (QJZ. 20170118) was preserved in the herbarium of Shenyang Pharmaceutical University. G



Article

data (Tables 1 and 2); HRESIMS m/z 561.2479 [M + Na]+ (calcd for C31H38O8Na+, 561.2464). (2S,3S,4R,9S,11R,15R)-15,17-Diacetoxy-3-hydroxy-14-oxolathyra-5E,12E-diene (4): white powder (MeOH); [α]25 D +90 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 216.6 (4.7) nm; IR (KBr) νmax 3396, 2924, 2851, 1740, 1647, 1380, 1231, 802 cm−1; 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data (Tables 1 and 2); HRESIMS m/z 441.2270 [M + Na]+ (calcd for C24H34O6Na+, 441.2253). (2S,3S,4S,9S,11R,15R)-15,17-Diacetoxy-3-[(2′R)-hydroxy-phenylacetyl]-14- oxolathyra-5E,12E-diene (5): white powder (MeOH); [α]25 D −40 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 254.8 (4.3) nm; ECD (MeOH) λmax (Δε), 202 (+14.11), 239 (−6.47), 277 (+8.44) nm; IR (KBr) νmax 3396, 2922, 2850, 1740, 1646, 1261, 802 cm−1; 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data (Tables 1 and 2); HRESIMS m/z 575.2642 [M + Na]+ (calcd for C32H40O8Na+, 575.2621). (2S,3S,4R,5R,9S,11R,15R)-3-Benzoyloxy-5,17-diacetoxy-15-hydroxy-14- oxolathyra-6E,12E-diene (6): white powder (MeOH); [α]25 D +17 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 228.8 (4.6) nm; IR (KBr) νmax 3436, 2923, 1741, 1629, 1278, 1115, 714 cm−1; 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data (Tables 1 and 2); HRESIMS m/z 561.2474 [M + Na]+ (calcd for C31H38O8Na+, 561.2464). X-ray Crystallographic Analysis of Compound 1. Single crystals of compound 1 were obtained from CH2Cl2 containing a small amount of MeOH at room temperature. The crystallography data were collected on a SuperNova, Dual, Cu at zero, AtlasS2 diffractometer using monochromatized Cu Kα (λ = 1.541 84 Å) radiation. The crystal was kept at 100.00(10) K during the data collection process. Structure determination and refinement were executed by using the SHELXL program. Crystallographic data were deposited at the Cambridge Crystallographic Data Centre (CCDC No. 1855220) and can be obtained free of charge from the CCDC Web site (www.ccdc.cam.ac.uk). Crystal data of 1: C31H38O6 (M = 506.61 g/mol), monoclinic, P21 (no. 4), a = 10.13676(11) Å, b = 17.28546(14) Å, c = 33.1549 (2) Å, β = 93.6550 (7)°, V = 5797.53 (9) Å3, Z = 8, T = 100.00(10) K, μ(Cu Kα) = 0.640 mm−1, Dcalc = 1.161 g/cm3, 49 755 reflections measured (5.342° ≤ 2θ ≤ 147.094°), 21881 unique (Rint = 0.0287, Rsigma = 0.0333). The final R1 was 0.0390 (I > 2σ(I)) and wR2 was 0.1019 (all data). The Flack and Hooft parameters were −0.04(4) and −0.02(4), respectively. ECD Calculations. To identify the absolute configuration of compound 5, ECD calculations were implemented with density functional theory (DFT) by using the Gaussian 09 program package.27 The conformational search of compound 5 was performed using the SPARTAN conformational search. Second, at the B3LYP/631G(d) level, each conformation was subjected to the Gaussian 09 program for further optimization by using the DFT method. Then, at the B3LYP/6-311++G(2d,p) level, ECD calculations for conformations were performed by using the TDDFT method with the CPCM model in methanol. Finally, the calculated ECD spectrum was generated by SpecDis 1.51.28 Cell Cultures. Mouse macrophage RAW264.7 cells were purchased from ATCC and were cultured in DMEM medium with 5% CO2/95% air (v/v) at 37 °C in a humidified incubator. All media were supplemented with penicillin/streptomycin (100 unit/mL, Hyclone) and 10% fetal bovine serum (Sigma). Determination of Nitrite. As the stable final product of NO oxidation, nitrite was measured in RAW264.7 cells to indicate the NO production. It was assayed by a method based on Griess reaction. Cells were inoculated into 96-well plates with 5000 cells per well and cultured overnight. The culture medium was replaced by new medium containing compounds (0−100 μM). After incubation for 3 h, 1 μg/ mL LPS was added into the medium and then incubated for 24 h. The supernatant samples were mixed with the same amount of Griess reagent at room temperature for 10 min, and the absorbance of the final product was measured at 540 nm using a plate reader. The nitrite concentration was calculated by the standard curve method. The inhibitory rate of the compounds at gradient concentration was

