Sesquiterpenoids from the aerial parts of Conyza japonica and their inhibitory activity against nitric oxide production

Journal Pre-proof Sesquiterpenoids from the aerial parts of Conyza japonica and their inhibitory activity against nitric oxide production

Long-Gao Xiao, Si-Chen Zhang, Yu Zhang, Lu Liu, Hong-Li Zhang, Qian Yu, Lin-Kun An PII:

S0367-326X(19)32360-3

DOI:

https://doi.org/10.1016/j.fitote.2020.104473

Reference:

FITOTE 104473

To appear in:

Fitoterapia

Received date:

3 December 2019

Revised date:

6 January 2020

Accepted date:

6 January 2020

Please cite this article as: L.-G. Xiao, S.-C. Zhang, Y. Zhang, et al., Sesquiterpenoids from the aerial parts of Conyza japonica and their inhibitory activity against nitric oxide production, Fitoterapia (2020), https://doi.org/10.1016/j.fitote.2020.104473

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© 2020 Published by Elsevier.

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Sesquiterpenoids from the aerial parts of Conyza japonica and their inhibitory activity against nitric oxide production a

a

a

a

a

Long-Gao Xiao , Si-Chen Zhang , Yu Zhang , Lu Liu , Hong-Li Zhang , Qian Yu

a,b,*

, Lin-Kun An

a,c,*

a

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China

b

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal

University, Guangxi, 541004, China c Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, Guangzhou, 510006, China

*

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Corresponding author. E-mail address: [email protected] (Q. Yu), [email protected] (L.-K. An).

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Author Contribution Statement Long-Gao Xiao: Conceptualization, Investigation, Writing - Original Draft. Si-Chen Zhang:

e-

Investigation. Yu Zhang: Visualization. Lu Liu: Formal analysis. Hong-Li Zhang: Investigation.

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Qian Yu: Writing- Reviewing and Editing. Lin-Kun An: Supervision, Funding acquisition.

Journal Pre-proof ABSTRACT: Four new sesquiterpenoids, conyterpenols A−D (1−4), along with nineteen known analogues (5−23) were isolated from the aerial parts of Conyza japonica. The structures of 1−4 were determined through spectroscopic analysis, while their absolute configurations were determined by comparison of calculated and experimental electronic circular dichroism (ECD) spectra. Conyterpenol D (4) was a new type of sesquiterpenoid with a seven- membered lactone ring. Compounds 1−23 were evaluated for their inhibitory activity against LPS- induced nitric oxide production in RAW264.7 macrophages and cytotoxicity against human hepatoma cell line (HepG2) and human breast adenocarcinoma cell line (MCF-7). Compounds 3, 4, and 12 displayed moderate

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inhibition against NO production with IC50 values in the range of 26.4−33.6 μM. And all compounds

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showed no obvious cytotoxicity against these two cancer cell lines at 100 μM.

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Keywords: Conyza japonica; sesquiterpenoids; anti-inflammatory activity; cytotoxicity.

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1. Introduction

Conyza japonica (Thunb.) Less., a species of family Compositae, is an annual or biennial herb widely distributed in the southern regions of China. The plant C. japonica has a long history of medicinal use for treating various diseases such as pneumonia, pleurisy, laryngitis and keratitis in children [1]. Previous phytochemical researches on C. japonica showed there are a variety of constituents such as strictic acids, flavonoids and their glycosides [2,3], phenylpropanoid glycosides [4], triterpenoid saponins [5], diterpenoids and their glycosides [6]. The research on the constituents of C. japonica has attracted our attention because its secondary metabolites mainly contain terpenoids, which show a variety of biological activities, including anti- inflammatory, antitumor,

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f

antioxidant and against acute gastric ulcer [7–10].

Our phytochemical research was carried out on the 95% EtOH extract of the aerial parts of C.

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japonica, which resulted in the isolation of four new sesquiterpenoids (1−4) along with nineteen known analogues. The isolated compounds were screened for their inhibitory effects against

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LPS-induced nitric oxide (NO) production in RAW264.7 macrophages and their cytotoxicity against

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HepG2 and MCF-7 cancer cell lines. Herein, we report the isolation, structural elucidation, and

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biological evaluation of these sesquiterpenoids.

