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Bioassay-guided isolation of cytotoxic constituents from the flowers of Aquilaria sinensis

Bioassay-guided isolation of cytotoxic constituents from the flowers of Aquilaria sinensis have been isolated from the flower buds of A. sinensis and 1 Introduction two compounds, aquilasides B and C, displayed moderate Cancer is a major public health problem and one of the cytotoxicity against SK-MEL cells with IC values of 17.0 leading causes of mortality and morbidity worldwide and 12.0 μM, respectively [7]. [1]. Chemical drugs, such as paclitaxel, are useful to In our screening for anticancer extracts of plants, the treat cancers. However, resistance to paclitaxel reduces extract (PXS65) of A. sinensis flowers was found to pos - the efficacy of chemotherapy and limits its clinical sess significant inhibitory activities against 16 cancer application [2]. Therefore, it is necessary to develop cell lines (Table  1), especially inhibiting lung cancer cell novel and effective therapeutic medicines or adjuvants lines, such as human lung adenocarcinoma SPC-A-1 for cancer. Natural plant resources are a rich source of (IC = 0.11  μg/mL), human lung squamous cell carci- anticancer agents. The discovery of new effective can - noma NCI-H520 (I C = 0.25  μg/mL), and human lung cer drugs and understanding their underlying mecha- adenocarcinoma A549 (IC = 0.44  μg/mL) cells. Then, nism is one of the most studied topics among biologists a bioassay-guided isolation of cytotoxic constituents and chemists. against A549, NCI-H520, SPC-A-1, paclitaxel-resistant Aquilaria sinensis (Lour.) Spreng. (y Th melaeaceae) is A549 (A549/Taxol), and human normal bronchial epithe- widely distributed in Hainan, Fujian, Yunnan, Guang- lial BEAS-2B cell lines was conducted (Fig.  1; Tables  2– dong, and Taiwan in China [3]. It has a particular eco- 5), which led to the isolation of four active compounds, nomic interest because it is the principal source of including a new cucurbitane-type triterpenoid (1) agarwood (chen-xiang in Chinese), namely, the resinous (Fig.  2). The bioassay results and the structural elucida - heartwood of the plant. As a traditional Chinese medi- tion of aquilarolide A (1) are reported. cine, agarwood has been widely investigated [4]. How- ever, there have been only a few studies on the chemical 2 Results and discussion constituents and bioactivities of A. sinensis flowers. The 2.1 Bioactivity‑guided fractionation and isolation volatile constituents from flowers of A. sinensis have been The 90% EtOH extract (PXS65) of A. sinensis flow - analyzed by GC–MS [5]. Flavonoids and their glycosides ers after water extraction was tested in  vitro for its are found in its flowers [6]. Benzophenone glycosides Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 3 of 9 Table 1 Cytotoxicity of the EtOH extract (PXS65) of A. sinensis cytotoxicity against 16 cancer cell lines, including SPC- after water extraction against 16 cancer cell lines and the normal A-1, NCI-H520, A549, human cervical cancer HeLa, human bronchial epithelial BEAS‑2B cell line human neuroblastoma SH-SY5Y, human ovarian car- cinoma SK-OV-3, human T-cell leukemia MT4, human No Cell lines IC (μg/mL) prostate cancer PC-3, human hepatoma SMMC-7721, PXS65 Cisplatin Paclitaxel human breast cancer MDA-MB-231, human small cell 1 SPC‑A‑1 0.11 ± 0.00 1.76 ± 0.40 < 0.007 lung cancer NCI-H446, human large cell lung carci- 2 NCI‑H520 0.25 ± 0.02 6.69 ± 1.46 < 0.007 noma NCI-H460, human colon cancer SW-480, human 3 A549 0.44 ± 0.00 3.83 ± 0.79 < 0.007 breast cancer MCF-7, human leukemia HL-60, and 4 HeLa 0.46 ± 0.01 1.14 ± 0.152 < 0.007 human colon cancer Caco2 cell lines, as well as the 5 SH‑SY5Y 0.48 ± 0.02 2.85 ± 0.272 0.008 ± 0.001 normal BEAS-2B cell line by 3-(4,5-dimethylthiazol- 6 SK‑ OV‑3 0.55 ± 0.03 3.67 ± 0.97 < 0.007 2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)- 2H-tetrazolium (MTS) assay and their IC values 7 MT4 0.59 ± 0.074 0.16 ± 0.01 < 0.008 (μg/mL) were determined (Table  1). PXS65 possessed 8 PC‑3 0.84 ± 0.01 1.38 ± 0.23 < 0.007 pronounced cytotoxic activity against SPC-A-1, NCI- 9 SMMC‑7721 1.36 ± 0.38 1.53 ± 0.11 0.15 ± 0.02 H520, A549, HeLa, SH-SY5Y, SK-OV-3, MT4, and 10 MDA‑MB‑231 1.39 ± 0.04 3.16 ± 0.97 < 0.007 PC-3 cells with I C values less than 1  μg/mL. Mean- 11 NCI‑H446 5.51 ± 0.41 3.78 ± 0.79 < 0.007 while, PXS65 exhibited weak inhibitory activity against 12 NCI‑H460 12.24 ± 0.63 5.29 ± 0.48 < 0.007 SW480, MCF-7, HL-60, HL-60, Caco2, and BEAS-2B 13 SW480 21.94 ± 1.18 2.00 ± 0.52 < 0.007 cells with IC values greater than 20 μg/mL. These data 14 MCF‑7 33.29 ± 2.23 2.00 ± 0.93 < 0.007 indicated that PXS65 had some selectivity for different 15 HL‑60 36.68 ± 1.20 1.97 ± 1.30 < 0.007 cancerous cell lines and the normal BEAS-2B cell line. 16 Caco2 > 40 3.24 ± 0.09 0.02 ± 0.02 It had better inhibition against lung cancer cell lines 17 BEAS‑2B 28.73 ± 1.42 9.14 ± 1.41 3.66 ± 0.30 than against other cancer cell lines (Table  1). Thus, the Fig. 1 Schematic diagram showing cytotoxic compounds from Aquilaria sinensis flowers by bioassay‑ guided isolation Yang et al. Natural Products and Bioprospecting (2022) 12:11 Page 4 of 9 Table 2 Cytotoxicity of the