Ent-kaurane diterpenoids from the cherries of Coffea arabica

Fitoterapia 132 (2019) 7–11

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Ent-kaurane diterpenoids from the cherries of Coffea arabica a,b

Xia Wang

a

, Xingrong Peng , Jing Lu

a,b

a,b

, Guilin Hu

, Minghua Qiu

a,b,⁎

T

a

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China b University of the Chinese Academy of Sciences, Beijing 100049, People's Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Coffea arabica Cherries Diterpenoids Biosynthesis Structure elucidation

Four new rearranged ent-kaurane diterpenoids named as caffruenol A (1), caffruenol B (2), caffruolide A (3), caffruolide B (4), along with eleven known analogues (5–15) were isolated from the cherries of Coffea arabica. The structures of new compounds were elucidated by extensive spectroscopic analysis and X-ray crystallography. Coffruenols A and B (1 and 2) possessed an ent-4(18)-kaurene framework and might play an important role on the biosynthesis of the rearranged diterpenes that occurred in C. arabica. Moreover, inhibitory effects of compounds 1–4 on nitric oxide (NO) production in lipopolysaccharide-activited RAW 264.7 macrophages were evaluated.

1. Introduction

2. Experimental part

Coffea arabica (Rubiaceae) is one of the most important species cultivated for drinking in genus Coffea [1]. Previous phytochemical investigations on green and roasted coffee beans of C. arbaica had resulted in the isolation of alkaloids, phenolic acids, flavonoids and diterpenoids [2–4]. Coffee diterpenoids are considered to be responsible for some good impacts of coffee consumption on human health, such as anti-hepatitis, anti-oxidant and anti-cancers [5–7], but also they were proved to have the potential to increase serum lipids [8]. Until now, more than 60 diterpenoids have been isolated from C. arabica, including a series of rearranged, oxygenated and degraded ent-kaurane derivatives [9–11]. Among them, 16-ent-kauren-19-ol showed cytotoxic effect against K569 cells (IC50: 6.8 μg/mL) and (2-desoxy-carboxyatractyligenin)-β-D-glucopyranoside showed the inhibition of mitochondrial adenine nucleotide translocase (IC50: 1.0 μM) [12,13]. Meanwhile, mozambioside and mascaroside were two diterpenoid glucosides which was contributed to the bitter taste of coffee brews [11,14]. Therefore, in the course of ongoing investigations on diterpenoids in C. arabica, four new rearranged ent-kaurane diterpenoids (1–4), and eleven known analogues (5–15) were isolated from the cherries of C.arabica (Fig. 1). Herein, we reported the isolation and structural identification of these new compounds and their inhibitory activities on NO production.

2.1. General Ultraviolet spectra were measured by UV-2401 PC spectrophotometers (Shimadzu, Japan). A Bruker Tensor-27 instrument (Bruker, German) was used for recording infrared spectra by using KBr pellets. A Jasco P-1020 polarimeter (Jasco, Japan) was used to obtain optical rotations and HRESIMS data were measured by an API QSTAR Pulsar spectrometer (Waters, UK). The Bruker DRX-600 instrument (Bruker, Switzerland) were used to detect 1D and 2D NMR spectra with TMS as internal standard for chemical shifts. Semi-preparative HPLC was performed on an Agilent HP1100 or 1260 series instrument with a UV L-2400 detector (Agilent, USA) and an ZORBAX SB C-18 column (5 μm, 9.4 mm × 250 mm, wavelength detection at 210 nm). TLC detection was performed on TLC plates (200–250 μm thickness, Qingdao Marine Chemical, Inc., China). The ordinary column chromatographic materials include Lichroprep RP-18 (40–63 μm, Fuji, Japan), Sephadex LH-20 (20–150 μm, Pharmacia, USA), Silical gel (200–300 mesh, Qingdao Marine Chemical, Inc., China) and Macroporous resin (0.3–1.25 mm, Mitsubishi Chemical Corporation, Japan). The industrial-grade methanol, chloroform, ethyl acetate, acetone, petroleum ether were purchased from Tianjing Chemical Reagents Co. (Tianjing, China). The analytical-grade acetonitrile were purchased from Aladdin Industrial Corporation (Shanghai, China).

