Abstract
Two unprecedented 2-(2-phenethyl)chromone dimers, aquicrassones A and B (1 and 2), were isolated by HPLC-MS guided method from the ethanol extract of red soil agarwood originating from Aquilaria crassna in Vietnam. Their structures were unambiguously elucidated by analysis of HR-ESI-MS, 1D and 2D NMR, CD data and by comparison with literature data. Compounds 1 and 2 were evaluated for their inhibition effect on nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells. Compound 2 displayed anti-inflammatory activity with an IC50 value of 20.36 ± 0.41 μM.
1. Introduction
Agarwood is valuable non-timber resinous wood from Aquilaria or Gyrinops species of the family Thymelaeaceae. It has been widely used for medicine, perfume, cosmetics and incense culture (Li et al., 2021a). Agarwood has been proven various bioactivities, such as anti-asthmatic, anti-microbial, anti-malaria, anti-cancer, anti-inflammatory, and cytotoxicity (Liu et al., 2021a, Millaty et al., 2020, Wang et al., 2021, Yang et al., 2022, Yu et al., 2020, Yu et al., 2022). Sesquiterpenoids and 2-(2-phenylethyl)chromone derivatives were identified as the main chemical constituents of agarwood, with 2-(2-phenylethyl)chromones being the main active ingredients (Li et al., 2019, Li et al., 2021a). Various types of dimeric 2-(2-phenylethyl)chromones were identified in agarwood originating from A. crassna and A. sinensis in recent years that exhibited anti-inflammatory, cytotoxicity, AChE inhibitory and gastric mucosal protection activities (Liu et al., 2021b, Xia et al., 2019, Xiang et al., 2020, Yang et al., 2018, Zhang et al., 2023).
The red soil agarwood in Vietnam is derived from A. crassna, which is unique and highly valued in the world market. (He et al., 2022). The quality of red soil agarwood is more comparable to the highest quality agarwood “Qi Nan”, when compared to ordinary agarwood (Yang et al., 2015). In our previous investigation on red soil agarwood, a series of 2-(2-phenylethyl)chromone dimers were isolated, and crassin N showed cytotoxicity against the human myeloid leukemia cell line (He et al., 2021, Liu et al., 2021b). Continuing the identification of bioactive dimeric 2-(2-phenethyl)chromones from red soil agarwood, two new 2-(2-phenethyl)chromone dimers, aquicrassones A and B (1 and 2) were successfully isolated under the guidance of HPLC-MS. Their structures were elucidated based on HR-ESI-MS, 1D and 2D NMR, CD data, and by comparison with literature data. This paper describes the isolation, structure elucidation and anti-inflammatory activity of 2-(2-phenethyl)chromone dimers.
2. Results and discussion