EtOH extract (PXS66) and petroleum ether‑soluble (PXS66‑1), EtOAc‑soluble (PXS66‑2), n‑BuOH‑soluble (PXS66‑3), and H O‑soluble (PSX66‑4) fractions Extracts/fractions IC (μg/mL) A‑549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B PXS66 2.04 ± 0.01 0.72 ± 0.06 1.59 ± 0.15 1.49 ± 0.20 30.97 ± 0.76 PXS66‑1 2.05 ± 0.08 1.39 ± 0.03 1.63 ± 0.05 1.87 ± 0.05 13.59 ± 0.57 PXS66‑2 0.17 ± 0.02 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 4.48 ± 0.16 PXS66‑3 13.56 ± 0.32 11.06 ± 0.81 8.30 ± 0.15 10.11 ± 0.31 > 100 PXS66‑4 > 100 > 100 > 100 > 100 > 100 Cisplatin 4.99 ± 0.08 3.12 ± 0.18 3.10 ± 0.13 4.29 ± 0.26 7.36 ± 0.56 Paclitaxel < 0.007 < 0.007 < 0.007 0.54 ± 0.09 1.85 ± 0.19 Fig. 2 Chemical structures of isolates 1–6 next bioactivity-guided separations were conducted Table 3 Cytotoxicity of the subfractions from the EtOAc‑soluble according to the cytotoxicities of the fractions against fraction (PXS66‑2) against the A549, NCI‑H520, SPC‑A‑1, A549/ Taxol, and BEAS‑2B cell lines lung cancer cell lines (A-549, NCI-H520, and SPC- A-1), as well as normal BEAS-2B cells and paclitaxel- Fractions IC (μg/mL) resistant lung cancer A549/Taxol cells. A549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B As shown in Table  2, the EtOAc-soluble fraction Fr. B‑1 0.02 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 5.54 ± 0.32 (PXS66-2) showed the most inhibitory activities against Fr. B‑2 2.16 ± 0.04 0.45 ± 0.03 1.76 ± 0.08 1.49 ± 0.11 49.83 ± 1.66 A-549 (IC = 0.17  μg/mL), NCI-H520 (IC = 0.08  μg/ 50 50 Fr. B‑3 13.36 ± 1.02 6.44 ± 0.55 7.85 ± 0.07 8.22 ± 0.55 92.79 ± 0.92 mL), SPC-A-1 (IC = 0.08  μg/mL), and A549/Taxol Fr. B‑4 5.84 ± 0.08 2.01 ± 0.14 9.57 ± 0.41 2.10 ± 0.06 > 100 (IC = 0.08 μg/mL) cells. The inhibitory activity against Fr. B‑5 35.10 ± 0.63 7.95 ± 0.07 29.72 ± 2.13 9.01 ± 0.21 > 100 A549/Taxol cells was better than that of paclitaxel Fr. B‑6 12.97 ± 0.21 5.50 ± 0.50 11.33 ± 0.93 7.80 ± 0.19 > 100 (IC = 0.54 μg/mL), with lower toxicity (IC = 4.48 μg/ 50 50 Cisplatin 3.02 ± 0.59 3.75 ± 0.52 3.06 ± 1.07 3.41 ± 0.47 > 12 mL) than that of paclitaxel (I C = 1.85  μg/mL) against Paclitaxel < 0.007 < 0.007 < 0.007 1.45 ± 0.12 2.04 ± 0.11 normal BEAS-2B cells. Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 5 of 9 PXS66-2 was fractionated by silica gel column chro- spectrum (Table  1) of 1 indicated the presence of nine matography to yield six further fractions (B-1 to B-6), methyl groups at δ 0.98, 1.05, 1.33, 1.42, 1.51, 1.53, which were also submitted to a cytotoxicity assay 1.54, 1.57, and 2.02 (methyl protons of an acetyl group) (Table  3). Fr. B-1 showed observably higher inhibi- ppm, a trans double bond at δ 7.05 (d, J = 15.7 Hz) and tory activities against these four lung cancer cell lines 6.44 (d, J = 15.7  Hz) ppm, and a trisubstituted double than other fractions with I C values less than or equal bond at δ 5.75 (br s) ppm. The C NMR spectrum 50 H to 0.02  μg/mL. Fr. B-1 was separated by reverse-phase (Table  1) of 1 displayed 31 carbon signals indicating (RP) C silica gel column chromatography to yield 12 the presence of four carbonyl groups (δ 211.7, 202.4, 18 C further fractions (B-1-1 to B-1-12), which were also 172.0, and 170.3), two double bonds (δ 152.1, 136.7, submitted to a cytotoxicity assay (Table  4). Frs. B-1-6 120.6, and 120.2), nine methyl groups (δ 31.5, 30.6, and B-1-7 showed observably higher inhibitory activity 26.5, 25.9, 23.9, 22.0, 19.9, 18.5, and 18.4), four meth- against these four lung cancer cell lines than other frac- ylenes, four sp methines, and six quaternary carbons. tions with IC values less than or equal to 0.02 μg/mL. These data showed a similar signal pattern with those Next, Frs. B-1-6 and B-1-7 were isolated and purified to of a lactone-type norcucurbitacin, neocucurbitacin E, yield six compounds (1–6) (Fig. 2). except for the double bond at Δ in 1 [8]. 1 1 Based on the H– H COSY correlations (Fig.  3), 2.2 S tructural elucidation of isolates 1–6 four connections, H -1/H-10, H-6/H -7/H-8, 2 2 In total, six secondary metabolites (Fig.  2), including H -15/H-16/H-17, and H-23/H-24, were deduced. The a new metabolite (1), were isolated from the cytotoxi- HMBC data revealed the lactone-type structure of ring cally active fractions of A. sinensis flowers as a result of A, similar to that of neocucurbitacin E [8], since H -1 chromatographic separations. The chemical structure (δ 2.50 and 2.16) was correlated to the carbon atoms of the new compound was elucidated by 1D and 2D at δ = 172.0 (C-2), 136.7 (C-5), and 47.7 (C-9) ppm, nuclear magnetic resonance (NMR) experiments as well as well as H -28 and H -29 to C-5 (Fig.  3) and H-6 to 3 3 as high-resolution electron ionization mass spectrom- C-4. According to the HMBC correlations from H -19 etry (HRESIMS) and electronic circular dichroism (ECD) to C-8, C-10, and C-11, from H -30 to C-8, C-13, and calculations. C-15, and from H -18 to C-12, C-14, and C-17, rings Compound 1 was isolated as a white amorphous B–D were deduced. Based on the HMBC correlations powder and exhibited a quasi-molecular ion peak at from H-16 to C-20, from H -21 and 20-OH to C-17 and m/z 567.2937 [M + Na] in HRESIMS, suggesting a C-22, from H-23 to C-25, from H-24 to C-22, and from