General experiments

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2.1.

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2. Experimental

Optical rotations were measured on a Perkin- Elmer 341 polarimeter (PerkinElmer, Inc. Shelton, USA). UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. CD spectra were obtained from an Applied Photophysics Chirascan spectrometer. IR spectra were determined by a Bruker Tensor 37 infrared spectrophotometer with KBr pellets. HRESIMS were measured on a Shimadzu LCMS-IT- TOF spectrometer. Nuclear magnetic resonance spectra were measured on a Bruker AM-400 or AM-500 spectrometer using tetramethylsilane as an internal reference. A SEP LC-52 equipped with UV-200 and a Dr. Maisch C18 column (250 × 10 mm, S-5 μm) was used for semi-preparative HPLC separation. RP-C18 silica gel (YMC, 50 μm), MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (100−200 and 200−300 Mesh, Qingdao Marine Chemical, Inc. China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography (CC). TLC was carried out on silica gel GF254 plates (Qingdao Marine Chemical, Inc. China). All solvents were analytical grade (Guangzhou Chemical Reagents Company, Ltd.). The RAW264.7 cells were purchased from the American Type Culture

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Collection. HepG2 and MCF-7 cell lines were purchased from China Center for Type Culture Collection in Wuhan.

2.2.

Plant material The aerial parts of C. japonica were collected in Quanzhou, Fujian Province, People's Republic

of China in May 2018, and identified by Dr. Gui- Hua Tang from the School of Pharmaceutical Sciences, Sun Yat-sen University. A voucher specimen (No. 2018051101) was deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.

2.3.

Extraction and isolation

f

The air-dried and powdered aerial parts of C. japonica (20.0 kg) were extracted with 95%

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EtOH (40 L × 3) at room temperature to obtain a crude extract (971 g), which was suspended in 10 L water and then extracted continuously with petroleum ether (10 L × 3) and ethyl acetate (10 L × 3) to

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afford the petroleum ether extract (324.9 g) and EtOAc extract (85.2 g). The petroleum ether extract

e-

was subjected to column chromatography over silica gel with petroleum ether−EtOAc mixture (gradient from 1:0 to 0:1, v/v) and CHCl3 −MeOH (gradient from 1:0 to 0:1, v/v) to give twelve

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fractions (Fr.1−Fr.12). Fr.3 was separated using silica gel column eluting with petroleum ether−EtOAc and further purified by using Sephadex LH-20 column (CH2 Cl2 −MeOH, 1:1, v/v) to

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afford 22 (13.0 mg). Fr.4 was separated using silica gel column eluting with petroleum ether−EtOAc

rn

further purified by using semi-preparative HPLC (MeOH/H2 O, 80%→100%, 3.0 ml/min) to afford 17 (20.8 mg, tR = 18.5 min). Fr.5 was separated using silica gel column eluting with petroleum

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ether−EtOAc and further purified by using semi-preparative HPLC (82% MeOH in H2 O, 3.0 ml/min) to yield 12 (9.4 mg, tR = 26.0 min). Fr.6 was separated by MCI gel column (MeOH/H2 O, 50%→100%, v/v) followed by silica gel column (petroleum ether−CH2 Cl2 , 2:1, v/v) and Sephadex LH-20 column (MeOH) to yield 16 (9.3 mg). Fr.7 was separated by MCI gel column (MeOH/H2 O, 50%→100%, v/v) followed by silica gel column (petroleum ether−acetone, 20:1, v/v) to yield 3 (4.7 mg). Fr.8 was separated by MCI gel column (MeOH/H2 O, 30%→100%, v/v) to afford Fr.8.1−8.2, which was analyzed by using TLC. Fr.8.1 was separated by Sephadex LH-20 column (CH2 Cl2 −MeOH, 1:1, v/v) and silica gel column (CH2 Cl2 −MeOH, 200:1, v/v) to yield 6 (33.2 mg). Fr.8.2 was separated by various columns and further purified by semi-preparative HPLC (65% MeOH in H2 O, 3.0 ml/min) to afford 7 (3.7 mg, tR = 14.5 min), 8 (4.3 mg, t R = 26.0 min), and 14 (9.3 mg, tR = 31.0 min). Fr.9 was separated using silica gel column eluting with CH2 Cl2 −MeOH mixture (gradient from 1:0 to 10:1, v/v) to give Fr.9.1−9.3. Fr.9.2 was fractionated by RP-C18 silica gel column (MeOH/H2 O, 50%→100%, v/v) to afford Fr.9.2.1−Fr.9.2.8, which was analyzed by