⁎ Corresponding author at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China. E-mail address: [email protected] (M. Qiu).

https://doi.org/10.1016/j.fitote.2018.08.023 Received 10 May 2018; Received in revised form 23 August 2018; Accepted 29 August 2018 Available online 07 September 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. Structures of ent-kaurane diterpenoids isolated from the cherries of C. arabica.

rate = 3.0 mL/min, UV 210, 280 nm) to get 6 (8 mg, tR = 22.4 min), 7 (5 mg, tR = 25.4 min), 8 (2 mg, tR = 30.6 min), 9 (4 mg, tR = 38.8 min). Fr. E-4 (1.6 g) was divided into four minor fractions by use of Sephadex LH-20 (5.0 × 200 cm, eluted with MeOH, 100%, 2 L), then Fr. E-4-3 was applied to RP C-18 (3.0 × 70 cm) and eluted in a gradient of MeOH/H2O (40 → 80%, v/v) to yield minor fractions, then Fr. E-4-32 was treat by reverse-phase semi-preparative HPLC (CH3CN/H2O: 45 → 60%, 30 min, flow rate = 3.0 mL/min, UV 210 nm) to gain 11 (5 mg, tR = 14.5 min). Fr. E-5 (1.8 g) was chromatographed on a silica gel column (5.0 × 50 cm), eluted with CHCl3/MeOH (20:1, v/v) to yield four minor fractions, then, Fr. E-5-3 was recrystallized to obtain 1 (12 mg). Fr. E-6 (930 mg) was subjected to silica gel column chromatography (3.0 × 50 cm, eluted with CHCl3/MeOH, 20:1, v/v) to afford four subfractions. Then, Fr. E-6-2 (180 mg) was applied to chromatography over RP C-18 (2.0 × 20 cm, eluted with MeOH/H2O, 30–60%, v/v) to obtain 2 (5 mg), 5 (3 mg) and 12 (5 mg). Fr. E-7 (1.6 g) was separated by use of Sephadex LH-20 (5.0 × 200 cm, eluted with MeOH, 100%, 2 L), then was chromatographed on a silica gel column (CHCl3/ MeOH, 20:1, v/v) to yield four minor fractions. Then, Fr. E-7-2 (400 mg) was treated by reverse-phase semi-preparative HPLC (CH3CN/ H2O: 40 → 70%, 60 min, flow rate = 3.0 mL/min, UV 210 nm) to get 13 (4 mg, tR = 34.2 min), 14 (9 mg, tR = 36.4 min) and 15 (5 mg, tR = 40.3 min).