molecular formula of C H O (calcd. for C H NaO , H -26 and H -27 to C-24, the side chain was confirmed 31 44 8 31 44 8 3 3 567.2934) and 10 degrees of unsaturation. The H NMR and was located at C-17 of ring D. The acetyl group Table 4 Cytotoxicity of the subfractions from the active fraction (Fr. B‑1) against the A549, NCI‑H520, SPC‑A‑1, A549/Taxol, and BEAS‑2B cell lines Fractions IC (μg/mL) A549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B Fr. B‑1–1 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–2 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–3 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–4 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–5 54.99 ± 2.81 41.33 ± 2.32 45.41 ± 0.91 52.57 ± 0.63 > 100 Fr. B‑1–6 0.02 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 1.34 ± 0.05 Fr. B‑1–7 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 1.42 ± 0.04 Fr. B‑1–8 0.08 ± 0.00 0.02 ± 0.00 0.06 ± 0.00 0.08 ± 0.00 4.95 ± 0.19 Fr. B‑1–9 0.35 ± 0.04 0.48 ± 0.03 0.35 ± 0.02 1.60 ± 0.06 13.20 ± 1.13 Fr. B‑1–10 1.72 ± 0.10 1.20 ± 0.09 1.10 ± 0.08 2.04 ± 0.06 25.50 ± 0.84 Fr. B‑1–11 11.04 ± 0.54 8.16 ± 0.46 8.27 ± 0.38 18.24 ± 0.61 26.46 ± 2.01 Fr. B‑1–12 55.00 ± 1.83 36.70 ± 0.83 37.84 ± 2.03 50.21 ± 2.03 > 100 Cisplatin 2.28 ± 0.57 3.11 ± 0.22 2.49 ± 0.07 4.13 ± 0.86 8.86 ± 1.32 Paclitaxel < 0.007 < 0.007 < 0.007 0.96 ± 0.05 1.94 ± 0.11 Yang et al. Natural Products and Bioprospecting (2022) 12:11 Page 6 of 9 Fig. 3 Key 2D NMR correlations of compound 1 was located at C-25 (δ 79.4) by comparing the chemi- cal shift of C-25 in 25-OH analogs (δ is approximately 71  ppm) and 25-OAc analogs (δ is approximately 79 ppm) [9]. Thus, the planar structure of 1 was deter- mined as shown in Fig. 3. The relative configuration of 1 was deduced by ROESY correlations (Fig.  3). Correlations of H-1β/H -19, H-7β/ H -19, H-8/H -18, H-8/H -19, H-12β/H -18, H-15β/H - 3 3 3 3 3 18, and H-16/H -18 indicated that these protons should be β-oriented, while correlations of H-10/H -30, H-12α/ H -30, H-17/H -30, H-15α/16-OH, and H-17/16-OH 3 3 showed that these protons should be α-oriented. The configuration of C-20 could not be determined by the ROESY spectrum. Accordingly, the ECD spectra of (8 S,9R,10R,13R,14S,16R,17R,20R)-1 and (8S,9R,10R,13R ,14S,16R,17R,20S)-1 were calculated (Fig.  4). The calcu - lated ECD spectrum of (8S,9R,10R,13R,14S,16R,17R,20 R)-1 was similar to the experimental ECD spectrum of Fig. 4 Experimental and calculated ECD spectra for compound 1 1. Thus, the absolute configuration of compound 1 was elucidated to be 8S,9R,10R,13R,14S,16R,17R,20R, named aquilarolide A. Table 5 Cytotoxicity of compounds isolated from the active fractions (Frs. B‑1–6 and B‑1–7) against the A549, NCI‑H520, SPC‑A‑1, A549/Taxol, and BEAS‑2B cell lines Compounds IC (μM) A549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B 1 0.35 ± 0.06 0.16 ± 0.01 0.56 ± 0.02 0.20 ± 0.01 17.93 ± 0.30 2 > 40 > 40 > 40 > 40 > 40 3 0.02 ± 0.00 0.001 ± 0.000 0.005 ± 0.000 0.002 ± 0.000 3.46 ± 0.13 4 0.03 ± 0.00 0.002 ± 0.000 0.016 ± 0.000 0.007 ± 0.001 14.42 ± 1.36 5 > 40 > 40 > 40 > 40 > 40 6 1.52 ± 0.06 1.84 ± 0.16 1.13 ± 0.03 0.91 ± 0.01 > 40 Cisplation 13.54 ± 0.64 11.95 ± 0.60 21.42 ± 0.35 14.95 ± 0.93 34.90 ± 1.16 Paclitaxel < 0.008 < 0.008 < 0.008 1.80 ± 0.13 > 5 Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 7 of 9 1 13 2.3 Cytotoxic results of isolates 1–6 Table 6 H and C NMR data of compound 1 in CDCl (δ in ppm, J in Hz) Isolates 1–6 were evaluated for their cytotoxici- ties against SPC-A-1, NCI-H520, A549, A549/Taxol, No δ (500 MHz) δ (126 MHz) H C and BEAS-2B cell lines (Table  5). Aquilarolide A (1), 1α 2.50, 1H, dd (16.2, 3.8) 30.3 cucurbitacin E (3), cucurbitacin B (4), and 7-hydroxy- 1β 2.16, 1H, dd (16.2, 13.9) 6-methoxy-2-[2-(4-methoxyphenyl)ethyl]-4H-1-benzo- 2 172.0 pyran-4-one (6) displayed observable cytotoxicity against 4 83.9 four tested cancer cell lines with IC values ranging 5 136.7 from 0.001 to 1.84 μM and against the normal BEAS-2B 6 5.75, 1H, br s 120.6 cell line with I C values ranging from 3.46 to > 40  μM. 7α 2.05, 1H, m 23.7 All four active compounds, with activity strengths 7β 2.40, 1H, m of 3 (IC = 0.002  μM) > 4 (IC = 0.007  μg/mL) > 1 50 50 8 2.02, 1H, m 42.1 (IC = 0.20  μM) > 6 (IC = 0.91  μM), had better inhibi- 50 50 9 47.7 tory activities against A549/Taxol cells than paclitaxel 10 2.76, 1H, m 33.2 (IC = 1.80 μM) (Table 5). 11 211.7 These active compounds belong to cucurbitane-type 12α 3.11, d (14.9) 48.7 triterpenoids (1, 3, and 4) and a 2-(2-phenylethyl)chr- 12β 2.68, d (14.9) omone (6). This result agrees with those reported in the 13 50.4 literature that cucurbitane-type triterpenoids were found 14 48.0 to be the main constituents contributing to the cytotoxic 15α 1.46, 1H, m 45.5 activities in A. sinensis fruits and peels [14, 15]. 15β 1.88, 1H, dd (13.2, 9.8) Both cucurbotacin E (3) and 23,24-dihydrocucurbita- 16 4.36, 1H, m 71.3 cin E (2) have a four-ringed core structure in the cucur- 17 2.45, 1H, br d (7.1) 58.2 bitane skeleton, except for the side chain with an olefinic 18 0.98, 3H, s 19.9 bond at C-23 in compound 3. Cucurbitacin E showed 19 1.05, 3H, s 18.5 significant cytotoxic activities against human lung can - 20 78.1 cer SPC-A-1, NCI-H520, A549, and A549/Taxol cell lines 21 1.42, 3H, s 23.9 with IC values less than or equal to 0.02 μM. However, 22 202.4 23,24-dihydrocucurbitacin E (2) was inactive. This indi - 23 6.44, 1H, d (15.7) 120.2 cated that the side chain with the olefinic bond at C-23 24 7.05, 1H, d (15.7) 152.1 seems to be key to the cytotoxic activity of this type of 25 79.4 compound. 