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using TLC. Fr.9.2.2 was further purified by silica gel column eluting with CH2 Cl2 to yield 23 (13.4 mg). Fr.9.2.4 was purified by silica gel column eluting with CH2 Cl2 −MeOH mixture to yield 19 (6.9 mg). Fr.9.2.5 was purified by semi-preparative HPLC (65% MeOH in H2 O, 3.0 ml/min) to afford 1 (4.3 mg, tR = 10.5 min). Fr.9.2.6 was separated by silica gel column to afford 20 (10.4 mg) and the rest was further purified by semi-preparative HPLC (65% MeOH in H2 O, 3.0 ml/min) to yield 9 (3.3 mg, tR = 14.7 min). Fr.9.2.7 was separated by various columns and further purified by semi-preparative HPLC (60% MeOH in H2 O, 3.0 ml/min) to afford 4 (3.7 mg, tR = 14.6 min), 2 (5.3 mg, tR = 20.9 min), and 15 (7.8 mg, tR = 22.6 min). Fr.9.3 was separated by RP-C18 silica gel column (MeOH/H2 O, 60% →100%, v/v) to afford Fr.9.3.1−Fr.9.3.6. Fr.9.3.3 was separated by silica gel

f

columns with different eluent to give 11 (3.2 mg) and 13 (4.5 mg). Fr.9.3.4 was purified with the

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similar procedure to give 10 (6.6 mg). Fr.10 was separated by MCI gel column (MeOH/H2 O,

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30%→100%, v/v) followed by silica gel column and semi-preparative HPLC (70% MeOH in H2 O, 3.0 ml/min) to yield 5 (4.1 mg, tR = 15.0 min). Fr.11 was fractionated by silica gel column to get

e-

Fr.11.1−Fr.11.8, which was analyzed by using TLC. Fr.11.4 was separated by various columns and

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further purified by semi-preparative HPLC (30% MeOH in H2 O, 3.0 ml/min) to yield 21 (4.7 mg, tR = 13.7 min). Fr.11.6 was separated by Sephadex LH-20 column (MeOH) and silica gel column

2.4.

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(CH2 Cl2 −MeOH, 50:1, v/v) to yield 18 (10.6 mg).

Spectral data of new compounds

Conyterpenol A (1): Colorless oil; [ ] D −34.5 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 202 (3.40)

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20

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nm, 194 (2.47) nm; IR (KBr) ν max 3422, 2958, 2873, 1705, 1459, 1380, 1259, 1031, 966, 694 cm−1 ; NMR (CD3 OD) data, see Tables 1 and 2; HRESIMS m/z 245.1524 [M + Na]+ (calcd for C 14 H22O2Na, 245.1512).

Conyterpenol B (2): Colorless oil; [ ] D −43.1 (c 0.26, MeOH); UV (MeOH) λmax (log ε) 202 (3.65) 20

nm, 193(2.79) nm; IR (KBr) ν max 3400, 2954, 2931, 2872, 1687, 1610, 1499, 1453, 1373, 1261, 1054, 702 cm−1 ; NMR (CDCl3 ) data, see Tables 1 and 2; HRESIMS m/z 259.1679 [M+Na]+ (calcd for C15 H24 O 2Na, 259.1669). Conyterpenol C (3): Colorless oil; [ ]D −27.3 (c 0.30, MeOH); UV (MeOH) λmax (log ε) 240 (3.58) 20

nm, 201(3.32) nm, 197(3.19) nm; IR (KBr) ν max 3483, 2957, 2928, 1645, 1455, 1385, 1283, 1040, 816 cm−1 ; NMR (CDCl3 ) data, see Tables 1 and 2; HRESIMS m/z 259.1668 [M+Na]+ (calcd for C15 H24 O 2Na, 259.1669). Conyterpenol D (4): Light yellow oil; UV (MeOH) λmax (log ε) 202 (3.95) nm, 317 (3.93) nm, 231