Compound 1 was isolated as a colorless gum. Its molecular formula was deduced as C36H34O10 based on a prominent ion peak at m/z 649.2048 [M + Na]+ in the HR-ESI-MS spectrum. The 1H, 13C NMR data (Table 1) and the combination of HSQC spectrum indicated the presence of a mono-substituent benzene [δC/H 129.3/6.99 (2 H, t, J = 7.3 Hz, H-2″, 6″), 129.5/7.18 (2 H, t, J = 7.3 Hz, H-3″, 5″), 127.4/7.11 (1 H, t, J = 7.8 Hz, H-4″)], two sets of trisubstituted benzene groups [δC/H 105.7/7.39 (1 H, br s, H-5′), 124.9/7.29 (1 H, m, H-7′), 120.7/7.48 (1 H, m, H-8′), δC 158.5 (C-6′), 152.7 (C-9′), 124.8 (C-10′)] and [δC/H 118.7/7.22 (1 H, br s, H-2‴), 113.8/6.92 (1 H, overlapped, H-5‴), 123.7/6.91 (1 H, overlapped, H-6‴), δC 134.1 (C-1‴), 150.0 (C-3‴), 150.2 (C-4‴)], two sets of unsaturated olefin signals [δC/H 114.2/6.08 (1 H, s, H-3), 110.1/6.13 (1 H, s, H-3′), δC 170.8 (C-2), 171.3 (C-2′)], four consecutive oxymethines [δC/H 66.8/4.80 (1 H, d, J = 4.4 Hz, H-5), 74.1/4.13 (1 H, dd, J = 4.4, 2.3 Hz, H-6), 70.9/4.38 (1 H, dd, J = 7.1, 2.3 Hz, H-7), 78.5/5.29 (1 H, d, J = 7.1 Hz, H-8)], four methylenes [δC/H 33.2/2.61 (2 H, overlapped, H-7″), 36.3/2.61 (2 H, overlapped, H-8″), 33.2/3.02 (2 H, overlapped, H-7‴), 36.8/3.02 (2 H, overlapped, H-8‴)], two carbonyl carbons [δC 181.7 (C-4), 180.2 (C-4′)] and two methoxy groups [δC/H 56.3/3.83 (3 H, s, 6′-OCH3), 56.5/3.77 (3 H, s, 4‴-OCH3)]. Combined with its molecular formula and 1D NMR data, compound 1 was inferred to be a 2-(2-phenylethyl)chromone dimer, and its structure was very similar to the known compound wallone D (Chen et al., 2023) based on similar chemical shifts and coupling constants (Table 1). Unit A of compound 1 was elucidated as the same planer structure as agarotetrol by 1H–1H COSY of H-5/H-6/H-7/H-8 and the key HMBC correlations from H-3 to C-10/C-8″, from H-5 to C-5/C-9, from H-8 to C-6/C-10, from H-7″ to C-2″/C-6″, from H-3″/5″ to C-1″, from H-2″/6″ to C-4″. Its unit B was established as 6-methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone based on their comparable 1H and 13C NMR data (Xia et al., 2019). The HMBC correlations from OCH3 (δH 3.83) to C-6′, from H-8′ to C-6′, and together with the NOE correlation between OCH3 (δH 3.83) and H-5′ (Fig. 3) confirmed the presence of 6′-OCH3. Similarly, the existence of 4‴-OCH3 was determined by the HMBC correlation from OCH3 (δH 3.77) to C-4‴, and NOE correlations between H-7‴ and H-2‴/H-6‴, OCH3 (δH 3.77) and H-5‴ in the ROESY spectrum. The key HMBC correlation from H-8 to C-3‴ and the NOE correlation between H-8/H-2‴ suggested that units A and B connected via the (C8-O-C3‴)-ether bond. The remaining substructures of compound 1 were confirmed by detailed analysis of its 2D NMR spectra.
Table 1. 1H and 13C NMR data for 1 and 2 (δ in ppm).