26 1.54, 3H, s 26.5 The inhibitory activities of cucurbitacins E (3) and B 27 1.57, 3H, overlapped 25.9 (4) were close to each other, with IC values less than 28 1.53, 3H, s 30.6 or equal to 0.03  μM. However, the inhibitory activity 29 1.51, 3H, s 31.5 of 1 was significantly weaker than that of compounds 3 30 1.33, 3H, s 18.4 and 4. The difference between 1 and 3 and 4 is ring A. 16‑ OH 1.75, 1H, d (6.7) This indicates that the structure of ring A is also key to 20‑ OH 4.28, 1H, s the cytotoxic activity of this type of compound. The cyto - 25‑ OAc 170.3 toxic potency of cucurbitacins in A549 cells was related 2.02, 3H, s 22.0 to multivariate factors, among which the electrophilicity of molecules played a pivotal role, according to the mul- tivariate structure–activity relationship (SAR) and quan- titative structure–activity relationship modeling (QSAQ) analyses of cucurbitacin derivatives [16]. The known compounds were identified as 23,24-dihy - drocucurbitacin E (2) [10], cucurbitacin E (3) [11], cucurbitacin B (4) [10], (−)-(2S)-5,4ʹ-dihydroxy-7- 3 Experimental section methoxyflavanone (5) [12], and 7-hydroxy-6-methoxy- 3.1 General experimental procedures 2-[2-(4-methoxyphenyl)ethyl]-4H-1-benzopyran-4-one The reagents and instrumentation utilized for extraction, (6) [13] by comparison of the obtained spectroscopic isolation, and structure characterization throughout this data with those published in the literature. study are described in Additional file 1. Yang et al. Natural Products and Bioprospecting (2022) 12:11 Page 8 of 9 3.2 C ollection of plant samples 60% MeOH-eluted portion (Fr. B-1–7) was purified by The flowers of Aquilaria sinensis were collected from column chromatography (silica gel; petroleum ether/ Menghai County, Yunnan Province, China, in 2019. A EtOAc, 5:1 → 0:1, v/v) to yield six further fractions voucher specimen (No. KIB001-003) was identified by (B-1-7-1–B-1-7-6). Fr. B-1-7-5 and Fr. B-1–7-6 were Ms. Jun Yang at Kunming Institute of Botany, Chinese recrystallized from MeOH to yield 3 (18.5 mg). Academy of Sciences. Aquilarolide A (1). White amorphous powder; [α] − 7.1 (c 0.10, MeOH); ECD (c 0.056, MeOH) λ (Δε) 334 (− 0.22), 298 (+ 2.68), 219 (− 1.67), 199 3.3 P reparation of extractions and fractions and isolation max (+ 5.67) nm; UV (MeOH) λ 282 (2.70), 229 (3.84) nm; of compounds max 1 13 H and C NMR data, see Table  6; ESI–MS m/z 567 Air-dried, powdered flowers (50.0  g) of A. sinensis were + + [M + Na] ; HRESIMS m/z 567.2937 [M + Na] (calcd. for extracted under ultrasound with H O (500  mL × 3) C H NaO , 567.2934) (Additional file 1). at 60 ℃ for 30  min. The remaining residue was fur - 31 44 8 ther extracted with 90% EtOH (500  mL × 3) at 60 ℃ for 30  min and the solvent was removed to yield crude 3.4 MTS assay for cytotoxicity extract PXS65 (2.4 g). The cytotoxicity activities were evaluated by MTS assay Air-dried, powdered flowers (1.2  kg) of A. sinensis as previously described [17]. were extracted under ultrasound with 90% EtOH (2 L × 4) at 60 ℃ for 30 min and the solvent was removed to yield crude extract PXS66 (174.2  g). PXS66 was 3.5 Computational methods suspended in water (500  mL) and then partitioned The absolute configuration of the new compound was in turn with petroleum ether (500  mL × 4), EtOAc determined by time-dependent density functional theory (500  mL × 4), and n-BuOH (500  mL × 4) to yield (TDDFT) calculations of ECD spectra according to our three fractions, PXS66-1 (21.1  g), PXS66-2 (23.0  g), previously published paper [18]. and PXS66-3 (54.0  g), respectively. The solvent in the remaining water phase was removed to yield PXS66-4 (54.9 g).4 Conclusion PXS66-2 (23.0  g) was subjected to column chroma In this study, bioassay-guided fractionation and purifica - tion were used to isolate the cytotoxic compounds of the tography (silica gel; CH Cl /MeOH, 1:0 → 0:1, v/v) 2 2 extract from A. sinensis flowers. First, the crude extract to yield six further fractions (B-1–B-6). Fr. B-1 was showed significant inhibitory activities against 16 cancer separated on an RP C silica gel column eluted with cell lines with the most significant activities against the MeOH/H O (5% → 100%) to yield twelve further frac- lung cancer SPC-A-1, NCI-H520, and A549 cell lines. tions (B-1-1–B-1-12). The 50% MeOH-eluted portion Second, all fractions, subfractions, and pure compounds (Fr. B-1–6) was purified by column chromatography were screened for their cytotoxic activity against lung (silica gel; petroleum ether/EtOAc, 5:1 → 0:1, v/v) to cancer SPC-A-1, NCI-H520, A549, and A549/Taxol cell yield six further fractions (B-1-6-1–B-1-6-6). Fr. B-1- lines and normal human bronchial epithelial BEAS-2B 6-1 was recrystallized from MeOH to yield 5 (92.2 mg). Fr. B-1-6-3 was purified by Sephadex LH-20 column cells. From the active fraction, six compounds, includ- chromatography (MeOH) and recrystallized from ing a new cucurbitane-type triterpenoid, aquilarolide A MeOH to yield 2 (11.0  mg). Fr. B-1-6-4 was recrystal (1), five known compounds, namely, 23,24-dihydrocu - curbitacin E (2), cucurbitacin E (3), cucurbitacin B (4), lized from MeOH to yield 3 (8.9  mg), and the remain- (−)-(2S)-5,4ʹ-dihydroxy-7-methoxyflavanone (5), and ing mother liquor was subjected to Sephadex LH-20 7-hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]- column chromatography (MeOH) to yield four fur- 4H-1-benzopyran-4-one (6), were identified. Compounds ther fractions (B-1-6-4-1–B-1-6-4-4). Fr. B-1-6-4-1 1, 3, 4, and 6 showed significant cytotoxicity activities (33.5  mg) was purified by semipreparative high-per - against these four human lung cancer cell lines. All four formance liquid chromatography (HPLC) (Welch active compounds, with activity strengths of 3 > 4 > 1 > 6, Ultimate AQ-C , 7.8 × 250  mm, MeOH/H O, 20:70, 18 2 had better inhibitory activities against A549/Taxol cells v = 2  mL/min) to yield 1 (3.1  mg, t = 24.543  min) and than paclitaxel. Further studies are needed to evaluate 4 (16.6  mg, t = 28.464  min). Fr. B-1–6-4–2 (72.6  mg) in vivo antitumor activities and clarify the mechanisms of was purified by semipreparative HPLC (Welch Ulti - these active compounds. mate AQ-C , 7.8 × 250  mm, MeOH/H O, 15:85, 18 2 v = 2 mL/min) to yield 6 (2.0 mg, t = 27.467  min). The R Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 9 of 9 11. Maatooq G, El‑Sharkawy S, Afifi MS, Rosazza JPN. Microbial transformation Supplementary Information of cucurbitacin E 2‑O‑β‑D ‑ glucopyranoside. J Nat Prod. 1995;58:165–71. The online version contains supplementary material available at https:// doi. 12. Valdés E, González C, Díaz K, Vásquez‑Martínez Y, Mascayano C, Torrent C, org/ 10. 1007/ s13659‑ 022‑ 00334‑3. Cabezas F, Mejias S, Montoya M, Martín C‑S, Muñoz MA, Joseph‑Nathan P, Osorio M, Taborga L. Biological properties and absolute configura‑ tion of flavanones from Calceolaria thyrsiflora Graham. Front Pharmacol. Additional file 1. General experimental procedures, computational meth‑ 2020;11:1125. ods for the ECD of compound 1, and NMR, HRESIMS, and ECD spectra of 13. Wu B, Kwon SW, Hwang GS, Park JH. Eight new 2‑(2‑phenylethyl)chr ‑ compound 1. omone (= 2‑(2‑phenylethyl)‑4H‑1‑benzopyran‑4‑ one) derivatives from Aquilaria malaccensis agarwood. Helv Chim Acta. 2012;95:1657–65. 14. Mei W‑L, Lin F, Zuo W ‑ J, Wang H, Dai H‑F. Cucurbitacins from fruits of Acknowledgements Aquilaria sinensis. Chin J Nat Med. 2012;10:234–7. This study was supported by Beijing Sino‑Science Aquilaria Technology Co., 15. Zhang X, Tao M‑H, Chen Y ‑ C, Gao X‑ X, Tan Y‑Z, Zhang W ‑M. Five cucurbi‑ Ltd., Beijing, China (Grant No. KET202101). tacins from Aquilaria sinensis peels and their cytotoxic activities. Nat Prod Res Dev. 2014;26:354–7. Author contributions 16. Silva IT, Carvalho A, Lang KL, Dudek SE, Masemann D, Duran FJ, Caro MSB, All authors read and approved the final manuscript. Rapp UR, Wixler V, Schenkel EP, Simões CMO, Ludwig S. In vitro and in vivo antitumor activity of a novel semisynthetic derivative of cucurbitacin B. Funding PLoS ONE. 2015;10:e0117794. Funding was provided by Beijing Sino‑Science Aquilaria Technology Co., Ltd., 17. Yang J, Su Y, Luo J‑F, Gu W, Niu H‑M, Li Y, Wang Y ‑H, Long C‑L. New amide Beijing, China (Grant No. KET202101). alkaloids from Piper longum fruits. Nat Prod Bioprospect. 2013;3:277–81. 18. Wei S‑ Y, Hu D‑B, Xia M ‑ Y, Luo J‑F, Yan H, Yang J‑H, Wang Y ‑S, Wang Declarations Y‑H. Sesquiterpenoids and 2‑(2‑phenylethyl)chromone derivatives from the resinous heartwood of Aquilaria sinensis. Nat Prod Bioprosp. Competing interests 2021;11:545–55. The authors declare that there are no conflicts of interest associated with this work. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Author details lished maps and institutional affiliations. Key Laboratory of Economic Plants and Biotechnology and Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China. School of Chemical Biology and Environment, Yuxi Normal University, Yuxi 653100, People’s Republic of China. College of Science, Yunnan Agricultural University, Kunming 650201, People’s Republic of China. Received: 9 February 2022 Accepted: 17 March 2022 References 1. El‑Hussein A, Manoto SL, Ombinda‑Lemboumba S, Alrowaili ZA, Mthunzi‑ Kufa P. A review of chemotherapy and photodynamic therapy for lung cancer treatment. Anti‑ Cancer Agents Med Chem. 2021;21:149–61. 2. Du J, Li J, Gao M, Guan Q, Liu T, Wu Y, Li Z, Zuo D, Zhang W, Wu Y. MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/Taxol cells and inhibits epithelial‑mesenchymal transition in A549/Taxol cells. Chem Biol Interact. 2020;323:109074. 3. Wang Y, Gilbert MG, Mathew B, Brickell CD, Nevling LI. Thymelaeaceae. In: Wu Z‑ Y, Raven PH, Hong D‑ Y, editors. Flora of China, vol. 13. Beijing: Science Press & Missouri Botanical Garden Press; 2007. p. 213–50. 4. Li W, Chen H‑ Q, Wang H, Mei W‑L, Dai H‑F. Natural products in agarwood and Aquilaria plants: chemistry, biological activities and biosynthesis. Nat Prod Rep. 2021;38:528–65. 5. Mei W‑L, Lin F, Dai H‑F. GC‑MS analysis of volatile constituents from flow‑ ers and fruits of Aquilaria sinensis. J Trop Subtrop Bot. 2009;17:305–8. 