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(3.61) nm; IR (KBr) ν max 3428, 2925, 2855, 1762, 1664, 1611, 1521, 1458, 1369, 1096, 961, 824 cm−1 ; NMR (CDCl3 and DMSO-d6 ) data see Tables 1 and 2; HRESIMS m/z 267.0999 [M+Na]+ (calcd for C 15 H16 O3Na, 267.0992). Table 1 1 H NMR spectrum data of compounds 1−4 (δ in ppm, J in Hz). a

3.84 d (11.7)

2

b

c

d

3

4

4

−

−

−

−

2.04 m

2.13 m

1.32 m

7.23 d (8.1)

7.24 d (7.8)

3

1.85 td (12.9, 4.6) 2.56 td (14.3, 6.9) 2.28 dd (14.3, 4.2)

1.50 m 1.77 m 1.44 m

1.66 overlapped 1.64 overlapped 2.00 dd (13.4, 7.2)

7.78 d (8.1)

7.78 d (7.8)

4

−

1.91 tt (11.0, 6.1)

−

−

−

5 6

2.22 m 5.70 s

1.94 dd (12.3, 3.0) 2.41 d (12.6) 2.29 dd (19.0, 4.2) 6.38 d (7.0)

7.78 d (8.1) 7.23 d (8.1)

7.78 d (7.8) 7.24 d (7.8)

7

2.45 dd (11.0, 4.8) 1.93 m 2.13 overlapped −

−

−

8

5.35 s

2.22 m 2.29 m

−

7.38 s

8.03 s

9 10

2.13 overlapped −

2.51 m −

− 2.70 d (15.3)

− 5.46 s

− 5.58 s

f

1

b

oo

2

−

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1

e-

No.

Pr

2.45 d (15.3)

2.22 m 1.02 m

1.65 dd (13.5, 6.8) 0.89 d (6.7)

1.73 dt (13.6, 6.7) 0.92 d (6.7)

− −

− −

13 14

1.02 m 0.69 s

0.82 d (6.7) 4.13 s

0.96 d (6.7) 1.86 s

1.55 s 1.55 s

1.38 s 1.38 s

1.02 s

1.09 s

2.38 s

2.30 s 5.08 s

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11 12

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15 OH-9

Recorded in CD 3OD at 500 M Hz.

b

Recorded in CDCl3 at 500 M Hz.

c

Recorded in CDCl3 at 400 M Hz.

d

Recorded in DM SO-d6 at 500 M Hz.

2.5.

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Anti-inflammatory assay Compounds 1-23 were tested for their anti- inflammatory activity in vitro by measuring the

LPS-induced NO production in RAW 264.7 mouse macrophages [11]. Cell viability was determined by MTT method at 60 μM, an initial concentration. Cells were treated with compounds in different concentrations with or without LPS (1 μg/mL) for 24 h in 96-well culture plates. The supernatant was collected and then mixed with an equal volume of Griess reagent I and Griess reagent II, the absorbance of the samples was read at 540 nm with a microplate reader. Quercetin was used as a positive control and DMSO was set as blank control in experiments. Experiments were operated in triplicate. The results were described as mean ± SD of three independent experiments.

2.6.

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MTT assay

Cytotoxic activity of the compounds against HepG2 and MCF-7 cell lines was analyzed by MTT assay as previously reported [12,13]. Briefly, the cancer cells were treated with the compounds at concentrations ranging from 10-8 to 10-4 M, after incubation for 72 h at 37 ℃, the MTT solution (20 μL, 2.5 mg/mL) in PBS was fed to each well of the culture plate. After 4 h incubation, the formazan crystal formed in the well was dissolved with 100 mL DMSO for optical density reading at 570 nm. Camptothecin was used as a positive control and DMSO was set as blank control in experiments, and the experiments were conducted for three independent replicates.

