Antioxidant and anti-inflammatory neolignans from the seeds of hawthorn

Antioxidant and anti-inflammatory neolignans from the seeds of hawthorn

Bioorganic & Medicinal Chemistry Letters 26 (2016) 5501–5506 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters jour...

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Bioorganic & Medicinal Chemistry Letters 26 (2016) 5501–5506

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Antioxidant and anti-inflammatory neolignans from the seeds of hawthorn Ying Peng c, Li-Li Lou a,b, Si-Fan Liu a,b, Le Zhou a,b, Xiao-Xiao Huang a,b,⇑, Shao-Jiang Song a,b,⇑ a

Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, PR China Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, PR China c School of Pharmacy, Shenyang Pharmaceutical University, PR China b

a r t i c l e

i n f o

Article history: Received 19 July 2016 Revised 2 October 2016 Accepted 6 October 2016 Available online 7 October 2016 Keywords: Hawthorn Crataegus pinnatifida Seeds Neolignan Antioxidant Anti-inflammatory

a b s t r a c t Seven new neolignans (1–2, 7–11) and five known compounds (3–6, 12) were isolated from the 70% EtOH extract of hawthorn seeds. Their structures were determined by spectroscopic analyses. The antioxidant and anti-inflammatory activities of all the isolates were investigated. Most of the isolates showed moderate radical scavenging activity in the DPPH assay and significant activities in the ABTS and FRAP assays. Furthermore, compounds 7–12 exhibited marked nitric oxide (NO) inhibition and compounds 1–4 had a potent necrosis factor-a (TNF-a) inhibitory effect. The results we obtained showed that hawthorn seeds can be regarded as a potential new and cheap source of antioxidants and inflammation inhibitors. Ó 2016 Elsevier Ltd. All rights reserved.

Crataegus pinnatifida Bge., (Rosaceae), also referred to as ‘Hawthorn’, is associated with about 280 wood plant species which are distributed in the Northern Hemisphere, mainly in China, Europe and North America.1 The pharmacological effects of hawthorn have mainly been attributed to polyphenolic compounds and their antioxidant ability.2 Recently, some studies have shown that the extracts of hawthorn fruits have the capacity to quench free radicals and inhibit the oxidation of low-density lipoprotein (LDL), as well as having anti-inflammatory properties.3,4 Our group is interested in isolating bioactive compounds from medicinal plants and new anti-inflammatory and anti-oxidant constituents have been discovered in hawthorn seeds, which should help us to improve human health. In this study, seven new neolignans (1–2, 7–11) and five known compounds (3–6, 12) were isolated from the 70% EtOH extract of hawthorn seeds.5 The isolation and structural elucidation of the isolated compounds (Fig. 1), as well as the evaluation of their antioxidant ability and inhibitory effects on LPS-induced NO and TNF-a production in macrophage RAW 264.7 cells are described here. The molecular formula of 16 was established as C21H28O9 on the basis of a quasi-molecular ion at m/z 447.1608 [M+Na]+ (calcd for ⇑ Corresponding authors. Tel.: +86 24 23986486 (X.-X.H.), +86 24 23986510 (S.-J.S.). E-mail addresses: [email protected] (X.-X. Huang), [email protected] (S.-J. Song). http://dx.doi.org/10.1016/j.bmcl.2016.10.012 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

C21H28O9Na, 447.1626) in its HRESIMS. The 1H NMR spectrum showed six aromatic proton signals revealing the presence of two 1,3,4-trisubstituted (ABX system) moieties. The proton signals established the presence of two 1,2,3-propane-triol moieties. In addition, a methoxyl group was attached to the aliphatic chain at d 3.23 and two methoxyls were attached to the aromatic ring at d 3.77 and 3.79. The 13C NMR spectrum of 1 showed twenty-one carbon signals. Aside from the three methoxy carbon signals, there were eighteen carbon signals, including twelve aromatic and six aliphatic carbons, and the HMBC correlations of H-7 with C-1, C-2, C-6, C-8 and C-9 and of H-70 with C-20 , C-60 and C-90 confirmed the presence of two phenyl propanoid units. This NMR spectroscopic data suggested that 1 was an 8-O-40 system neolignan (Fig. 2). In order to further determine the relative configuration of C7,8, compounds 1, 2 and 11 were analyzed by 1H NMR in CDCl3. In the 1H NMR spectra of 1 in CDCl3, a small coupling constant J7,8 = 4.2 Hz was observed, and the relative configuration of C-7 and C-8 of 1 was determined to be in the erythro-form.7 The CD spectrum of 1 showed negative Cotton effects at 220–240 nm, which indicated that compound 1 had a 7S,8R-configuration according to the study of a related system.8 The relative configuration of C-70 and C-80 in 1 was determined to be threo from its 13C NMR data at d C 75.1 (C-70 ) and 77.4 (C-80 ) and the value of DdC80 –C70 >2.0 ppm.9 Consequently, the full structure of 1 was deduced and it was named Crataegusnin A.