2.2. Coffee cherries material The air-dried cherries of C. arabica cultivated in Yunnan province (China) were harvested in July 2016 and were identified by Prof. Hongbo Zhang, Dehong Institute of Tropical Agriculture. A specimen NO. KCF1606 was deposited in State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The powder of air-dried coffee cherries (24 kg) was extracted by methanol at 80 °C for three times (3 h for each time). The combined methanol extract was evaporated under reduced pressure. Then, the 2.5 kg residue was suspended in water and extracted with petroleum ether, ethyl acetate (EtOAc) and n-butanol, in turn. The EtOAc layer (200 g) was separated on a macroporous resin column (20.0 × 120 cm) and eluted in a step gradient manner with MeOH/H2O (0:100, 20:80, 40:60, 60:40, 80:20, 100:0, v/v) to yield six fractions: Fr. A (10 g), Fr. B (24 g), Fr. C (25 g), Fr. D (38 g), Fr. E (16 g), Fr. F (30 g), respectively. Fr. E (16 g) was then further subjected to silica gel column chromatography (15.0 × 80 cm), eluting in a gradient system of CHCl3/MeOH (100:0 → 1:2, v/v) to yield seven sub-fractions (Fr. E-1–Fr. E-7) on the basis of TLC analysis. Fr. E–1 (420 mg) was applied to a silica gel column (5.0 × 50 cm) and eluted with CHCl3/acetone (80:1 → 20:1, v/ v) to get 10 (11 mg). Fr. E-2 (760 mg) was subjected to chromatography over silica gel (5.0 × 80 cm), eluting with a CHCl3/acetone (50:1 → 10:1, v/v) gradient system to afford four minor fractions (Fr. E-2-1–Fr. E-2-4), followed with recrystallization of Fr. E-2-2 to obtain 4 (6 mg). Then, Fr. E-2-4 was separated by preparative thin layer chromatography (P-TLC, eluting with CHCl3/MeOH, 30:1, v/v) to gain 3 (2 mg). Fr. E-3 (2 g) was separated by use of Sephadex LH-20 (5.0 × 200 cm, eluted with MeOH, 100%, 2 L) and divided into three fractions (Fr. E-31−Fr. E-3-3). After that, Fr. E-3-2 (700 mg) was chromato-graphed on a silica column (CHCl3/MeOH), then separated by reverse-phase semipreparative HPLC (CH3CN/H2O: 30 → 80%, 50 min, flow

2.4. Spectral data Caffruenol A (1): colorless prisms, [α]24 D − 84.2 (c = 0.2, MeOH); UV (MeOH) λmax (log ε): 203 (3.69), 300 (1.95) nm; IR (KBr) vmax: 3420, 3060, 2950, 1675, 1456, 1390, 1200, 1080, 830 cm−1; HRESIMS m/z 327.2294 [M + Na]+ (calcd for C20H32O2 Na, 327.2295); 1H and 13 C NMR data shown in Table 1. Crystal data of 1: C20H32O2, M = 304.45, orthorhombic, space group P212121, a = 7.4712 (2) Å, b = 10.5461 (3) Å, c = 21.7260 (6) Å, α = β = γ = 90°, V = 1711.88.4 (8) Å3, Z = 4, d = 1.181 mg/cm3, crystal dimensions 0.870 × 0.470 × 120 mm was used for measurements on a Bruker APEX DUO diffractometer with a graphite 8

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Table 1 The 1H NMR (600 MHz) and Position

13

C NMR (150 MHz) data of compounds 1–4 (δH in ppm, J in Hz). NMR solvents was aCDCl3, bMeOD, cC5D5N.

1a 13

C

2b 1

13

H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

40.1 (t) 22.9 (t) 29.6 (t) 145.5 (s) 52.9 (d) 23.2 (t) 37.3 (t) 44.6 (s) 53.6 (d) 41.4 (s) 19.2 (t) 31.0 (t) 38.1 (d) 39.9 (t) 45.1 (t) 43.3 (d) 67.6 (t)

1.86 1.66 2.64 – 1.67 1.66 0.99 – 1.15 – 1.63 1.50 2.07 1.50 1.59 1.97 3.40

(m), 1.42 (m) (m), 1.46 (m) (dd, 11.9, 4.2), 1.69 (m)

18 19 20 OMe-1

118.8 (d) 59.0 (t) 15.1 (q) –

5.20 (t, 7.2) 4.19 (t, 7.2) 0.79 (s) –

(m) (m),1.46 (m) (m), 1.84 (m) (d, 6.8) (m), 1.52 (m) (m), 1.41 (m) (s-like) (m), 0.98 (m) (m), 0.91 (dd, 13.3,5.4) (p, 7.7) (dd, 7.7, 5.4)