4c 140.2

4d 139.2

2 3

29.8 38.5

32.9 23.4

40.0 34.9

129.7 127.2

129.4 126.8

4

212.3

49.4

85.7

126.5

126.5

5 6

50.1 21.5

47.2 129.3

52.0 27.5

127.2 129.7

126.8 129.4

7

140.8

140.6

139.1

129.8

127.4

8

115.6

26.1

137.4

135.1

137.7

9 10 11 12

38.1 40.7 34.9 20.6

38.9 215.1 30.9 21.8

203.0 59.3 36.9 17.4

146.3 122.3 70.9 168.2

144.7 125.2 69.0 168.4

13 14

20.1 10.4

18.3 67.6

18.2 22.2

30.6 30.6

30.1 30.1

15

−

19.4

20.1

21.6

21.0

b c d

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a

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3b 40.9

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2b 59.8

e-

1a 76.3

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No. 1

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Table 2 13 C NMR spectrum data of compounds 1−4 (δ in ppm).

Recorded in CD 3OD at 125 M Hz. Recorded in CDCl3 at 125 M Hz. Recorded in CDCl3 at 100 M Hz.

Recorded in DM SO-d6 at 125 M Hz.

3. Results and discussion The petroleum ether extract from the aerial parts of C. japonica was fractionated by silica gel CC and further purified by various columns to obtain twenty-three sesquiterpenoids, including four new sesquiterpenoids, conyterpenols A−D (1−4) (Fig. 1) and nineteen known sesquiterpenoids (5−23)

(Fig.

S1),

which

were

identified

1β,6α-dihydroxy-4(15)-eudesmane (6) [15],

as

1β,11-dihydroxy-5-eudesmene

(5)

[14],

6β ,14-epoxyeudesm-4(15)-en-1β-ol (7)

[16],

3-epichenopotriol (8) [17], 7-epi-eudesm-4(15)-ene-1α,6α-diol (9) [18], 4(15)-eudesmene-1β,5α-diol (10) [19], oplodiol (11) [20], schisansphenins A (12) [21], 10β,15-hydroxy-α-cadinol (13) [22],

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artabotrol (14) [23], (+)-aphanamol I (15) [24], macrocarp-11(15)-en-8-ol (16) [25], spathulenol (17) [26], 4α,7α-aromodendranediol (18) [27], oplopanone (19) [20], (7R*)-opposit-4(15)-ene-1β,7-diol (20) [16], dehydrovomifoliol (21) [28], inflatenone (22) [29], pubescone (23) [30], respectively, by comparing the detailed NMR spectroscopic data with those reported in the literature.

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Fig. 1. Structures of compounds 1–4.

Compound 1 was obtained as colorless oil. Its HRESIMS gave an ion peak at m/z 245.1524 [M

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+ Na]+, suggesting a molecular formula of C14 H22 O2 (calcd for C 14 H22O2Na, 245.1512) with four

carbonyl group (1705 cm−1 ). The 1 H,

13

e-

degrees of unsaturation. The IR spectrum showed absorption of hydroxy group (3422 cm−1 ) and C NMR (Tables 1 and 2) and HSQC spectra showed one

Pr

carbonyl carbon at δC 212.3 (C-4), one double bond [δH 5.35 (1H, s, H-8), δC 115.6 (C-8), 140.8 (C-7)], one oxymethine group [δH 3.84 (1H, d, J = 11.7 Hz, H-1), δC 76.3 (C-1)], three methyl

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groups [δH 1.02 (3H, m, H-12), δH 1.02 (3H, m, H-13), δH 0.69 (3H, s, H-14), δC 20.6 (C-12), δC 20.1

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(C-13), δC 10.4 (C-14)]. The carbonyl group, and one double bond unit accounted for two degrees of

