Four new dimeric 2-(2-phenylethyl)chromones (1–4) were isolated from the agarwood of Aquilaria crassna in Laos. All structures were determined based on extensive spectroscopic methods including IR, UV, 1D and 2D NMR, ECD, and HRMS. The structures of compounds 1–3 were characterized by two C-O-C linkages between the two chromone moieties, while compound 4 featured one C-O-C linkage and one CC bond connecting with two chromone moieties. Compounds 1 and 3 showed weak cytotoxicity against human hepatocellular carcinoma BEL-7402 cell line with IC50 values of 44.68 and 42.10 μΜ.
Graphical abstract
The structures of four new compounds and the sample of agarwood agarwood of Aquilaria crassna in Laos.
Introduction
Agarwood is the resinous heartwood from Aquilaria or Gyrinops species (Thymelaeaceae), which also called eaglewood, gaharu, jinko, aloeswood, kanankoh, kyara, jinkoh, chenxiang or oud in different cultures [1,2]. Aquilaria crassna Pierre ex Lecomte, one of the important resources of agarwood in the world market, mainly grow in Laos, Thailand, Cambodia and Vietnam [3].
2-(2-Phenylethyl)chromone, an important type of characteristic component in agarwood, which was also the main class of active ingredient of agarwood [1,2,4]. So far, >150 2-(2-phenylethyl)chromone derivatives have been identified from agarwood of some Aquilaria species [[4], [5], [6], [7], [8]], while, vast majority of these 2-(2-phenylethyl)chromones were monomers, not polymers. Since Japanese researchers acquired a few 2-(2-phenylethyl)chromone polymers from agarwood in Kalimantan in the 1980s [[9], [10], [11], [12]], there haven't been identified any new 2-(2-phenylethyl)chromone polymer from agarwood until 2017. From the year 2017 to now, 30 new dimeric 2-(2-phenylethyl)chromones have been identified from agarwood of Aquilaria sinensis and Aquilaria crassna respectively [[5], [6], [7], [8]], which could be a breakthrough on the phytochemical study of agarwood. Due to the great separation potential of chromone polymer from agarwood, further studies on this type of compounds could be performed seriously in the future.
As an indispensable part of our study on bioactive chemical constituents of agarwood from A. crassna, previous investigation on agarwood of A. crassna from Laos, led to the isolation of a series of 2-(2-phenylethyl)chromone derivatives [5,6,13,14] and some eremophilane sesquiterpenes [[13], [14], [15]]. During our ongoing research for bioactive 2-(2-phenylethyl)chromone derivatives, four dimeric chromones (1–4) were obtained from agarwood of A. crassna. All dimeric chromones were evaluated for their cytotoxic activity against three human cancer cell lines in vitro. Compounds 1 and 3 showed weak cytotoxicity against human hepatocellular carcinoma BEL-7402 cell line with IC50 values of 44.68 and 42.10 μΜ. This paper described the structural elucidation of the four new dimeric 2-(2-phenylethyl)chromones (1–4), as well as their cytotoxic activities.
Section snippets
General
HRESIMS were measured with an API QSTAR Pulsar mass spectrometer (Bruker). 1D and 2D NMR spectra were recorded on Bruker AV III spectrometer (Bruker) (1H NMR at 500 MHz and 13C NMR at 125 MHz), TMS as the internal standard. Optical rotation was measured on a Rudolph Autopol III polarimeter. UV spectra were performed on a Shimadzu UV-2550 spectrometer (Beckman, America). IR absorptions were obtained on a Nicolet 380 FT-IR instrument (Thermo) using KBr pellets. CD data were collected using a
Results and discussion
The EtOAc extract of the agarwood A. crassna was separated successively by repeated column chromatography and semi-preparative HPLC purification to afford four new bi-2-(2-phenylethyl)chromone derivatives (1–4) (Fig.1).
Compound 1 was obtained as white amorphous powder. Its molecular formula was found to be C35H30O9 on the basis of HREIMS (m/z 617.1828 [M + Na]+, calcd for C35H30NaO9, 617.1782). Its IR spectrum showed absorptions of OH groups (3438 cm−1) and α, β-unsaturated carbonyl groups
Acknowledgements
This work was financially supported by Central Public-interest Scientific Institution Basal Research Fund for Innovative Research Team Program of CATAS (17 CXTD-15), Innovative Research Team Grant of the Natural Science Foundation of Hainan Province (No.2017CXTD020), and China Agriculture Research System (CARS-21).