6. Chu C‑ W, Li W‑ J, Li H‑ T, Huang J‑ C, Chung M‑I, Chen C‑ Y. Flavonoids from the flowers of Aquilaria sinensis. Chem Nat Compd. 2016;52:497–8. 7. Yuan H, Zhao J, Wang M, Khan SI, Zhai C, Xu Q, Huang J, Peng C, Xiong G, Wang W. Benzophenone glycosides from the flower buds of Aquilaria sinensis. Fitoterapia. 2017;121:170–4. 8. Zhang X, Li H, Wang W, Chen T, Xuan L. Lipid‑lowering activities of cucur ‑ bitacins isolated from Trichosanthes cucumeroides and their synthetic derivatives. J Nat Prod. 2020;83(12):3536–44. 9. Jacobs H, Singh T, Reynolds WF, McLean S. Isolation and C‑NMR assign‑ ments of cucurbitacins from Cayaponia angustiloba, Cayaponia racemosa, and Gurania subumbellata. J Nat Prod. 1990;53:1600–5. 10. Ryu SY, Lee SH, Choi SU, Lee CO, No Z, Ahn JW. Antitumor activity of Trichosanthes kirilowii. Arch Pharm Res. 1994;17:348–53. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Natural Products and Bioprospecting Springer Journals

Bioassay-guided isolation of cytotoxic constituents from the flowers of Aquilaria sinensis

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Abstract

have been isolated from the flower buds of A. sinensis and 1 Introduction two compounds, aquilasides B and C, displayed moderate Cancer is a major public health problem and one of the cytotoxicity against SK-MEL cells with IC values of 17.0 leading causes of mortality and morbidity worldwide and 12.0 μM, respectively [7]. [1]. Chemical drugs, such as paclitaxel, are useful to In our screening for anticancer extracts of plants, the treat cancers. However, resistance to paclitaxel reduces extract (PXS65) of A. sinensis flowers was found to pos - the efficacy of chemotherapy and limits its clinical sess significant inhibitory activities against 16 cancer application [2]. Therefore, it is necessary to develop cell lines (Table  1), especially inhibiting lung cancer cell novel and effective therapeutic medicines or adjuvants lines, such as human lung adenocarcinoma SPC-A-1 for cancer. Natural plant resources are a rich source of (IC = 0.11  μg/mL), human lung squamous cell carci- anticancer agents. The discovery of new effective can - noma NCI-H520 (I C = 0.25  μg/mL), and human lung cer drugs and understanding their underlying mecha- adenocarcinoma A549 (IC = 0.44  μg/mL) cells. Then, nism is one of the most studied topics among biologists a bioassay-guided isolation of cytotoxic constituents and chemists. against A549, NCI-H520, SPC-A-1, paclitaxel-resistant Aquilaria sinensis (Lour.) Spreng. (y Th melaeaceae) is A549 (A549/Taxol), and human normal bronchial epithe- widely distributed in Hainan, Fujian, Yunnan, Guang- lial BEAS-2B cell lines was conducted (Fig.  1; Tables  2– dong, and Taiwan in China [3]. It has a particular eco- 5), which led to the isolation of four active compounds, nomic interest because it is the principal source of including a new cucurbitane-type triterpenoid (1) agarwood (chen-xiang in Chinese), namely, the resinous (Fig.  2). The bioassay results and the structural elucida - heartwood of the plant. As a traditional Chinese medi- tion of aquilarolide A (1) are reported. cine, agarwood has been widely investigated [4]. How- ever, there have been only a few studies on the chemical 2 Results and discussion constituents and bioactivities of A. sinensis flowers. The 2.1 Bioactivity‑guided fractionation and isolation volatile constituents from flowers of A. sinensis have been The 90% EtOH extract (PXS65) of A. sinensis flow - analyzed by GC–MS [5]. Flavonoids and their glycosides ers after water extraction was tested in  vitro for its are found in its flowers [6]. Benzophenone glycosides Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 3 of 9 Table 1 Cytotoxicity of the EtOH extract (PXS65) of A. sinensis cytotoxicity against 16 cancer cell lines, including SPC- after water extraction against 16 cancer cell lines and the normal A-1, NCI-H520, A549, human cervical cancer HeLa, human bronchial epithelial BEAS‑2B cell line human neuroblastoma SH-SY5Y, human ovarian car- cinoma SK-OV-3, human T-cell leukemia MT4, human No Cell lines IC (μg/mL) prostate cancer PC-3, human hepatoma SMMC-7721, PXS65 Cisplatin Paclitaxel human breast cancer MDA-MB-231, human small cell 1 SPC‑A‑1 0.11 ± 0.00 1.76 ± 0.40 < 0.007 lung cancer NCI-H446, human large cell lung carci- 2 NCI‑H520 0.25 ± 0.02 6.69 ± 1.46 < 0.007 noma NCI-H460, human colon cancer SW-480, human 3 A549 0.44 ± 0.00 3.83 ± 0.79 < 0.007 breast cancer MCF-7, human leukemia HL-60, and 4 HeLa 0.46 ± 0.01 1.14 ± 0.152 < 0.007 human colon cancer Caco2 cell lines, as well as the 5 SH‑SY5Y 0.48 ± 0.02 2.85 ± 0.272 0.008 ± 0.001 normal BEAS-2B cell line by 3-(4,5-dimethylthiazol- 6 SK‑ OV‑3 0.55 ± 0.03 3.67 ± 0.97 < 0.007 2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)- 2H-tetrazolium (MTS) assay and their IC values 7 MT4 0.59 ± 0.074 0.16 ± 0.01 < 0.008 (μg/mL) were determined (Table  1). PXS65 possessed 8 PC‑3 0.84 ± 0.01 1.38 ± 0.23 < 0.007 pronounced cytotoxic activity against SPC-A-1, NCI- 9 SMMC‑7721 1.36 ± 0.38 1.53 ± 0.11 0.15 ± 0.02 H520, A549, HeLa, SH-SY5Y, SK-OV-3, MT4, and 10 MDA‑MB‑231 1.39 ± 0.04 3.16 ± 0.97 < 0.007 PC-3 cells with I C