1

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unsaturation. The remaining two degrees of unsaturation were due to a bicyclic carbon skeleton. The H- 1 H COSY correlations between H-1/H-2/H-3, H-5/H-6, H-8/H-9, H-11/H-12, H-11/H-13 as

drawn in red bold bonds (Fig. 2) built up four spin-spin coupling systems, which could be linked together via three quaternary carbons (C-4, C-7, and C-10) by the HMBC correlations from H-2 and H-5 to C-4 (δC 212.3), H-6, H-9, and H-13 to C-7 (δC 140.8), H-2, H-8, and H-14 to C-10 (δC 40.7). Following the establishment of the planar structure, the relative configuration of compound 1 was then assigned by analysis of NOESY spectrum (Fig. 3). The NOESY correlations between H-1/H-5, H-5/H-2a (δH 2.04), and H-14/H-2b (δH 1.85) indicated that Me-14 and H-2b were on the same side of the ring, while H-4, H-5, and H-2a on the opposite side. In order to establish the absolute configuration of compound 1, the ECD spectrum of 1 was calculated by time-dependent density functional theory (TDDFT) means at the B3LYP/6–311+G(d) level in MeOH [31]. The results exhibited that the experimental CD curve matched well with the calculated ECD of (1R,5R,10R)- isomer

(Fig.

4).

Therefore,

the

compound

1

could

be

identified

(1R,5R,10R)-1-hydroxy-7-isopropyl-10-methyl-1,2,5,6,9,10-hexahydronaphthalen-4(2H)-one

as and

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the trivial name conyterpenol A was given for compound 1.

e-

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Fig. 2. The key 1 H-1 H COSY and HMBC correlations of compounds 1–4.

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Fig. 3. The key NOESY correlations of compounds 1−3.

Compound 2 was obtained as colorless oil. Its HRESIMS spectrum gave an ion peak at m/z

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259.1679 [M + Na]+ suggesting a molecular formula of C 15 H24O2 (calcd for C 15 H24O2Na, 259.1669)

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with four degrees of unsaturation. The IR spectrum revealed the presence of hydroxyl group (3400 cm−1 ) and carbonyl group (1687 cm−1 ). The 1 H,

13

C NMR (Tables 1 and 2) and HSQC spectra of 2

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showed one carbonyl carbon at δC 215.1 (C-10), one double bond [δH 5.70 (1H, s, H-6), δC 129.3 (C-6) and 140.6 (C-7)], one hydroxymethyl group [δH 4.13 (2H, s, H-14), δC 67.6 (C-14)], three methyl groups [δH 0.89 (3H, d, J = 6.7 Hz, H-12), δH 0.82 (3H, d, J = 6.7 Hz, H-13), δH 1.02 (3H, s, H-15), δC 21.8 (C-12), δC 18.3 (C-13), δC 19.4 (C-15)]. The NMR data suggested that compound 2 was an isodaucane-type sesquiterpenoid. The COSY spectrum showed correlations between H-2/H-3/H-4, H-4/H-11/ H-13, H-12/H-11/H-13, H-5/H-6, H-8/H-9 as drawn in red bold bonds (Fig. 2). The HMBC spectrum showed correlations from H-3 and H-6 to C-1 (δC 59.8), H-6 and H-14 to C-8 (δC 26.1), H-6 to C-14 (δC 67.6), H-14 to C-7 (δC 140.6), H-8, H-9, and H-15 to C-10 (δC 215.1). The NMR spectra indicated that compounds 2 and 15 shared the same planar structure with slight difference in the

13

C NMR spectrum caused by variation in configuration. The relative configuration

of compound 2 was determined by NOESY spectra (Fig. 3). The NOESY correlations between H-15/H-3b (δH 1.77), H-15/H-4, and H-5/H-3a (δH 1.44) indicated that Me-15, H-3b, and H-4 were on the same side of the ring, while H-5 and H-3a on the opposite side. The absolute configuration of

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2 was identified as (1S,4S,5R)- isomer by comparison of the calculated and experimental ECD spectra

(Fig.

4).

Thus,

the

compound

2

could

be

defined

as

(1S,4S,5R)-1- methyl-4- isopropyl-7-hydroxymethyl-1,2,3,5,8,9-hexahydroazulen-10(1H)-one (Fig. 1),

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rn

al

Pr

e-

pr

oo

f

and conyterpenol B as trivial name.

Fig. 4. Experimental and calculated ECD spectra for 1−3 in MeOH.