Two new chromone derivatives, 7-hydroxy-2-[2-(3'-methoxy-4'-hydroxyphenyl)-ethyl]chromone (1), and 6,7-dimethoxy-2-[2-(3'-hydroxyphenyl)-ethyl]chromone (2) were isolated from the EtOH extract of agarwood of Aquilaria sinensis, together with eleven known analogues. Their structures were established by detailed HR-ESIMS, 1D and 2D NMR spectroscopic analysis, as well as comparison with the literature data. Selected the isolates (1, 2, 4–8, 10, 11) were tested for their antitumor activities against SMMC-7721, MGC-803 and OV-90 cell lines using the MTT method with cisplatin and paclitaxel as the positive control. All the tested compounds showed weak cytotoxic activities with IC50 values ranged from 18.82 to 37.95 µg/ml.
Four new bi-phenylethylchromones (1–4) were isolated from the EtOAc extract of artificial agarwood induced by holing method originating from Aquilaria sinensis (Lour.) Gilg. The structures of new compounds were unambiguously elucidated by one- and two-dimensional NMR and HRESIMS measurements, and the absolute configuration was determined by analysis of circular dichroism (CD) spectra. All compounds were tested for acetylcholinesterase (AChE) inhibitory activity using modified Ellman's colorimetric method and α-glucosidase inhibitory activity using PNPG method. Compounds 2–4 exhibited different levels of inhibitory activity against AChE with the inhibition ratios in the range of 10–45%. However, none of the compounds was active against the α-glucosidase.
Graphical abstract
Four new bi-phenylethylchromones (1–4) were isolated from the EtOAc extract of artificial agarwood induced by holing method originating from Aquilaria sinensis (Lour.) Gilg.
Introduction
Agarwood is the fragrant resinous heartwood harvested from the Aquilaria species of the family Thymelaeaceae, which was mostly distributed in China, Japan, India and Southeast Asian countries [1], [2] and was widely used in incenses, perfumes, traditional medicines and other products in the world market [3]. Aquilaria sinensis (Lour.) is the main plant resource of Chinese agarwood, and the wild agarwood was close to exhausted due to the condition particularity of formation and a long period of time required for agar to accumulate in the wound tissues of trees [4], [5], coupled with the increase of commercial demand resulted in the anthropogenic deforestation. Fortunately, the holing method has widely been accepted recently to produce artificial agarwood attributed to the good quality of the product and the lasting aroma of its ether extract [5], [6]. Despite the commercial and biological significance of artificial agarwood, only a few researches on chemical constituents was conducted, which led to a series of sesquiterpenoids and 2-(2-phenylethyl)chromones [7], [8], [9], [10], [11].
Though 2-(2-phenylethyl)chromones were widely distributed in agarwood, and was considered as one of diagnostic components of agarwood quality [8], [9], [12], [13], the research progress about phenylethylchromone polymers (including dimer and trimer) was slow that no more than eleven analogues have been reported since the first one was isolated from the Kalimantan agalwood named “Jinko” and characterized by Iwagoe et al. in 1986 [14], [15], [16], [17], [18].
With the aim to study the chemical constituent of artificial agarwood, as well as to expand the diversity of bioactivities and chemical structures of bi-phenylethylchromones, the ongoing chemical investigation on agarwood induced by artificial holing was carried out, which led to the isolation and identification of four new bi-phenylethylchromone derivatives (1–4). Their structures were elucidated by extensive UV, IR, 1D and 2D NMR, and the absolute configuration was determined by electronic circular dichroism (ECD) spectroscopic analysis. All compounds were tested for AChE and α-glucosidase inhibitory activity.
Section snippets
General
1D and 2D NMR spectra were recorded on Bruker AV III spectrometer (Bruker) at 500 MHz (1H NMR) or 125 MHz (13C NMR), using TMS as an internal standard. HRESIMS spectra were measured with an API QSTAR Pulsar mass spectrometer (Bruker). CD data were collected using a JASCO J-715 spectrophotometer. Optical rotations were measured on a Rudolph Autopol III polarimeter. Semi-preparative HPLC was carried out using a Agilent Technologies 1260 Infinity equipped with a Agilent DAD G1315D detector (λ = 220
Results and discussion
Chemical examination of EtOAc extract of artificial agarwood originating from A. sinensis resulted in the isolation and identification of four new bi-phenylethylchromones.
Compound 1 was obtained as a pale yellow amorphous solid. The molecular formula of 1 was established as C37H36O12 on the basis of a prominent ion peak at m/z 695.2100 [M + Na]+ (calcd. 695.2099) observed in the HRESIMS spectrum. The IR spectrum of 1 showed the presence of hydroxyl groups (3414 cm− 1), and α, β-unsaturated carbonyl
Conflict of interest
The authors declare that this article has no conflict of interest.
Acknowledgments
This research was financially supported by Special Fund for Agro-scientific Research in the Public Interest (201303117), Major Technology Project of Hainan Province (ZDKJ2016004-03), Natural Science Fund of Hainan Province (20168367), Special Fund for The Young Talents's Science and Technology Project of Hainan Association for Science and Technology (HAST201628), and Special Fund for Key Research Project of Hainan Province in 2016 (ZDYF2016210).