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HO 1

R 7

H3 CO 3 HO

1 HO

5

8

9

OCH3 3'

O

5 OH

1'

2

R 5'

OCH3 3 HO 1 9 7 8 R1 O

OH

7' 8' 9' R3

5'

7 1'

H3 CO

1 : R 1 =OCH3 ; R 2=H; R 3=OH; 7S,8R; 7',8'-threo 2 : R 1 =OH; R2 =H; R 3=OCH 3; 7R,8R; 7',8'-threo 3: R 1 =OH; R2 =H; R 3=OH; 7S,8R; 7',8'-threo 4: R 1 =OH; R2 =H; R 3=OH; 7R,8R; 7',8'-threo 5: R 1 =OH; R2 =OCH3 ; R3 =OH; 7S,8R; 7',8'-threo 6: R 1 =OH; R2 =OCH3 ; R3 =OH; 7R,8S; 7',8'-threo

9'

3'

OH 8' 1''

OCH3 3'' OH 5''

R2

7 : R 1 =OH; R2 =OCH3 ; 7R,8S; 7',8'-threo 8 : R 1 =OH; R2 =OCH3 ; 7S,8R; 7',8'-threo 9: R 1 =OH; R2 =OCH2 CH3 ; 7S,8S; 7',8'-threo 10: R1 =OH; R 2=OCH 2CH 3; 7S,8S; 7',8'-erythro 11: R 1 =OCH3 ; R 2=OCH 2CH 3; 7S,8S; 7',8'-threo 12: R1 =OH; R 2=OH; 7S,8R; 7',8'-erythro

Figure 1. Structures of compounds 1–12.

OCH 3 O

H 3CO

OH

OCH3 OH OH

OH

HO

O

2

OCH 3 HO HO

OH

OH OCH 3 7 HO

OCH 3 OH

OCH3

O H 3CO

HO

OH OCH 3

OH

HO

OH

OH

HO

1 HO

OCH3

H 3CO

OCH 3

OCH 3

HO

OH

OCH 3

OH HO

O OCH2 CH3

H3 CO 9

OH H 3CO

O

OCH2 CH3

H3 CO 11

Figure 2. Key HMBC correlations of compounds 1, 2, 7, 9 and 11.

Comparison of the NMR data of 210 with that of 1 suggested that their structures were closely related, and they differed only in the substitution position of the methoxyl group attached to the aliphatic chain. The HMBC correlations (Fig. 2) of these methoxyls also confirmed their positions. Some literature data reported that the effect of systematic errors [DdC8–C7 (threo) > DdC8–C7 (erythro)] can be used to differentiate threo and erythro aryl glycerols without substituent(s) at C-7 or/and C-8 of the glycerol moiety as well as the erythro and threo 8-O-40 isomers.8,11 The threo relative configuration at C-7 and C-8 in 2 was suggested by comparing the 13C NMR spectra with those of compounds 3 and 4. The coupling constant (J7,8 = 7.6 Hz, measured in CDCl3) also indicated that 2 has a 7,8-threo configuration.11 Furthermore, the negative CD effect at 220–240 nm for 2 supported a 7R,8R-configuration.8 In addition, the coupling constant between H-70 and H-80 (J = 8.1 Hz in CDCl3) suggested a threo conformation of C-70 /C-80 .12 Consequently, the structure of 2 was established and it was named Crataegusnin B. In the 1H NMR spectrum of 7,13 three ABX spin system signals were observed, as well as four methoxy group signals. The 13C NMR and HSQC spectra revealed the presence of twenty-eight carbon resonances, due to four methyls, two methylenes, thirteen methines, and nine quaternary carbons. Detailed analysis of the NMR data of 7 showed many similarities to those of leptolepisol

D, an 8-O-40 system neolignan from Euonymus acanthocarpus, and clearly showed that 7 shared the same basic parent structure with the known compound.14 The difference was the addition of a methoxyl attached to the aliphatic chain at dH 3.08 (3H, s). Analysis of the HMBC spectrum (Fig. 2) then established the connectivity of the functional groups. In our study, the DdC8–C7 value of 7 was the same as 3, thus, the relative configuration at C-7/C-8 of 7 was determined to be in the erythro-form.8,11 Since the CD spectrum of 1 showed a positive Cotton effect at 220–240 nm, this indicated that 7 had the 8S-configuration according to a study of a related system.8 On the basis of the bulkiness rule for secondary alcohols,15 a negative Cotton effect at 352 nm in the Rh2(OCOCF3)4-induced CD spectrum indicated the 7R configuration for 1, which was in agreement with that due to the 7,8-erythro and 8S configurations assigned above. The 70 ,80 -threo relative configuration was suggested by comparing the 13C NMR spectra with those of the threo and erythro isomers,14,16,17 since the DdC80 –C70 value of 7 was larger than that of 10, and the same as 8, 9 and 11. Consequently, the structure of 7 was assigned and it was named Crataegusnin C. The NMR data of 818 closely resembled that of 7, indicating that they shared the same planar structure and differed only in their configuration. The relative configuration of C-7 and C-8 in 8 was

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Y. Peng et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5501–5506 Table 1 H NMR data of compounds 1, 2 and 7–11 (at 400 MHz)