C

3a 1

13

H

41.6 (t) 23.8 (t) 30.4 (t) 144.9 (s) 54.0 (d) 24.1 (t) 41.2 (t) 45.6 (s) 55.7 (d) 42.5 (s) 19.8 (t) 27.0 (t) 46.2 (d) 38.2 (t) 53.7 (t) 82.8 (s) 66.8 (t)

1.60 1.54 2.65 – 1.70 1.64 1.85 – 1.16 – 1.62 1.64 2.02 1.92 1.56 – 3.71

(m), 1.54 (m) (m), 1.63 (m) (m), 1.65 (m)

120.2 (d) 59.0 (t) 15.7 (q) –

5.13 (t, 6.9) 4.11 (t, 6.9) 0.84 (s) –

(m) (m), 1.54 (m) (m), 0.99 (td, 13.1, 4.5) (d, 6.2) (m), 1.51 (m), 1.49 (s-like) (d, 11.5), (m), 1.40

(m) (m) 1.64 (m) (m)

(d, 11.3), 3.61 (d, 11.3)

C

4c 1

H

13

1

77.8 (d) 105.3 (d) 152.1 (s) 159.1 (s) 39.3 (d) 21.4 (t) 38.9 (t) 44.0 (s) 43.5 (d) 44.5 (s) 18.1 (t) 25.8 (t) 45.2 (d) 37.1 (t) 53.1 (t) 81.6 (s) 66.0 (t)

3.75 5.93 – – 2.78 1.90 1.66 – 2.01 – 1.61 1.65 2.10 1.95 1.73 – 3.83 3.71 5.81 – 0.95 3.37

C

36.0 (t) 34.8 (t) 105.5 (s) 173.8 (s) 47.2 (d) 22.2 (t) 39.4 (t) 44.5 (s) 52.9 (d) 43.5 (s) 19.6 (t) 31.5 (t) 38.5 (d) 37.6 (t) 45.5 (t) 43.7 (d) 66.7 (t)

1.49 2.50 – – 2.52 1.48 1.48 – 1.23 – 1.50 1.65 2.38 1.82 1.63 2.19 3.67

(m), 1.75 (m) (m), 1.94 (m)

112.0 (d) 171.4 (s) 14.3 (q) –

5.57 (s) – 0.95 (s) –

(d, 11.8) (m), 1.37 (m) (m), 1.35 (m) (d, 8.4) (m), 1.64 (m) (m), 1.44 (m) (m) (m), 1.12(m) (m), 1.11(m) (m) (d, 6.0)

112.0 (d) 170.0 (s) 16.0 (q) 57.0 (q)

H (d, 6.1) (dd, 6.1, 1.8)

(m) (m), 1.64 (m) (m), 2.78 (m) (d, 7.8) (m), 1.54 (m), 1.55 (s-like) (d, 11.9), (m), 1.54

(m) (m) 1.70 (m) (m)

(d, 11.0), (d, 11.0) (s-like) (s) (s)

showed the presence of one methyl (δH 0.79, s, H3–20), one olefinic [δH 5.20 (t, J = 7.0 Hz, H-18)] and two methylenes [δH 3.40 (dd, J = 7.7 Hz and 5.3 Hz, H2–17), δH 4.19 (d, J = 7.2 Hz, H2–19)] proton singnals. In the 13C-DEPT NMR (Table 1) spectra, the observed 20 carbon signals consist of one methyl, eleven methylenes (including two oxygenated), four methines (including one olefinic), and four quateranary carbon (including one olefinic), which implied that 1 was a diterpenoid. However, the 1He1H COSY correlation of an oxygenated methylene protons (δH 4.19) with an olefinic proton (δH 5.20) in 1 suggested that 1 possessed an unusual C=CH–CH2–OH moiety and the key HMBC correlations from δH 1.67 (H-5) and δH 2.64 (H-3) to the olefinic carbon δC 145.5 (C-4) suggested that the double bond was located at C-4(18), which was different with the known rearranged diterpenoid skeletons that have been isolated from C.arabica. Moreover, the HMBC correlations from an oxygenated methylene protons (δH 3.40) to C-16 (δC 43.3) and C-15 (δC 45.1), together with the 1He1H COSY correlation of δH 3.40/H-16 (δH 1.97) demonstrated that a hydroxyl group was also located at C-17 in 1.Eventually, the single-crystal X-ray crystallographic analysis (Fig. 2) unambiguously confirmed the ent-4(18)-kauren-19-ol skeleton of 1 and clarified the absolute configuration of C-16 as R. Thus, compound 1 was established as 16(R)-17,19-dihydroxy-4(18)-entkauren-ol. Caffruenol B (2) was obtained as white amorphous powder. The HRESIMS and 13Cspectrum established the molecular formula of 2 as C20H32O3, showing one more oxygen atom than 1. The extremely similar 1D NMR spectra between 1 and 2 demonstrated that they have the same basic structure, except for an aliphatic methine signal (δC 43.3) in 1 were replaced by an oxygenated quaternary carbon signal (δC