| Pos. | 1a | 2b | ||
|---|---|---|---|---|
| δH (J in Hz) | δC, type | δH (J in Hz) | δC, type | |
| 2 | 170.8, C | 169.3, C | ||
| 3 | 6.08, s | 114.2, CH | 6.13, s | 113.7, CH |
| 4 | 181.7, C | 180.7, C | ||
| 5 | 4.80, d (4.4) | 66.8, CH | 4.96, d (3.7) | 65.7, CH |
| 6 | 4.13, dd (4.4, 2.3) | 74.1, CH | 4.38, d (3.7, 2.0) | 69.2, CH |
| 7 | 4.38, dd (7.1, 2.3) | 70.9, CH | 4.19, d (2.0, 6.3) | 72.3, CH |
| 8 | 5.29, d (7.1) | 78.5, CH | 5.18, d (6.3) | 79.6, CH |
| 9 | 162.9, C | 159.3, C | ||
| 10 | 122.6, C | 121.2, C | ||
| 2′ | 171.3, C | 168.1, C | ||
| 3′ | 6.13, s | 110.1, CH | 6.07, s | 109.8, CH |
| 4′ | 180.2, C | 178.3, C | ||
| 5′ | 7.39, br s | 105.7, CH | 7.50, d (3.1) | 104.9, CH |
| 6′ | 158.5, C | 157.0, C | ||
| 7′ | 7.29, m | 124.9, CH | 7.23, dd (9.1, 3.1) | 123.9, CH |
| 8′ | 7.48, m | 120.7, CH | 7.33, d (9.1) | 119.3, CH |
| 9′ | 152.7, C | 151.4, C | ||
| 10′ | 124.8, C | 124.3, C | ||
| 1″ | 141.0, C | 131.3, C | ||
| 2″ | 6.99, d (7.3) | 129.3, CH | 6.99, d, (8.6) | 129.2, CH |
| 3″ | 7.18, t (7.3) | 129.5, CH | 6.77, d, (8.6) | 114.2, CH |
| 4″ | 7.11, t (7.8) | 127.4, CH | 158.4, C | |
| 5″ | 7.18, t (7.3) | 129.5, CH | 6.77, d, (8.6) | 114.2, CH |
| 6″ | 6.99, d (7.3) | 129.3, CH | 6.99, d, (8.6) | 129.2, CH |
| 7″ | 2.61, overlapped | 33.2, CH2 | 2.80, m | 31.9, CH2 |
| 8″ | 2.61, overlapped | 36.3, CH2 | 2.76, m | 35.9, CH2 |
| 1‴ | 134.1, C | 132.9, C | ||
| 2‴ | 7.22, br s | 118.7, CH | 7.06, d (1.9) | 119.8, CH |
| 3‴ | 150.0, C | 147.8, C | ||
| 4‴ | 150.2, CH | 149.4, C | ||
| 5‴ | 6.92, overlapped | 113.8, CH | 6.87, d (8.3) | 112.7, CH |
| 6‴ | 6.91, overlapped | 123.7, CH | 6.91, dd (8.3, 1.9) | 123.9, CH |
| 7‴ | 3.02, overlapped | 33.2, CH2 | 2.97, m | 32.5, CH2 |
| 8‴ | 3.02, overlapped | 36.8, CH2 | 2.87, m | 36.2, CH2 |
| 6′-OCH3 | 3.83, s | 56.3, OCH3 | 3.87, s | 56.1, OCH3 |
| 4″-OCH3 | 3.74, s | 55.4, OCH3 | ||
| 4‴-OCH3 | 3.77, s | 56.5, OCH3 | 3.85, s | 56.1, OCH3 |
- a
Recorded in methanol-d4; a 1H (600 MHz) and 13C NMR (150 MHz).
- b
Recorded in CDCl3; b 1H (500 MHz) and 13C NMR (125 MHz).
The coupling constants of H-7/H-8 (3J7,8 = 7.1 Hz) and H-6/H-7 (3J6,7 = 2.3 Hz) implied that H-7 and H-8 are located at axial bond and H-6 is located at equatorial bond (Fig. 1). H-5 is located at equatorial bond for unobserved NOE correlation between H-5/H-7, which was further established by the similarity of coupling constant and chemical shift to that of wallone D (Chen et al., 2023). In addition, the cotton effects of 1 [338 (−0.18), 307 (+2.69), 282 (−6.15) nm] were identical to those of wallone D [332 (−1.76), 297 (+5.68), 251 (−43.68) nm], implying that they had the same absolute configuration as 5 S,6 R,7 R,8 S. Therefore, the structure of compound 1 was elucidated as shown in Fig. 1, and named as aquicrassone A.