values less than 1  μg/mL. Mean- 11 NCI‑H446 5.51 ± 0.41 3.78 ± 0.79 < 0.007 while, PXS65 exhibited weak inhibitory activity against 12 NCI‑H460 12.24 ± 0.63 5.29 ± 0.48 < 0.007 SW480, MCF-7, HL-60, HL-60, Caco2, and BEAS-2B 13 SW480 21.94 ± 1.18 2.00 ± 0.52 < 0.007 cells with IC values greater than 20 μg/mL. These data 14 MCF‑7 33.29 ± 2.23 2.00 ± 0.93 < 0.007 indicated that PXS65 had some selectivity for different 15 HL‑60 36.68 ± 1.20 1.97 ± 1.30 < 0.007 cancerous cell lines and the normal BEAS-2B cell line. 16 Caco2 > 40 3.24 ± 0.09 0.02 ± 0.02 It had better inhibition against lung cancer cell lines 17 BEAS‑2B 28.73 ± 1.42 9.14 ± 1.41 3.66 ± 0.30 than against other cancer cell lines (Table  1). Thus, the Fig. 1 Schematic diagram showing cytotoxic compounds from Aquilaria sinensis flowers by bioassay‑ guided isolation Yang et al. Natural Products and Bioprospecting (2022) 12:11 Page 4 of 9 Table 2 Cytotoxicity of the EtOH extract (PXS66) and petroleum ether‑soluble (PXS66‑1), EtOAc‑soluble (PXS66‑2), n‑BuOH‑soluble (PXS66‑3), and H O‑soluble (PSX66‑4) fractions Extracts/fractions IC (μg/mL) A‑549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B PXS66 2.04 ± 0.01 0.72 ± 0.06 1.59 ± 0.15 1.49 ± 0.20 30.97 ± 0.76 PXS66‑1 2.05 ± 0.08 1.39 ± 0.03 1.63 ± 0.05 1.87 ± 0.05 13.59 ± 0.57 PXS66‑2 0.17 ± 0.02 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 4.48 ± 0.16 PXS66‑3 13.56 ± 0.32 11.06 ± 0.81 8.30 ± 0.15 10.11 ± 0.31 > 100 PXS66‑4 > 100 > 100 > 100 > 100 > 100 Cisplatin 4.99 ± 0.08 3.12 ± 0.18 3.10 ± 0.13 4.29 ± 0.26 7.36 ± 0.56 Paclitaxel < 0.007 < 0.007 < 0.007 0.54 ± 0.09 1.85 ± 0.19 Fig. 2 Chemical structures of isolates 1–6 next bioactivity-guided separations were conducted Table 3 Cytotoxicity of the subfractions from the EtOAc‑soluble according to the cytotoxicities of the fractions against fraction (PXS66‑2) against the A549, NCI‑H520, SPC‑A‑1, A549/ Taxol, and BEAS‑2B cell lines lung cancer cell lines (A-549, NCI-H520, and SPC- A-1), as well as normal BEAS-2B cells and paclitaxel- Fractions IC (μg/mL) resistant lung cancer A549/Taxol cells. A549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B As shown in Table  2, the EtOAc-soluble fraction Fr. B‑1 0.02 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 5.54 ± 0.32 (PXS66-2) showed the most inhibitory activities against Fr. B‑2 2.16 ± 0.04 0.45 ± 0.03 1.76 ± 0.08 1.49 ± 0.11 49.83 ± 1.66 A-549 (IC = 0.17  μg/mL), NCI-H520 (IC = 0.08  μg/ 50 50 Fr. B‑3 13.36 ± 1.02 6.44 ± 0.55 7.85 ± 0.07 8.22 ± 0.55 92.79 ± 0.92 mL), SPC-A-1 (IC = 0.08  μg/mL), and A549/Taxol Fr. B‑4 5.84 ± 0.08 2.01 ± 0.14 9.57 ± 0.41 2.10 ± 0.06 > 100 (IC = 0.08 μg/mL) cells. The inhibitory activity against Fr. B‑5 35.10 ± 0.63 7.95 ± 0.07 29.72 ± 2.13 9.01 ± 0.21 > 100 A549/Taxol cells was better than that of paclitaxel Fr. B‑6 12.97 ± 0.21 5.50 ± 0.50 11.33 ± 0.93 7.80 ± 0.19 > 100 (IC = 0.54 μg/mL), with lower toxicity (IC = 4.48 μg/ 50 50 Cisplatin 3.02 ± 0.59 3.75 ± 0.52 3.06 ± 1.07 3.41 ± 0.47 > 12 mL) than that of paclitaxel (I C = 1.85  μg/mL) against Paclitaxel < 0.007 < 0.007 < 0.007 1.45 ± 0.12 2.04 ± 0.11 normal BEAS-2B cells. Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 5 of 9 PXS66-2 was fractionated by silica gel column chro- spectrum (Table  1) of 1 indicated the presence of nine matography to yield six further fractions (B-1 to B-6), methyl groups at δ 0.98, 1.05, 1.33, 1.42, 1.51, 1.53, which were also submitted to a cytotoxicity assay 1.54, 1.57, and 2.02 (methyl protons of an acetyl group) (Table  3). Fr. B-1 showed observably higher inhibi- ppm, a trans double bond at δ 7.05 (d, J = 15.7 Hz) and tory activities against these four lung cancer cell lines 6.44 (d, J = 15.7  Hz) ppm, and a trisubstituted double than other fractions with I C values less than or equal bond at δ 5.75 (br s) ppm. The C NMR spectrum 50 H to 0.02  μg/mL. Fr. B-1 was separated by reverse-phase (Table  1) of 1 displayed 31 carbon signals indicating (RP) C silica gel column chromatography to yield 12 the presence of four carbonyl groups (δ 211.7, 202.4, 18 C further fractions (B-1-1 to B-1-12), which were also 172.0, and 170.3), two double bonds (δ 152.1, 136.7, submitted to a cytotoxicity assay (Table  4). Frs. B-1-6 120.6, and 120.2), nine methyl groups (δ 31.5, 30.6, and B-1-7 showed observably higher inhibitory activity 26.5, 25.9, 23.9, 22.0, 19.9, 18.5, and 18.4), four meth- against these four lung cancer cell lines than other frac- ylenes, four sp methines, and six quaternary carbons. tions with IC values less than or equal to 0.02 μg/mL. These data showed a similar signal pattern with those Next, Frs. B-1-6 and B-1-7 were isolated and purified to of a lactone-type norcucurbitacin, neocucurbitacin E, yield six compounds (1–6) (Fig. 2). except for the double bond at Δ in 1 [8]. 