Two rare flavonoid-2-(2-phenylethyl)chromones and five new dimeric 2-(2-phenylethyl)chromones were isolated from ethanol extract of agarwood ofAquilaria wallaby LC-MS-guided fractionation procedure. Their structures were established based on extensive spectroscopic methods including HRESIMS, 1D and 2D NMR, as well as by comparison with the literature. Compound1showed cytotoxic activity against five human cancer cell lines with IC50 values ranging from 13.40 to 28.96 μM with cisplatin as the positive control.
Agarwood is the resinous heartwood containing fragrant resin obtained from the trees of Aquilaria and Gyrinops species after they get physical injury or microbial attack [1,2]. To date, twenty-one species in the genus Aquilaria and nine species in the genus Gyrinops have been documented scattered extensively in south China, Myanmar, Thailand, Vietnam, India, and other Southeast Asia countries [2,3]. Aquilaria walla is recorded mainly distributing in the wet and intermediate zones of Sri Lanka, and minority in southwest India [4]. Its agarwood appears usually black with small white spots and has unique cool fragrance [5].
Modern phytochemistry studies indicated that 2-(2-phenylethyl)chromones are one of the main characteristic components in agarwood exhibiting various of biological activities [[6], [7], [8], [9], [10]]. Previous chemical studies on A. walla have led to identify three new 2-(2-phenylethyl)chromones, especially including an unprecedented flavonoid-2-(2-phenylethyl)chromone, which displayed significant cytotoxicity versus five human cancer cell lines [5,[11], [12], [13]]. In order to look for more interesting structures from agarwood of A. walla, the continuing research was carried out, and two rare flavonoid-2-(2-phenylethyl)chromones (1,2) and five new dimeric 2-(2-phenylethyl)chromones (3–7) were further isolated. In vitro cytotoxic assessment using MTT method showed that compound 1 presented cytotoxic activity versus the same five human cancer cell lines with IC50 values ranging from 13.40 to 28.96 μM.
Section snippets
Results and discussion
Compound 1 was obtained in the form of yellow gum. Its molecular formula was established as C35H32O12 based on the HRESIMS pseudomolecular ion peak at m/z 667.1791 [M + Na]+ (calcd. For C35H32NaO12, 667.1786) and 13C NMR data, indicating 20 indices of hydrogen deficiency. The 1H NMR data of 1 (Table 1) exhibited resonances characteristic for a set of para-substituted phenyl group [δH 6.89 (2H, d, J = 8.5 Hz, H-2′, 6′), δH 6.65 (2H, d, J = 8.6 Hz, H-3′, 5′)], a set of ABX coupling aromatic
General
The HRESIMS were measured with an API QSTAR Pulsar mass spectrometer (Bruker, Germany). 1H, 13C, and 2D NMR spectra were measured on Bruker AV III spectrometer (Bruker, Germany) at 500 MHz (1H) and 125 MHz (13C). Chemical shifts were referenced to the solvent residual peaks. Optical rotations were measured on a Modular Circular Polarimeter 500 polarimeter (Anton Paar, Austria). ECD and UV spectra were recorded on a MOS-500 spectrometer (Biologic, France). Analytic HPLC was performed with an
Conclusion and discussion
Two rare flavonoid-2-(2-phenethyl)chromones (1, 2) and five new dimeric 2-(2-phenylethyl)chromones (3–7) were identified from agarwood of A. walla. By review bi-2-(2-phenylethyl)chromone derivatives from agarwood from different original plants, only three flavonoid-2-(2-phenlethyl)chromone derivatives have been isolated from agarwood of A. walla including the one reported in our previous publication [5], and none of them have been reported from agarwood from other species so far. Accordingly,
CRediT authorship contribution statement
Leyao Chen was responsible for investigation and writing-original draft; Huiqin Chen was responsible for project administration and writing-review ans editing; Caihong Cai, Jingzhe Yuan and Cuijuan Gai were responsible for investigation; Shoubai Liu was responsible for project administration; Wenli Mei was responsible for funding acquisition, writing-review and editing; Haofu Dai was responsible for funding acquisition and supervision.
Declaration of Competing Interest
There are no conflicts of interest to declare.
Acknowledgments
This work was financially supported by National Key Research and Development Program (2018YFC1706404), and Agriculture Research System of China of MOF and MARA (cars-21).
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.
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).
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.
Compound 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).
The 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 C36H34NaO10m/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 C37H36NaO11m/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).
C bond connecting with two chromone moieties. Compounds 1 and 3 showed weak cytotoxicity against human hepatocellular carcinoma BEL-7402 cell line with IC50 values of 44.68 and 42.10 μΜ.