monochromator (Ф/ω scans, 2θmax = 67.68°), Cu Kα radiation. The total number of reflections measured was 9846, of which 3058 independent reflections were observed (|F|2 ≥ 2σ|F|2). Final indices: R1 = 0.0390, wR2 = 0.1163 (w = 1/σ|F|2),S = 1.130. Caffruenol (2): white amorphous powder, [α]24 D − 24.1 (c = 0.1, MeOH); UV (MeOH) λmax (log ε): 204 (3.14), 303 (1.87) nm; IR (KBr) vmax: 3415, 3030, 2933, 1667, 1430, 1210, 1150, 1020, 830 cm−1; HRESIMS m/z 343.2243 [M + Na]+ (calcd for C20H32O3 Na, 343.2244); 1H and 13C NMR data shown in Table 1. Caffruolide A (3): white amorphous powder, [α]24 D − 208.9 (c = 0.1, MeOH); UV (MeOH) λmax (log ε): 271 (3.94), 195 (3.64) nm; IR (KBr) vmax: 3410, 2934, 1746, 1675, 1603, 1460, 1450, 1259, 1208, 1020, 675 cm−1; HRESIMS m/z 355.1882 [M + Na]+ (calcd for C20H28O4Na, 355.1880); 1H and 13C NMR data shown in Table 1. Caffruolide B (4): white amorphous powder, [α]24 D − 30.2 (c = 0.1, MeOH); UV (MeOH) λmax (log ε): 214 (3.28), 300 (1.87) nm; IR (KBr) vmax: 3417, 2920, 1775, 1445, 1380, 1266, 1206, 1020 cm−1; HRESIMS m/z 383.1827 [M + Na]+ (calcd for C21H28O5Na, 383.1829); 1H and 13 C NMR data shown in Table 1. 2.5. Lipopolysaccahride (LPS)-induced NO production Raw 264.7 macrophage cells (purchased from Shanghai institute of biochemistry and cell biology, CAS) were cultured on 96-well plates to evaluate the production of NO at a density of 1 × 105 cells/mL. After 2 h, the cells were pretreated with 25 μM compounds 1–4 and stimulated for 24 h with or without 1 μg/mL LPS. Nitrite concentration was measured in the supernatant of RAW 264.7 cells by Griess reaction. Each 100 μL of culture supernatant was mixed with an equal volume of Griess reagent (1.0% sulfanilamide, 0.1% naphthylenediamine and 5% phophoric acid) and incubated at room temperature for 10 min. The optical density of 550 nm (OD550) was measured with a microplate reader (Bio-Rad 680). The concentrations of NO were determined by a sodium nitrite standard curve. 3. Results and discussion Caffruenol A (1) was isolated as colorless prisms and the molecular formula C20H32O2 was assigned by HRESIMS spectrum, representing five degrees of unsaturation. The 1H NMR (Table 1) spectrum of 1