1

H

1

2 5 6 7 8 9

6.93 6.79 6.73 4.89 4.36 3.81 3.62 6.96 6.80 6.80 4.52 3.64 3.33 3.46

20 50 60 70 80 90 200 500 600 3-OCH3 30 -OCH3 300 -OCH3 7-OCH3 70 -OCH3 70 -OCH2CH3

2 (br s) (m) (dd, 8.0, 1.1) (d, 6.0) (m) (m) (m) (br s) (m) (m) (d, 6.0) (m) (m) (dd, 11.2, 4.0)

3.77 (3H, s) 3.79 (3H, s)

7.04 6.76 6.87 4.90 4.34 3.77 3.52 7.00 7.06 6.85 4.15 3.67 3.32 3.47

7 (br s) (d, 8.1) (dd, 8.1, 1.0) (d, 5.6) (m) (dd, 11.9, 4.0) (dd, 11.9, 5.5) (d, 1.6) (d, 8.5) (dd, 8.5, 1.6) (d, 6.5) (m) (m) (dd, 11.3, 4.0)

3.89 (3H, s) 3.84 (3H, s)

8

9

6.96 6.66 6.74 5.27 4.23 3.65 3.50 6.50 6.81 6.52 4.31 2.92 3.74

(d, 1.7) (d, 8.1) (dd, 8.1, 1.7) (d, 4.1) (m) (m) (m) (d, 1.2) (d, 8.8) (dd, 8.8, 1.2) (d, 7.8) (m) (2H, m)

6.97 6.67 6.74 4.68 4.23 3.56 3.49 6.50 6.83 6.55 4.31 2.91 3.74

(d, 1.7) (d, 8.1) (dd, 8.1, 1.7) (d, 4.1) (m) (m) (m) (d, 1.7) (d, 8.4) (dd, 8.4, 1.7) (d, 7.8) (m) (2H, m)

6.48 6.52 6.39 3.71 3.57 3.58

(d, 1.8) (d, 8.1) (dd, 8.1, 1.8) (3H, s) (3H, s) (3H, s)

6.46 6.52 6.39 3.71 3.55 3.57

(d, 1.7) (d, 8.1) (d, 8.1, 1.7) (3H, s) (3H, s) (3H, s)

10

7.02 6.76 6.85 4.87 4.24 3.70 3.45 6.60 6.91 6.66 4.46 3.00 4.09 3.92 6.52 6.01 6.51 3.82 3.71 3.69

(d, 1.5) (d, 8.1) (dd, 8.1, 1.5) (overlap) (m) (m) (m) (d, 1.8) (d, 8.3) (dd, 8.3, 1.8) (d, 8.5) (m) (dd, 10.9, 5.7) (dd, 10.9, 7.1) (d, 1.9) (d, 8.1) (d, 8.1, 1.9) (3H, s) (3H, s) (3H, s)

7.03 6.76 6.86 4.88 4.26 3.70 3.45 6.68 6.96 6.66 4.66 2.85 4.10 3.94 6.54 6.66 6.56 3.83 3.73 3.67

11 (d, 1.2) (d, 8.1) (dd, 8.1, 1.2) (d, 5.8) (m) (m) (m) (d, 1.4) (d, 8.2) (dd, 8.2, 1.4) (d, 4.9) (m) (m) (dd, 10.7, 7.3) (d, 1.2) (d, 8.2) (d, 8.2, 1.2) (3H, s) (3H, s) (3H, s)

3.23 (3H, s) 3.24 (3H, s)

3.08 (3H, s)

6.96 6.78 6.80 4.41 4.29 3.63 3.41 6.57 6.85 6.65 4.44 2.99 3.93 4.10 6.51 6.62 6.52 3.69 3.69 3.81 3.24

(d, 1.6) (d, 8.1) (dd, 8.1, 1.6) (d, 5.7) (m) (m) (m) (d, 1.8) (d, 8.3) (dd, 8.3, 1.8) (d, 8.6) (m) (dd, 10.9, 7.1) (dd, 10.9, 5.6) (br s) (d, 7.9) (d, 8.1, 1.8) (3H, s) (3H, s) (3H, s) (3H, s)

3.08 (3H, s) 3.37 (2H, m) 1.19 (3H, t, 7.0)

3.36 (2H, m) 1.15 (3H, t, 7.0)

3.39 (2H, m) 1.20 (3H, t, 7.0)

Coupling constants (J) in Hz are given in parentheses; chemical shift values are expressed in ppm.

Table 2 C NMR data of compounds 1, 2 and 7–11 (at 100 MHz)

13

No.