calculated, and the inhibitory effect of the compounds on LPSinduced NO production was evaluated by IC50 value. Cell Survival Assay. RAW264.7 cells were treated with compound 1 (0−100 um M) for 24 h, and the cell viability was measured. The supernatant of the well was collected, and the CCK8 (Cell Counting Kit-8) assay was performed according to the manufacturer’s instructions. ELISA. To determine the production of cytokines IL-1β and IL-6, RAW264.7 cells were treated with compound 1 at 2.5, 5, 10 μM for 3 h and then excited with 1 μg/mL LPS for 24 h. Collected supernatants were used to determine IL-1β and IL-6 by the commercial ELISA kits. NF-κB/p65 Nuclear Translocation Immunofluorescence. RAW264.7 cells were cultured to 70% confluence, pretreated with DMSO, treated with 5 or 10 μM compound 1 for 2 h, and activated by 1 μg/mL LPS for 12 h. The treatment was stopped by washing with phosphate-buffered saline (PBS). Freshly prepared 4% paraformaldehyde in PBS was used to fix the cells for 10 min; then the cells were washed 3 times with PBS and permeabilized for 10 min in 0.25% Triton X-100 in PBS. After blocking 1 h with 5% bovine serum albumin at room temperature, P65 antibody at 1:1000 dilution was added and incubated overnight at 4 °C. After washing the glass chambers, secondary antibody (AlexaFluor488) was added at 1:500 dilution for 1 h in the dark. Finally, the nuclei were stained with DAPI at 37 °C in the dark for 30 min. Western Blot Assay. The protein expressions of iNOS, NF-κB, IκBα, and p-IκBα were assayed by Western blotting. Cell pellets were collected and resuspended in RIPA lysis buffer (containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.1 mM PMSF). The cell pellet was resuspended by vortexing, kept on ice for 20 min, and centrifuged 30 min at 20000g. The supernatants were subjected to Western blot analysis. A BCA protein assay kit was used to determine the protein content of samples. Each sample with the same protein content was separated by 10% SDS-PAGE. The gels were transferred to nitrocellulose membranes electrophoretically, which were already blocked with 5% skim milk. The membranes were sequentially incubated with primary antibodies and secondary antibodies, followed by chemiluminescence detection. Statistical Analysis. The statistical analysis was performed by GraphPad Prism 4.0 (GraphPad Software Inc., USA). All data were displayed as mean ± SD for three independent tests. Then, through Tukey’s multiple comparison experiment, three or more groups were compared by one-way analysis of variance.

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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.8b00600.

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Tables S1 and S2, 1D and 2D NMR, HRESIMS, UV, and IR spectra for new compounds (1−6) (PDF) X-ray crystallographic data (CIF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-24-23986463. E-mail: [email protected]. (H. Li). *Tel: +86-24-23986463. E-mail: [email protected]. (L.-X. Chen). ORCID

Hua Li: 0000-0003-1903-836X Li-Xia Chen: 0000-0003-2196-1428 Author Contributions ⊥

C.-Y. Zhang and Y.-L. Wu contributed equally to this work.

Notes

The authors declare no competing financial interest. H



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Article

Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.02, Gaussian, Inc: Wallingford, CT, 2009. (28) Torsten, B.; Anu, S.; Yasmin, H.; Gerhard, B. Chirality 2013, 25, 243−249.

ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (Nos. U1803122, U1703111, 81473254, 81773594, and 81773637).

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Anti-inflammatory Lathyrane Diterpenoids from Euphorbia lathyris

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