Compound 3 was obtained as colorless oil. Its HRESIMS spectrum gave an ion peak at m/z 259.1668 [M + Na]+ suggesting a molecular formula of C 15 H24O2 (calcd for C 15 H24O2Na, 259.1669) with four degrees of unsaturation. The two distinct absorption bands at 3483 and 1645 cm −1 in the IR spectrum demonstrated the existence of hydroxyl and α, β-unsaturated carbonyl groups. The 1 H,

13

C

NMR (Tables 1 and 2) and HSQC spectra of 3 showed one carbonyl carbon δC 203.0 (C-9), one

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double bond [δH 6.38 (1H, d, J = 7.0 Hz, H-7), δC 137.4 (C-7), and 139.1 (C-8)], one oxygenated quaternary carbon δC 85.7 (C-4), four methyl groups [δH 0.92 (3H, d, J = 6.7 Hz, H-12), δH 0.96 (3H, d, J = 6.7 Hz, H-13), δH 1.86 (3H, s, H-14), δH 1.09 (3H, s, H-15), δC 17.4 (C-12), δC 18.2 (C-13), δC 22.2 (C-14) , δC 20.1 (C-15)]. The NMR data suggested that compound 3 was a daucane-type sesquiterpenoid and closely comparable to the known compound Trichocarotin A [32]. The difference between the two compounds is that compound 3 bears a methylene group instead of the oxymethine group in Trichocarotin A. The COSY correlations between H-3/H-2 as drawn in red bold bonds (Fig. 2) and the HMBC correlations from H-3 to C-1 (δC 40.9), H-3 to C-11 (δC 36.9) also confirmed this variety. Thus, the planar structure of 3 was elucidated as shown in Fig. 1. The relative

f

configuration of compound 3 was determined by NOESY spectra (Fig. 3). The NOESY correlations

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between H-5/H-11, H-11/H-2b (δH 1.32), and, H-15/H-2a (δH 1.66) indicated that Me-15 and H-2a

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were on the same side of the ring, while the isopropyl, H-5 and H-2b on the opposite side. By comparison of the experimental and calculated ECD spectra (Fig. 4), compound 3 could be identified

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as (1S,4S,5S)-1,8-dimethyl-4- isopropyl-4-hydroxy-1,2,3,5,6,10-hexahy-droazulen-9(1H)-one (Fig. 1),

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and conyterpenol C as trival name.

Compound 4 was obtained as light- yellow oil. The HRESIMS spectrum gave an ion peak at m/z 267.0999 [M + Na]+ suggesting a molecular formula of C 15 H16O3 (calcd for C 15 H16 O3 Na, 267.0992)

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with eight degrees of unsaturation. The IR spectrum showed the presence of hydroxyl group (3428

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cm−1 ), carbonyl group (1762 cm−1 ), and aromatic ring (1664, 1611, 1521, and 1457 cm−1 ). Its 1 H, 13 C

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NMR (Tables 1 and 2), DEPT and HSQC spectra showed one carbonyl carbon at δC 168.2 (C-12), one benzene ring with para-disubstitution [δH 7.23 (2H, d, J = 8.1 Hz, H-2 and H-6), δH 7.78 (2H, d, J = 8.1 Hz, H-3 and H-5), δ C 129.7 (C-2 and C-6), δC 127.2 (C-3 and C-5)], two double bonds [δH 7.38 (1H, s, H-8), δC 129.8 (C-7) and δC 135.1 (C-8), δH 5.46 (1H, s, H-10), δC 146.3 (C-9) and δC 122.3 (C-10)], one oxygenated quaternary carbon at δC 70.9 (C-11), and three methyl groups [δH 1.55 (6H, s, H-13 and H-14), δH 2.38 (3H, s, H-15), δC 30.6 (C-13 and C-14), δC 21.6 (C-15)]. The remaining one degree of unsaturation required a cyclic ring. The signal of C-11 (δC 70.9) appeared in higher field than that of general oxygenated quaternary carbons. There was an ester carbonyl group at δC 168.2 (C-12), suggesting the cyclic carbon skeleton might be a lactone ring. The HMBC correlations from H-15 to C-1 (δC 140.2) and C-2/6 (δC 129.7) suggested that the Me-15 connected with C-1. The correlations from H-3 and H-5 to C-7 (δC 129.8), H-8 to C-4 (δC 126.5) confirmed that the benzene ring connected with the lactone ring by C-7. The HMBC correlation from OH-9 to C-10 (δC 125.2) confirmed that the hydroxyl group linked to C-9. Furthermore, HMBC spectrum showed the correlations from H-8 to C-9 (δ C 146.3) and C-12 (δC 168.2), H-10 to C-8 (δC 135.1) and C-13