1

2

7

8

9

10

11

1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 100 200 300 400 500 600 3-OCH3 30 -OCH3 300 -OCH3 7-OCH3 70 -OCH3 70 -OCH2CH3

131.0 112.3 148.8 147.3 118.4 122.1 83.7 85.3 62.2 137.9 112.4 151.5 148.6 115.7 120.3 75.1 77.4 64.2

133.8 111.7 148.8 147.1 115.8 120.7 74.0 87.1 62.0 134.4 112.6 151.7 149.4 118.6 121.5 85.3 77.0 63.9

56.4 56.4

55.3 55.5

133.2 111.4 146.9 145.4 114.6 119.5 71.5 83.6 59.9 132.6 111.6 149.0 147.0 114.5 120.0 84.2 53.6 62.3 131.5 113.4 146.6 144.6 114.7 121.5 55.5 55.4 55.4

133.1 111.4 146.7 145.5 114.7 119.5 71.5 83.5 59.8 132.7 112.0 148.9 146.9 114.5 119.7 84.2 53.6 62.3 131.4 113.4 145.5 144.6 114.7 121.5 55.5 55.4 55.5

133.7 111.7 148.8 147.0 115.8 120.7 73.9 87.0 61.8 136.3 113.0 151.0 148.6 118.0 121.3 85.4 56.0 65.0 132.6 114.3 148.4 146.0 115.7 122.6 56.3 56.4 56.3

133.8 111.7 148.8 147.1 115.8 120.7 74.0 87.0 61.9 137.0 112.5 151.2 148.5 118.0 121.0 82.4 56.5 64.4 131.9 114.8 148.2 146.2 115.5 123.4 55.5 55.4 55.5

130.9 112.1 149.1 147.5 115.9 121.6 84.1 85.5 62.0 135.9 113.1 151.0 148.9 117.6 121.1 85.5 56.1 65.1 132.6 114.3 148.4 146.1 115.7 122.5 56.3 56.3 56.4 57.2

56.1

56.1

57.0 57.1

determined to be erythro by its 13C NMR data at dC 71.5 (C-7) and 83.5 (C-8) and the value of DdC8–C7 = 12.0 ppm. The negative CD effect of compound 8 at 220–240 nm, was due to a 7S,8R-configuration.8 This was also supported by the positive Cotton effect in the Rh2(OCOCF3)4-induced CD spectra of 8 (351 nm).11,15 The threo configuration between two chiral centers at the C-70 and C-80 positions was determined by comparing the NMR spectra with those of the erythro and threo isomers.14,16,17 Therefore, compound 8 was identified as an optical isomer of 7 and was named Crataegusnin D.

65.2; 15.6

65.4; 15.6

65.2; 15.6

The 1H and 13C NMR spectra of 919 showed similarities to those of 7, except for the presence of MeO-70 , which was replaced by an ethoxy group in 9. These minor structural changes were further supported by HSQC correlations and HMBC correlations. The threo configuration between the two chiral centers at C-70 and C-80 positions was determined by comparing the NMR spectra with those of the threo and erythro isomers.14,16,17 The DdC8–C7 value of 9 was larger than that of 3, 7, 8, and the same as 4, thus, the relative configuration at C-7/C-8 of 9 was determined to be in the threo-form.

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The positive Cotton effect at 220–240 nm in the CD spectrum of 9 supported a 7S,8S-configuration8 which was also supported by the positive Cotton effect in the Rh2(OCOCF3)4-induced CD spectra of 9 (350 nm).11,15 Therefore, the structure of 9 was identified and it was named Crataegusnin E. A comparison of the HRESIMS and NMR spectroscopic data of 1020 with those of 9 revealed that the only difference between them was the 13C NMR signals of C-700 (85.4 in 9 and 82.4 in 10) suggesting that they have the same planer structure. The chemical shifts of C-70 and C-80 suggested a 70 ,80 -erythro relative configuration.14,16,17 In addition, the relative configuration of C-7/C-8 was suggested to be threo according to the DdC8–C7 value of 10. The positive CD effect at 220–240 nm of 10, supported a 7S,8S-configuration,8 which was also verified by a positive Cotton effect at 350 nm in the Rh2(OCOCF3)4-induced CD spectra of 10. Therefore, compound 10 was identified as a diastereo-isomer of 9 and named Crataegusnin F. The 1H and 13C NMR data of compound 1121 were similar to those of compound 9, except for the addition of a methoxyl group at the C-7 position. These changes were also supported by HSQC and HMBC correlations. The coupling constant (J7,8 = 7.3 Hz, measured in CDCl3) also indicated that 9 has a 7,8-threo configuration.11 Furthermore, the positive CD effect at 229 nm of 11 supported a 7S,8S-configuration.8 In addition, the threo configuration between the two chiral centers at the C-70 and C-80 positions was determined by comparing the NMR spectra.14,16,17 Thus, the structure of 11 was identified and it was named Crataegusnin G. The known compounds were identified as 70 ,80 -threo,7S,8R-1-[4[(2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)

Table 3 Antioxidant activities of compounds 1–12 Compound

DPPH (IC50, lM)

ABTS (IC50, lM)

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

108.7 ± 1.5 113.7 ± 2.0 82.4 ± 2.5 89.0 ± 3.3 138.0 ± 2.6 113.6 ± 3.1 43.6 ± 1.8 67.2 ± 2.4 56.8 ± 3.5 79.6 ± 3.9 81.6 ± 3.0 68.6 ± 3.8 30.2 ± 0.6