Fig. 2. The key HMBC and 1He1H COSY correlations of compound 1, and its structure determined by single-crystal X-ray analysis. 9

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five methines (including one olefinic) and six quaternary carbons (including one oxygenated, one olefinic and one carbonyl). The 1D NMR data of 3 showed extreme similarities to those of tricalysiolide B (6) [15], except for an oxygenated quaternary carbon signal in 6 were replaced by an aliphatic methine signal (δC 43.7) in 3. The 1He1H COSY correlations of the methine proton (δH 2.19) with δH 3.67 (H2–17) and δH 1.63 (H-15a) demonstrated that C-16 was the methine in 3 (Fig. 4). The relative configuration of 3 was confirmed by the ROESY correlations of OH-3 (δH 9.50)/H-5 (δH 2.52), H-9 (δH 1.23)/H-15a (δH1.63) and H-15a/H-16 (δH 2.19), which indicated that OH-3 and H-16 were βoriented (Fig. 4). Thus, the structure of 3 was determined as shown in Fig. 1. Caffruolide B (4) was obtained as white amorphous powder, whose molecular formula was determined to be C21H28O5 from the HRESIMS data. The 1H NMR spectrum (Table 1) exhibited one singlet methyl (δH 0.95), one methoxyl (δH 3.37, s, OMe-1), one oxymethylene [δH 3.83 (d, J = 11.0 Hz, H-17a), 3.71 (d, J = 11.0 Hz, H-17b)], one oxymethine [δH 3.75 (d, J = 11.0 Hz, H-1)] and two olefinic [δH 5.93 (dd, J = 6.0 Hz and 1.8 Hz, H-2), δH 5.81 (s, H-18)] proton singals. The 13CDEPT (Table 1) spectra of 4 displayed 21 carbon resonances, belonging to two methyls (including one oxymethyl), seven methylenes (including one oxygenated), six methines (including two olefinic) and six quaternary carbons (including one oxygenated, two olefinic and one carbonyl). Based on the 1D NMR, HSQC and 1He1H COSY spectra, the basic structure of 4 was the same as tricalysiolide E (8), except for an additional methoxy group was located at C-1 in 4, which was confirmed by the HMBC correlations from the methoxyl protons (δH 3.37) to C-1 (δC 77.8), C-2 (δC 105.3), C-3(δC 152.1) and C-10 (δC 44.5) (Fig. 4). The relative configuration of OMe-1'β and OH-16'α was established by the ROESYcorrelations of H-1 (δH 3.75)/H3–20 (δH 0.95), H-20 (δH 0.95)/ H-12a (δH 1.44) and H-12b (δH 1.65)/H2–17 (δH 3.83, 3.71) (Fig. 4). Therefore, the structure of 4 was elucidated as shown. The known ent-kaurane diterpenoids were elucidated as tricalysiolide A (5) [15], tricalysiolide B [15] (6), tricalysiolide C (7) [15], tricalysiolide E (8) [15], 16α, 17-dihydroxy-ent-kauran-19-al (9) [16], 16β-17-hydroxy-ent-kauran-19-oic acid (10) [17], 16α,17-dihydroxyent-kauran-19-oic acid (11) [18], 9β,16α,17-trihydroxy-ent-kauran-19oic acid (12) [19], 16β-7,17-dihydroxy-ent-kauran-19-oic-methyl ester (13) [20], 16α,17-dihydroxy-9(11)-ent-kauren-19-oic acid (14) [21], (2β,4β,15α)-15-hydroxy-2-{[2-O-(3-methyl-1-oxo-butyl)]-β-D-glucopyrnosyl]oxy}-18-nor-ent-kaur-16-en-18-oic acid (15) [13] on the basis of comparison of their spectroscopic data with those reported in the literatures. Eight of them, compounds 5–11 and 13 were first isolated from genus Coffea. These rearranged diterpenes with ent-4(18)-kauren-19-ol skeleton have been isolated for the first time. We deduced that compounds 1 and 2 could be the biosynthetic precursor of other rearranged ent-kaurane

Fig. 3. The key HMBC, 1He1H COSY and ROESY correlations of compound 2.