Fig. 1Compound 2 was obtained as a white powder. Its molecular formula was established as C37H36O11 based on the HR-ESI-MS spectrum with a peak at m/z 679.2145 [M + Na]+. The 1H, 13C NMR and the combination of HSQC spectroscopic data revealed two ABC coupling aromatic systems [δC/H 104.9/7.50 (1 H, d, J = 3.1 Hz, H-5′), 123.9/7.23 (1 H, dd, J = 9.1, 3.1 Hz, H-7′), 119.3/7.33 (1 H, d, J = 9.1 Hz, H-8′), δC 157.0 (C-6′), 151.4 (C-9′), 124.3 (C-10′)] and [δC/H 119.8/7.06 (1 H, d, J = 1.9 Hz, H-2‴), 112.7/6.87 (1 H, d, J = 8.3 Hz, H-5‴), 123.9/6.91 (1 H, dd, J = 8.3, 1.9 Hz, H-6‴), δC 132.9 (C-1‴), 147.8 (C-3‴), 149.4 (C-4‴)], a set of para-substituted phenyl [δC/H 129.2/6.99 (2 H, d, J = 8.6 Hz, H-2″, 6″), 114.2/6.77 (2 H, d, J = 8.6 Hz, H-3″, H-5″), δC 131.3 (C-1″), 158.4 (C-4″)], two sets of unsaturated olefin signals [δC/H 113.7/6.13 (1 H, s, H-3), 109.8/6.07 (1 H, s, H-3′), δC 169.3 (C-2), 168.1 (C-2′)], four consecutive oxymethines [δC/H 65.7/4.96 (1 H, d, J = 3.7 Hz, H-5), 69.2/4.38 (1 H, d, J = 3.7, 2.0 Hz, H-6), 72.3/4.19 (1 H, d, J = 6.3, 2.0 Hz, H-7), 79.6/5.18 (1 H, d, J = 6.3 Hz, H-8)], four methylenes [δC/H 31.9/2.80 (2 H, m, H-7″), 35.9/2.76 (2 H, m, H-8″), 32.5/2.97 (2 H, m, H-7‴), 36.2/2.87 (2 H, m, H-8‴)], two carbonyl carbons [δC 180.7 (C-4), 178.3 (C-4′)] and three methoxys [δC/H 56.1/3.87 (3 H, s, 6′-OCH3), 55.4/3.74 (3 H, s, 4″-OCH3), 56.1/3.85 (3 H, s, 4‴-OCH3)]. The above information indicated that compound 2 was a dimeric 2-(2-phenylethyl)chromone, including a 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone moiety (unit A) and a 2-(2-phenylethyl)chromone (unit B) moiety. Its 1D NMR data (Table 1) were similar to those of aquicrassone A (1), except for the observation of an additional methoxy group. The extra methoxy group of 2 was attached at C-4″, which was deduced by the HMBC correlation from OCH3 (δH 3.74) to C-4″ and by NOE correlations between OCH3 (δH 3.74) and H-3″/H-5″ in the ROESY spectrum. Detailed analysis of the 2D NMR spectra of 2 revealed that its remaining substructures were identical to those of aquicrassone A (1). Based on the above evidence, the planar structure of compound 2 was depicted (Fig. 2, Fig. 3).
Fig. 2
Fig. 3The coupling constants (3J7,8 = 6.3 Hz, 3J6,7 = 2.0 Hz, 3J5,6 = 3.7 Hz) together with the NOE correlation between H-5/H-7, suggested a chair conformation of the cyclohexane ring with H-5, H-7, H-8 located at axial bond, while H-6 located at equatorial bond. Therefore, the relative configuration of 2 was the same as the known aquilasinenone A (Kuang et al., 2019). The absolute configuration of 2 was assigned to be 5 R,6 R,7 R,8 S due to the same cotton effects as these of crassin D (Yang et al., 2017). Hence, the absolute configuration of 2 was identified as depicted (Fig. 1) and named as aquicrassone B.
In addition, the anti-inflammatory activity of compounds 1 and 2 were evaluated in vitro. The results showed that compound 2 demonstrated significant inhibition of NO production in LPS-stimulated RAW264.7 cells with IC50 values of 20.36 ± 0.41 μM. Quercetin was used as positive control with IC50 value of 10.09 ± 2.93 μM. Compounds 1 and 2 did not show toxicity at a concentration of 50 μM with LPS treatment for 24 h, measured by MTT method.