10.8 ± 0.2 12.9 ± 1.0 9.5 ± 0.2 10.7 ± 0.2 11.8 ± 0.3 10.7 ± 0.5 5.9 ± 0.7 7.8 ± 0.8 7.9 ± 0.4 7.1 ± 0.3 6.3 ± 0.5 4.4 ± 0.3 18.2 ± 0.4

IC50 values represent the means ± SEM of three parallel measurements.

ethoxy]-3-methoxyphenyl]-1,2,3-propanetriol (3),22 70 ,80 -threo,7R, 8R-1-[4-[(2-hydroxy-2-(4-hydroxyl-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3-methoxyphenyl]-1,2,3-propanetriol (4),22 0 0 7 ,8 -threo,7S,8R-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)1-(hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol (5),23 70 ,80 -threo-7R,8S-1-[4-[2-Hydroxy-2-(4-hydroxy3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol (6)23 and leptolepisol D (12)14 by comparing their physical and spectroscopic data with values reported in the literature. Some of the literatures have reported that the hydroxyl group at C-7 of phenylpropanoids may be substituted by ethoxyl, which are artificial products produced in the separation process.24 But there is no literature to prove that the hydroxyl groups at C-7 of neolignans are replaced by the 7-methoxyl/ethoxyl. In addition, there are two hydroxyl groups on benzyl of compounds 1–10, but only one hydroxyl group is replaced by methoxyl or ethoxyl, the other is not. Therefore, we believe that these compounds are more likely to be natural products. Phenolic compounds are widely known to act as free radical scavengers due to their reducing properties, acting as hydrogen or electron donating agents, singlet oxygen quenchers and metal chelators.25 The purpose of this assay was to compare the antioxidant activity values obtained by different methods (DPPH, ABTS, FRAP) to determine the antioxidant ability based on the single electron transfer reaction, displayed through a change in colour as the oxidant is reduced.26 The DPPH radical-scavenging assay is usually used to evaluate the abilities of new compounds to capture free radicals by producing the reduced form DPPH-H through a hydrogen-donating action. ABTS+ is another synthetic radical and is more versatile than DPPH and the ABTS+ model can be used to assess the scavenging activity for both polar and nonpolar samples. With the different mechanisms of these free radical scavenging reactions, these neolignans also showed different activities.27,28 The results of the radical scavenging ability of all the isolates are summarized in Tables 3 and 4. It was clear that some of the tested compounds (7–10, 12) showed more potent antioxidant activity in the DPPH assay,29 although they were slightly weaker than the positive control (Trolox), and other compounds only showed weak antioxidant activity. In the ABTS assay,30 most of the compounds showed significant antioxidant activity, with compounds 7–12 being the most potent, while the other compounds also exhibited potent ABTS radical scavenging activity, comparable to that of the standard compound. A comparison of the structures of 7–12 with those of 1–6 indicated that the additional benzene group at position C-80 appeared to increase the DPPH and ABTS radical scavenging activities. In addition, we observed that all compounds exhibited stronger antioxidant

Table 4 FRAP values (lmol/L) of compounds 1–12 with different concentrations (lg/mL) Sample

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

Concentration 375 (lg/mL)

750 (lg/mL)

1500 (lg/mL)

3000 (lg/mL)

29.9 ± 1.5 32.5 ± 1.8 9.4 ± 0.9 12.0 ± 1.4 37.1 ± 1.6 35.9 ± 2.0 60.8 ± 2.3 45.8 ± 1.1 33.5 ± 1.6 38.6 ± 2.1 34.5 ± 2.5 29.1 ± 0.9 51.7 ± 1.4

50.5 ± 0.8 54.1 ± 2.0 16.0 ± 0.8 18.5 ± 1.3 60.0 ± 1.5 58.1 ± 1.0 89.5 ± 2.2 73.0 ± 3.5 65.1 ± 2.8 62.7 ± 3.6 66.3 ± 2.1 42.2 ± 1.5 96.9 ± 2.0

74.1 ± 0.9 78.3 ± 2.7 29.4 ± 1.6 26.0 ± 3.8 89.1 ± 4.0 84.6 ± 4.3 120.6 ± 5.5 105.8 ± 4.8 108.0 ± 3.7 96.8 ± 2.2 99.2 ± 3.1 89.9 ± 2.8 184.8 ± 2.6

105.2 ± 1.2 102.2 ± 3.6 55.8 ± 2.7 66.3 ± 4.1 127.4 ± 1.9 118.6 ± 2.3 116.2 ± 1.8 128.0 ± 3.5 135.1 ± 4.0 134.3 ± 3.1 129.2 ± 1.8 132.1 ± 2.7 171.7 ± 3.3

Date were presented as means ± SEM of three parallel measurements

Y. Peng et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5501–5506 Table 5 Anti-inflammatory activities of compounds 1–12 Compound

NO (IC50, lM)

TNF-a (IC50, lM)