Fig. 4. The key HMBC, ROESY and 1He1H COSY correlations of compounds 3 and 4.

82.8) in 2. The HMBC correlations of the oxygenated quaternary carbon with protons of δH 3.71, 3.61 (H2–17), δH 1.40 (H-15b), δH 1.92 (H-14a) and δH 2.02 (H−13) (Fig. 3) confirmed that the oxygenated quaternary carbon in 2 was C-16. Furthermore, the ROESY correlations of H3–20 (δH 0.84)/H-12b (δH1.49), H-12a (δH 1.64)/H2–17 (δH 3.71, 3.61), suggested that the OH-16 in 2 was α-oriented (Fig. 3). Therefore, caffruenol B was identified as 16α,17,19-trihydroxy-4(18)-ent-kauren-ol. Caffruolide A (3) was isolated as white amorphous powder. Its molecular formula was determined to be C20H28O4 from the [M + Na]+ ion peak at m/z 355.1882 (calcd for C20H28O4Na, 355.1880) in HRESIMS spectrum, suggesting seven degrees of unsaturation. The IR spectrum indicated that 3 possessed hydroxyl (3410 cm−1) and α,βunsaturated lactone (1746 cm−1) groups. The 1H NMR (Table 1) spectrum showed the characteristic signals of one methyl (δH 0.75, s, H3–20), one oxymethylene [δH 3.67, d, J = 6.0 Hz, H2–17], and one olefinic (δH 5.80, s, H-18) protons. The 13C NMR (Table 1) spectrum suggested 3 was a diterpenoid derivative with 20 carbons, which was assigned as one methyl, eight methylenes (including one oxygenated),

Scheme 1. The plausible biosynthetic pathway of the ent-4(18)-kaurene diterpene. 10

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diterpenoids by comparing their structures reported in the present study. Herein, we proposed a plausible biosynthetic pathway for the ent-4(18)-kaurene diterpenes from the titled plant (Scheme 1). Moreover, compounds 3 and 4 were also new rearranged ent-kaurane diterpenoids which might be related to cafestol and kahweol [22], respectively. This type of diterpenoids had only been previously isolated from the plants of Tricalysia (Rubiaceae) [15]. Compounds 1–4 were evaluated for their inhibitory effects on NO production in LPS-activated RAW 264.7 macrophages at the concentration of 25 μM and they showed no inhibitory effect.

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Conflict of interest We declare that there is no conflict of interest. Acknowlegements This project was supported financially by the National Natural Science Foundation of China, China (No. 31670364), Project of Key New Productions of Yunnan Province, China (No. 2015BB002 and 2016HE004), the STS Programme of Chinese Academy of Sciences, China (KFJ-SW-STS-143-8), Special Fund Project of Pu’er municipal government, China (2017), as well as Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China, China (P2015-ZZ09). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2018.08.023. References [1] P. Esquivel, V.M. Jiménez, Functional properties of coffee and coffee by-products, Food Res. Int. 46 (2012) 488–495. [2] M.E.D.C. Moreira, D.F. Dias, V.S. Gontijo, F.C. Vilela, A. Giusti-Paiva, M.H.D. Santos, Anti-inflammatory effect of aqueous extracts of roasted and green Coffea arabica L, J. Funct. Foods 5 (2013) 466–474. [3] J.L. Shao, D.S. Yang, J.L. Fan, L.J. Du, L. Wang, L.X. Wang, Comparative analysis on trigonelline, chlorogenic acid and caffeine content in coffee and its product, J. Shanxi Agric. Sci. 44 (2016) 158–163. [4] T. Kurzrock, K. Speer, Diterpenes and diterpene esters in coffee, Food Rev. Int. 17

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