3. Experimental
3.1. General
UV and CD spectra data were recorded on a MOS-500 spectrometer (Biologic, France). Optical rotations were measured on a Modular Circular Polarimeter 500 polarimeter (Anton Paar, Austria). NMR spectra (1D and 2D NMR) of compounds 1 and 2 were recorded on Bruker Avance III-500 instrument (Bruker, Germany) and Quantum-IPlus 600 (Q.One, China) with TMS as an internal standard, respectively. HR-ESI-MS analyses were obtained on an ESI-Q-TOF Pulsar mass spectrometer (Bruker, Germany). HPLC-MS (Bruker Compact, Germany) was used to guide the separation of 2-(2-phenethyl)chromone dimers. Analytic HPLC was carried out on C18 column (250 mm × 4.6 mm, 5 µm, flow rate 1 mL/min) (YMC, Japan) and 5PFP column (250 mm × 4.6 mm, 5 µm, flow rate 1 mL/min) with an Agilent Technologies 1260 Infinity II equipped with an Agilent DAD G1315D detector (Agilent, USA). Semi-preparative HPLC was performed on C18 column (250 mm × 10 mm, 5 µm, flow rate 4 mL/min), 5PFP column (250 mm × 10 mm, 5 µm, flow rate 4 mL/min) and πNAP column (250 mm × 10 mm, 5 µm, flow rate 4 mL/min) using a Lab Alliance (λ = 210 nm and 254 nm). Silica gel (60 – 80, 200 – 300 mesh, Qingdao Marine Chemical Co. Ltd., China), Sephadex LH-20 (Merck, Germany) and ODS gel (20 – 45 μm, Fuji Silysia Chemical Co. Ltd., America) were used for open-column chromatography. TLC analyses were performed on precoated silica gel GF254 plates (Qingdao Marine Chemical Ltd., China), and spots were visualized under UV light at 254 nm and detected by spraying with 5% H2SO4 in EtOH followed by heating.
3.2. Plant material
Red soil agarwood was bought from Guangxi province, China, in November 2017. The original plant material was identified as Aquilaria crassna by Prof. Haofu Dai from the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where its voucher specimen (No. YNHT2017) was deposited.
3.3. Extraction and isolation
Air-dried red soil agarwood (1.5 kg) was exhaustively extracted three times by refluxing with 95% EtOH, and the EtOH extract (330.6 g) was suspended in water (1.0 L) and partitioned successively with EtOAc (2.5 L × 3) and n-BuOH (2.5 L × 3). The EtOAc soluble section (131.0 g) was fractioned by silica gel vacuum liquid chromatography (VLC) eluting with a gradient solvent of CHCl3/MeOH (v/v, 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1, each 8.0 L) to obtain sixteen fractions Fr.1 to Fr.16. Fr.14 was found to be rich in bi-phenylethylchromones based on HPLC-UV and HPLC-MS data analysis. Fr.14 (20.0 g) was further subjected to ODS gel column chromatography (MeOH/H2O, v/v 3:7–10:0) to yield fifteen fractions (Fr.14–1–Fr.14–15).
Fr.14–5 (300.5 mg) was separated by semi-preparative HPLC with πNAP column (C2H3N/H2O, v/v 39:61) to afford three subfractions (Fr.14–5-1 to Fr.14–5-3). Fr.14–5-3 (16.7 mg) was purified by semi-preparative HPLC with C18 column (MeOH/H2O, v/v 60:40) to give compound 1 (13.5 mg, tR 33.0 min).