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

>100 >100 >100 >100 >100 >100 97.3 62.9 76.3 66.4 80.5 50.5 55.1 —

76.1 47.9 84.4 94.2 >100 >100 >100 >100 >100 >100 >100 >100 — 69.2

activities in the FRAP assay,31 with values higher than that of the positive control Trolox. This finding supported the hypothesis that lignans from Crataegus pinnatifida industrial by-products can be regarded as potential natural antioxidants. NO and TNF-a are key mediators for the pathogenesis of osteoarthritis, inflammatory bowel diseases, psoriasis, ulcerative colitis and rheumatoid arthritis. Hence, the inhibition of NO and TNF-a will be an important strategy for the treatment of these inflammatory conditions.32,33 We evaluated the anti-inflammatory activity of the isolated compounds using exposure to LPS-induced NO34 and TNF-a35 production in the mouse macrophage RAW264.7 cell line. At a concentration of 100 lM, the MTT assay36 results showed that compounds 1–12 did not affect cell viability. Thus, the effects of compounds 1–12 on LPS-induced production of the inflammatory mediators NO and TNF-ain RAW 264.7 cells were evaluated at concentrations lower than 100 lM. In Table 5, compounds 7–12 showed potent inhibition of NO production. In particular, the activity of 12 was more potent than that of the positive control, minocycline (IC50, 55.1 lM), in inhibiting NO production with an IC50 value of 50.5 lM. In addition, compounds 1–4 exhibited a moderate TNF-a inhibitory effect, with IC50 values of 76.1, 47.9, 84.4 and 94.2 lM, respectively, and the activity of 2 was greater than that of the positive control silybin. Interestingly, consideration of the structures of 1–6 versus 7–12 suggested that 80 -OH was replaced by a 3-methoxy-4-hydroxy benzoic group resulting in an increase in the NO inhibitory activity and a decrease in the TNF-a inhibitory activity of 8-O-40 neolignans. Acknowledgements Financial support from the National Natural Science Foundation of China (81502954) and the Project of Innovation Team of Liaoning Foundation (20120016) is gratefully acknowledged. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.10. 012. References and notes 1. Edwards, J. E.; Brown, P. N.; Talent, N.; Dickinson, T. A.; Shipley, P. R. Phytochemistry 2012, 79, 5. 2. Liu, T.; Cao, Y.; Zhao, M. Food Chem. 2010, 119, 1656. 3. Chu, C. Y.; Lee, M. J.; Liao, C. L.; Lin, W. L.; Yin, Y. F.; Tseng, T. H. J. Agric. Food Chem. 2003, 51, 7583. 4. Kao, E. S.; Wang, C. J.; Lin, W. L.; Yin, Y. F.; Wang, C. P.; Tseng, T. H. J. Agric. Food Chem. 2005, 53, 430.