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(δC 30.6), H-13 to C-11 (δC 70.9). Hence, 4 could be identified as 5-hydroxy-7,7-dimethyl-3-(p-tolyl) oxepin-2(7H)-one (Fig. 1), and conyterpenol D as trivial name. Plausible biosynthetic pathways for the conyterpenol D was postulated as shown in Fig. 5. Conyterpenol D was probably biosynthesized from α-curcumene by epoxidation, hydrolysis, dehydration, dehydrogenation, oxidation, intramolecular esterification, and ketone carbonyl

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enolization reactions.

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Fig. 5. Hypothetical biosynthesis of conyterpenol D.

The anti- inflammatory activity of the isolated sesquiterpenoids was evaluated by measuring

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their inhibition against LPS- induced NO production in RAW264.7 cells using quercetin as the

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positive control. The IC 50 values, defined as the concentration of compounds to inhibit 50% NO production, were summarized in Table 3. The positive control quercetin had IC 50 value of 13.8 M. Most compounds showed weak inhibitory activity with IC 50 > 60 M. Compounds 3, 4, and 12 showed moderate inhibitory activity with IC 50 values from 26.4 to 33.6 M. The cytotoxicity of the isolated compounds was also evaluated in HepG2 and MCF-7 cancer cell lines using the MTT method. All compounds showed no obvious cytotoxicity at 100 μM. Table 3 Inhibitory effects of the isolated compounds on the LPS-induced NO production in RAW264.7 cells. a compounds IC50 (μM) cell viability (%) b 3

33.6 ± 1.5

59.6

4

26.4 ± 0.1

64.3

12 Quercetin

33.5 ± 0.4 13.8 ± 0.4

60.7 55.1

a

The IC50 values of the compounds not shown were more than 60 μM .

b

The cell viability after treatment with compounds at 60 μM .

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4. Conclusions

Twenty-three sesquiterpenoids, including four new sesquiterpenoids, named conyterpenols A−D (1−4), were isolated from the aerial parts of C. japonica. Conyterpenol D (4) was a new type of sesquiterpenoid with a seven membered lactone ring and benzene ring. All isolates showed multiple carbon skeletons, mainly including eudesmane, cadinane, isodaucane, daucane and aromadendrane. This work enriches the chemical diversity of C. japonica. Compounds 3, 4, and 12 displayed moderate inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages with IC50 values of 33.6 ± 1.5 μM, 26.4 ± 0.1 μM, and 33.5 ± 0.4 μM respectively, suggesting that they

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possess potential anti-inflammatory activity.

Declaration of Competing Interest

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The authors declare no conflict of interest.

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Acknowledgments

This work is supported by the National Natural Science Foundation of China (No. 81703668), Guangdong Natural Science Fund (Nos. 2019A1515011317 and 2017A030310396), Guangdong

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Provincial Key Laboratory of Construction Foundation (No. 2017B030314030), and State Key

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Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal

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University) (CMEMR2017-B04).

Appendix A. Supplementary data Experimental details including IR, CD, HRESIMS, and NMR (1 H NMR, 13 C NMR, DEPT135, 1

H- 1 H COSY, HSQC, and HMBC) spectra of compounds 1−4, and NOESY spectra of compounds

1−3.

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Graphical abstract

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Highlights

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Four new sesquiterpenoids were isolated from the aerial parts of Conyza japonica (Thunb.) Less..



A new type of sesquiterpenoid with seven-membered lactone ring and benzene ring.

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Structure of new compounds were elucidated through various spectroscopic analysis. The inhibitory activity of isolated compounds against LPS-induced NO production in

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RAW264.7 macrophages was evaluated.

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

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5