Fr.14–6 (697.4 mg) was separated on Sephadex LH-20 gel column chromatography (3 × 150 cm) eluting with MeOH to obtain ten subfractions (Fr.14–6-1 to Fr.14–6-10). Fr.14–6-4 (169.5 mg) was silica gel vacuum liquid chromatography (VLC) eluting with a gradient solvent of CHCl3/MeOH (v/v, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:1, each 0.5 L) to yield nine parts Fr.14–6-4–1 to Fr.14–6-4–9. Fr.14–6-4–3 (17.1 mg) was purified by semi-preparative HPLC with 5PFP column (MeOH/H2O, v/v 65:35) to obtain three fractions Fr.14–6-4–3-1 to Fr.14–6-4–3-3. Fr.14–6-4–3-3 (6.1 mg) was further purified by semi-preparative HPLC with C18 column (MeOH/H2O, v/v 65:35) to give compound 2 (2.7 mg, tR 24.0 min).
The purities of compounds 1 and 2 were determined by HPLC with C18 column (MeOH/H2O, v/v 65:35). The results showed that their purities are at least 95% (HPLC-UV, 254 nm).
3.3.1. Aquicrassone A (1)
Colorless gum; [α]− 84 (c 0.10, MeOH); UV (MeOH) λmax (log ε): 265 (4.11), 322 (3.83) nm; CD (MeOH) λmax (Δε) 338 (−0.18), 307 (+2.69), 282 (−6.15), 240 (+5.47), 220 (+10.04) nm; 1H and 13C NMR data see Table 1; HR-ESI-MS m/z 649.2048 [M + Na]+ (calcd for C36H34NaO10 m/z 649.2044).
3.3.2. Aquicrassone B (2)
White powder; [α]− 40 (c 0.10, MeOH); UV (MeOH) λmax (log ε): 265 (4.12), 322 (3.89) nm; CD (MeOH) λmax (Δε) 335 (−0.07), 308 (+0.25), 282 (−1.66), 210 (+11.38) nm; 1H and 13C NMR data see Table 1; HR-ESI-MS m/z 679.2145 [M + Na]+ (calcd for C37H36NaO11 m/z 679.2150).
3.4. Biological activity
Compounds 1 and 2 were detected for their inhibitory effects on NO production in LPS-stimulated Mouse mononuclear macrophages (RAW264.7) (The Stem Cell Bank of the Chinese Academy of Sciences) using the Griess assay as described previously (Liu et al., 2021a). Quercetin and media with DMSO were used as positive control and negative control, respectively. The cells were cultured in DMEM medium (Thermo Fisher Technologies) in a humidified 5% CO2/95% air atmosphere at 37 °C. Compounds 1 and 2 were diluted in half by five concentration gradients (25 µM, 12.5 µM, 6.25 µM, 3.125 µM, 1.562 µM). RAW264.7 cells (5 × 104 cells/mL) were seeded into 96-well plates and stimulated with 500 ng/mL LPS (Sigma Company, USA). After incubation for 24 h, 100 µL of the supernatant from each well together with 100 µL (40 mg/mL) of Griess reagent (Sigma Company, USA) were added to a new 96-well flat-bottomed cell culture plate. The absorbance of every well was measured using ELISA reader (Thermo Scientific, USA) at 540 nm, and the IC50 value of the tested compounds was calculated.
4. Conclusion
Two new 2-(2-phenylethyl)chromone dimers were characterized from Aquilaria crassna in Vietnam, named aquicrassone A (1) and aquicrassone B (2). Their linkage positions of units A and B are at C-8 and C3‴ (C8-O-C3‴), which is a new linkage type of 2-(2-phenylethyl)chromone dimers from A. crassna. In previous studies, only three similar dimers were reported from A. sinensis (Li et al., 2019, Li et al., 2021b). Besides, aquicrassone B (2) demonstrated significant inhibition of NO production in LPS-stimulated RAW264.7 cells with IC50 values of 20.36 ± 0.41 μM.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by the Natural Science Foundation of Hainan Province (320RC714), Major Technology Project of Hainan Province (ZDKJ2021031), National Natural Science Foundation of China (32171824), and the earmarked fund for CARS-Chinese Materia Medica (CARS-21).
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