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5. The hawthorn seeds were collected in June 2011 from Hebei Province (China), and identified by Prof. Jin-Cai Lu (Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, P. R. China). The voucher specimen (No. 20110701) was deposited at the Herbarium of Shenyang Pharmaceutical University, Liaoning, P. R. China. The seeds of hawthorn (30 kg) were powdered, and extracted with 70% EtOH three times (40 L, 3 h for each extraction). The solvent was concentrated under reduced pressure to obtain a residue (1500 g) which was suspended in H2O (20 L) and partitioned with ethyl acetate (3  20 L) and n-BuOH (3  20 L). The n-BuOH extract (610 g) was suspended in H2O (5 L) and then chromatographed on a D101 macroporous resin column using H2O–EtOH (100:0, 70:30, 40:60 and 5:95) as the eluent. The H2O–EtOH (70:30) fraction (186 g) was chromatographed on a silica gel column and eluted with a gradient of CH2Cl2/MeOH to afford six fractions (1–6). Fraction 2 (20.2 g) was further purified by ODS column chromatography (CC) using MeOH–H2O as the mobile phase and a gradient (95:5, 90:10, 80:20, 70:30, 60:40, 50:50) to afford six fractions (F2-1–F2-6) based on HPLC analysis. F2-2 (4.5 g) was separated by silica gel CC (2.5  50.0 cm, CH2Cl2/MeOH 20:1–3:1, v/v), preparative HPLC (6 mL/min) and semipreparative HPLC (2.5 mL/min) respectively, to yield compounds 1 (30 mg), 2 (6 mg), 3 (53 mg), 4 (62 mg), 5 (33 mg) and 6 (34 mg). Similarly, F2-3 (3.8 g) was vacuum chromatographed on a silica gel column (2.5  50.0 cm, CH2Cl2/MeOH 15:1–2:1, v/v), preparative HPLC (6 mL/min) and semipreparative HPLC (2.5 mL/min) to obtain compounds 7 (7 mg), 8 (10 mg), 9 (22 mg), 10 (14 mg), 11 (3 mg) and 12 (4 mg). 6. Crataegusnin A (1) was a yellow oil; [a]20 D 7.0 (c 0.10, MeOH); UV (MeOH) kmax (log e): 228 (0.68), 287 (0.31); IR mmax (KBr): 3384, 2945, 2833, 1653, 1642, 1126, 1031, 649 cm1; CD [CH3OH, nm (e)]: 229 nm (1.02); HRESIMS at m/z 447.1608 [M+Na]+ (calcd for C21H28O9Na, 447.1626); 1H and 13C NMR (in CD3OD), see Tables 1 and 2. 7. Braga, A. C. H.; Zacchino, S.; Badano, H.; Sierra, M. G.; Rúveda, E. A. Phytochemistry 1984, 23, 2025. 8. Gan, M.; Zhang, Y.; Lin, S.; Liu, M.; Song, W.; Zi, J.; Yang, Y.; Fan, X.; Shi, J.; Hu, J.; Sun, J.; Chen, N. J. Nat. Prod. 2008, 71, 647. 9. Wang, L.; Li, F.; Yang, C. Y.; Khan, A. A.; Liu, X.; Wang, M. K. Fitoterapia 2014, 99, 92. 10. Crataegusnin B (2) was a yellow oil; [a]20 D 13.2 (c 0.12, MeOH); UV (MeOH) kmax (log e): 229 (0.72), 286 (0.32); IR mmax (KBr): 3374, 2944, 2832, 1454, 1124, 1031, 619 cm1; CD [CH3OH, nm (e)]: 229 nm (1.82); HRESIMS at m/z 447.1626 [M+Na]+ (calcd for C21H28O9Na, 447.1626); 1H and 13C NMR (in CD3OD), see Tables 1 and 2. 11. Xiong, L.; Zhu, C.; Li, Y.; Tian, Y.; Lin, S.; Yuan, S.; Hu, J.; Hou, Q.; Chen, N.; Yang, Y.; Shi, J. J. Nat. Prod. 2011, 74, 1188. 12. Spassov, S. L. Tetrahedron 1969, 25, 3631. 13. Crataegusnin C (7) was a yellow oil; [a]20 D +2.6 (c 0.10, MeOH); UV (MeOH) kmax (log e): 228 (0.67), 286 (0.29); IR mmax (KBr): 3422, 2940, 1661, 1594, 1463, 1273, 1126, 1032, 647 cm1; CD [CH3OH, nm (e)]: 233 nm (4.03); HRESIMS at m/z 533.2009 [M+Na]+ (calcd for C28H34O10Na, 533.2044); 1H and 13C NMR (in DMSO-d6), see Tables 1 and 2. 14. Zhu, J. X.; Ren, J.; Qin, J. J.; Cheng, X. R.; Zeng, Q.; Zhang, F.; Yan, S. K.; Jin, H. Z.; Zhang, W. D. Arch. Pharmacal Res. 2012, 35, 1739. 15. Frelek, J.; Szczepek, W. J. Tetrahedron: Asymmetry 1999, 10, 1507. 16. Hsiao, J. J.; Chiang, H. C. Phytochemistry 1995, 39, 899. 17. Rayanil, K. O.; Nimnoun, C.; Tuntiwachwuttikul, P. Phytochem. Lett. 2012, 5, 59. 18. Crataegusnin D (8) was a yellow oil; [a]20 D +15.0 (c 0.15, MeOH); UV (MeOH) kmax (log e): 227 (0.77), 287 (0.32); IR mmax (KBr): 3373, 2944, 2832, 1453, 1384, 1120, 1031, 619 cm1; CD [CH3OH, nm (e)]: 231 nm (3.92); HRESIMS at m/z 533.2013 [M+Na]+ (calcd for C28H34O10Na, 533.2044); 1H and 13C NMR (in DMSO-d6), see Tables 1 and 2. 20 19. Crataegusnin E (9) was a yellow oil; [a]D +16.2 (c 0.10, MeOH); UV (MeOH) kmax (log e): 226 (0.63), 285 (0.26); IR mmax (KBr): 3382, 2944, 2832, 1637, 1454, 1 1387, 1271, 1121, 1031, 619 cm ; CD [CH3OH, nm (e)]: 232 nm (0.99); HRESIMS at m/z 567.2169 [M+Na]+ (calcd for C29H36O10Na, 567.2201); 1H and 13 C NMR (in CD3OD), see Tables 1 and 2. 20. Crataegusnin F (10) was a yellow oil; [a]20 D +2.2 (c 0.10, MeOH); UV (MeOH) kmax (log e): 225 (0.72), 283 (0.29); IR mmax (KBr): 3364, 2944, 2832, 1453, 1118, 1 1031, 645 cm ; CD [CH3OH, nm (e)]: 238 nm (4.22); HRESIMS at m/z 567.2165 [M+Na]+ (calcd for C29H36O10Na, 567.2201); 1H and 13C NMR (in CD3OD), see Tables 1 and 2. 21. Crataegusnin G (11) was a yellow oil; [a]20 D +18.5 (c 0.10, MeOH); UV (MeOH) kmax (log e): 223 (0.80), 285 (0.34); IR mmax (KBr): 3375, 2943, 2832, 1650, 1453, 1 1270, 1126, 1031, 645 cm ; CD [CH3OH, nm (e)]: 238 nm (3.62); HRESIMS at m/z 581.2501 [M+Na]+ (calcd for C30H38O10Na, 581.2357); 1H and 13C NMR (in CD3OD), see Tables 1 and 2. 22. Greca, M. D.; Ferrara, M.; Fiorentino, A.; Monaco, P.; Previtera, L. Phytochemistry 1998, 49, 1299. 23. Li, L.; Seeram, N. P. J. Agric. Food Chem. 2011, 59, 7708. 24. Yang, X. W.; Zhao, P. J.; Ma, Y. L.; Xiao, H. T.; Zuo, Y. Q.; He, H. P.; Li, L.; Hao, X. J. J. Nat. Prod. 2007, 70, 521. 25. Riachi, L. G.; Maria, C. A. B. D. Food Chem. 2015, 176, 72. 26. Müller, L.; Fröhlich, K.; Böhm, V. Food Chem. 2011, 129, 139. 27. Hieda, Y.; Anraku, M.; Choshi, T.; Tomida, H.; Fujioka, H.; Hatae, N.; Hori, O.; Hirose, J.; Hibino, S. Bioorg. Med. Chem. Lett. 2014, 24, 3530. 28. Mistry, B. M.; Patel, R. V.; Keum, Y. S.; Kim, D. H. Bioorg. Med. Chem. Lett. 2015, 25, 5561. 29. The test samples and DPPH were all dissolved in ethanol. A series (5, 10, 20, 40 lL) of test samples (0.4 mM) and 20 lL 2 mM DPPH were added to 96-well

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plates. Ethanol was added to obtain a final volume of 200 lL and after incubating for 30 min in the dark at 37 °C, the OD values was measured using a Varioskan flash multimode reader at 517 nm. Ethanol was used instead of samples as the blank control. 30. A solution consisting of 7 mM ABTS and 2.4 mM potassium persulfate was reacted in the dark at room temperature for 12 h before being used. Then, it was mixed with ethanol to obtain an OD value of 0.700 at 734 nm. Different concentrations of test samples (100 lL) and ABTS+ solution (150 lL) were added to each well of the 96-well plates, and ethanol was used as a blank. After incubating at 37 °C for 30 min, the OD value was measured at 734 nm. The free radical scavenging capability was calculated using the equation below and expressed as the percentage inhibition rate of free radial scavenging compared with the blank. 31. The FRAP procedure was based on the method described in a previous report with some modifications. The FRAP reagent contained 25 mL sodium acetate (300 mM in acetic acid, pH 3.6), 2.5 mL TPTZ solution (10 mM in 40 mM HCl) and 2.5 ml FeCl36H2O solution (20 mM). To measure the FRAP value, 5 lL of each sample (3000, 1500, 750, 375 lg/mL) was mixed with 145 lL FRAP working reagent. Then, the mixture was reacted at 37 °C for 30 min and a series of FeSO47H2O aqueous solutions (5, 10, 50, 100, 250, 400, 450 lM) were to construct a calibration curve. The absorbance of the reaction solution was recorded at 593 nm. The FRAP values of the tested compounds were expressed as concentrations (lM) of Fe2+. 32. Pu, W.; Lin, Y.; Zhang, J.; Wang, F.; Wang, C.; Zhang, G. Bioorg. Med. Chem. Lett. 2014, 24, 5432.

33. Patel, N. K.; Bairwa, K.; Gangwal, R.; Jaiswal, G.; Jachak, S. M.; Sangamwar, A. T.; Bhutani, K. K. Bioorg. Med. Chem. Lett. 1952, 2015, 25. 34. NO produced by the cells was determined by assaying the levels of NO2 using Griess reagent. RAW264.7 cells were plated at a density of 5  105/mL in a 96well plate and pre-treated with test samples at concentrations of 100, 50, 10 and 1 lM. After the cells were incubated with samples and stimulated with LPS (100 ng/mL) for 24 h, the cell culture medium were collected to determine NO. Briefly, a 100 lL aliquot of each sample was added to an equal volume of Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% N-naphthyl-ethylenediamine dihydrochloride) in a 96-well plate, and then incubated at 37 °C for 10 min. After incubation, the absorbance was recorded at 540 nm on a Varioskan flash instrument. The amount of NO in the sample was calculated using a NaNO2 standard curve and minocycline was used as a positive control. 35. The levels of TNF-a were determined using an ELISA kit (R&D company, Minnesota, USA) according to the manufacturer’s instructions. TNF-a was determined from a standard curve and silybin was used as a positive control. 36. Mouse macrophage RAW264.7 cells were cultured in DMEM (including 10% FBS, 100 U/mL penicillin and 100 lg/mL streptomycin) at 37 °C in a 5% CO2 atmosphere. Cells (5  105 cells/well) with or without test samples, were cultured in a 96-well microplate. Untreated cells were used as the control and cell viability was evaluated by MTT assay. Cells were treated with test samples (100 lM) for 1 h and stimulated with LPS (100 ng/mL) for 24 h, and, then, the cells were incubated with MTT (0.25 mg/mL) followed by incubation for 4 h in 5% CO2, at 37 °C. The formation of formazan was recorded at 540 nm using a Varioskan flash instrument.