1 1 Based on the H– H COSY correlations (Fig.  3), 2.2 S tructural elucidation of isolates 1–6 four connections, H -1/H-10, H-6/H -7/H-8, 2 2 In total, six secondary metabolites (Fig.  2), including H -15/H-16/H-17, and H-23/H-24, were deduced. The a new metabolite (1), were isolated from the cytotoxi- HMBC data revealed the lactone-type structure of ring cally active fractions of A. sinensis flowers as a result of A, similar to that of neocucurbitacin E [8], since H -1 chromatographic separations. The chemical structure (δ 2.50 and 2.16) was correlated to the carbon atoms of the new compound was elucidated by 1D and 2D at δ = 172.0 (C-2), 136.7 (C-5), and 47.7 (C-9) ppm, nuclear magnetic resonance (NMR) experiments as well as well as H -28 and H -29 to C-5 (Fig.  3) and H-6 to 3 3 as high-resolution electron ionization mass spectrom- C-4. According to the HMBC correlations from H -19 etry (HRESIMS) and electronic circular dichroism (ECD) to C-8, C-10, and C-11, from H -30 to C-8, C-13, and calculations. C-15, and from H -18 to C-12, C-14, and C-17, rings Compound 1 was isolated as a white amorphous B–D were deduced. Based on the HMBC correlations powder and exhibited a quasi-molecular ion peak at from H-16 to C-20, from H -21 and 20-OH to C-17 and m/z 567.2937 [M + Na] in HRESIMS, suggesting a C-22, from H-23 to C-25, from H-24 to C-22, and from molecular formula of C H O (calcd. for C H NaO , H -26 and H -27 to C-24, the side chain was confirmed 31 44 8 31 44 8 3 3 567.2934) and 10 degrees of unsaturation. The H NMR and was located at C-17 of ring D. The acetyl group Table 4 Cytotoxicity of the subfractions from the active fraction (Fr. B‑1) against the A549, NCI‑H520, SPC‑A‑1, A549/Taxol, and BEAS‑2B cell lines Fractions IC (μg/mL) A549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B Fr. B‑1–1 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–2 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–3 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–4 > 100 > 100 > 100 > 100 > 100 Fr. B‑1–5 54.99 ± 2.81 41.33 ± 2.32 45.41 ± 0.91 52.57 ± 0.63 > 100 Fr. B‑1–6 0.02 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 1.34 ± 0.05 Fr. B‑1–7 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 1.42 ± 0.04 Fr. B‑1–8 0.08 ± 0.00 0.02 ± 0.00 0.06 ± 0.00 0.08 ± 0.00 4.95 ± 0.19 Fr. B‑1–9 0.35 ± 0.04 0.48 ± 0.03 0.35 ± 0.02 1.60 ± 0.06 13.20 ± 1.13 Fr. B‑1–10 1.72 ± 0.10 1.20 ± 0.09 1.10 ± 0.08 2.04 ± 0.06 25.50 ± 0.84 Fr. B‑1–11 11.04 ± 0.54 8.16 ± 0.46 8.27 ± 0.38 18.24 ± 0.61 26.46 ± 2.01 Fr. B‑1–12 55.00 ± 1.83 36.70 ± 0.83 37.84 ± 2.03 50.21 ± 2.03 > 100 Cisplatin 2.28 ± 0.57 3.11 ± 0.22 2.49 ± 0.07 4.13 ± 0.86 8.86 ± 1.32 Paclitaxel < 0.007 < 0.007 < 0.007 0.96 ± 0.05 1.94 ± 0.11 Yang et al. Natural Products and Bioprospecting (2022) 12:11 Page 6 of 9 Fig. 3 Key 2D NMR correlations of compound 1 was located at C-25 (δ 79.4) by comparing the chemi- cal shift of C-25 in 25-OH analogs (δ is approximately 71  ppm) and 25-OAc analogs (δ is approximately 79 ppm) [9]. Thus, the planar structure of 1 was deter- mined as shown in Fig. 3. The relative configuration of 1 was deduced by ROESY correlations (Fig.  3). Correlations of H-1β/H -19, H-7β/ H -19, H-8/H -18, H-8/H -19, H-12β/H -18, H-15β/H - 3 3 3 3 3 18, and H-16/H -18 indicated that these protons should be β-oriented, while correlations of H-10/H -30, H-12α/ H -30, H-17/H -30, H-15α/16-OH, and H-17/16-OH 3 3 showed that these protons should be α-oriented. The configuration of C-20 could not be determined by the ROESY spectrum. Accordingly, the ECD spectra of (8 S,9R,10R,13R,14S,16R,17R,20R)-1 and (8S,9R,10R,13R ,14S,16R,17R,20S)-1 were calculated (Fig.  4). The calcu - lated ECD spectrum of (8S,9R,10R,13R,14S,16R,17R,20 R)-1 was similar to the experimental ECD spectrum of Fig. 4 Experimental and calculated ECD spectra for compound 1 1. Thus, the absolute configuration of compound 1 was elucidated to be 8S,9R,10R,13R,14S,16R,17R,20R, named aquilarolide A. Table 5 Cytotoxicity of compounds isolated from the active fractions (Frs. B‑1–6 and B‑1–7) against the A549, NCI‑H520, SPC‑A‑1, A549/Taxol, and BEAS‑2B cell lines Compounds IC (μM) A549 NCI‑H520 SPC‑ A‑1 A549/Taxol BEAS‑2B 1 0.35 ± 0.06 0.16 ± 0.01 0.56 ± 0.02 0.20 ± 0.01 17.93 ± 0.30 2 > 40 > 40 > 40 > 40 > 40 3 0.02 ± 0.00 0.001 ± 0.000 0.005 ± 0.000 0.002 ± 0.000 3.46 ± 0.13 4 0.03 ± 0.00 0.002 ± 0.000 0.016 ± 0.000 0.007 ± 0.001 14.42 ± 1.36 5 > 40 > 40 > 40 > 40 > 40 6 1.52 ± 0.06 1.84 ± 0.16 1.13 ± 0.03 0.91 ± 0.01 > 40 Cisplation 13.54 ± 0.64 11.95 ± 0.60 21.42 ± 0.35 14.95 ± 0.93 34.90 ± 1.16 Paclitaxel < 0.008 < 0.008 < 0.008 1.80 ± 0.13 > 5 Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 7 of 9 1 13 2.3 Cytotoxic results of isolates 1–6 Table 6 H and C NMR data of compound 1 in CDCl (δ in ppm, J in Hz) Isolates 1–6 were evaluated for their cytotoxici- ties against SPC-A-1, NCI-H520, A549, A549/Taxol, No δ (500 MHz) δ (126 MHz) H C and BEAS-2B cell lines (Table  5). Aquilarolide A (1), 1α 2.50, 1H, dd (16.2, 3.8) 30.3 cucurbitacin E (3), cucurbitacin B (4), and 7-hydroxy- 1β 2.16, 1H, dd (16.2, 13.9) 6-methoxy-2-[2-(4-methoxyphenyl)ethyl]-4H-1-benzo- 2 172.0 pyran-4-one (6) displayed observable cytotoxicity against 4 83.9 four tested cancer cell lines with IC values ranging 5 136.7 from 0.001 to 1.84 μM and against the normal BEAS-2B 6 5.75, 1H, br s 120.6 cell line with I C values ranging from 3.46 to > 40  μM. 7α 2.05, 1H, m 23.7 All four active compounds, with activity strengths 7β 2.40, 1H, m of 3 (IC = 0.002  μM) > 4 (IC = 0.007  μg/mL) > 1 50 50 8 2.02, 1H, m 42.1 (IC = 0.20  μM) > 6 (IC = 0.91  μM), had better inhibi- 50 50 9 47.7 tory activities against A549/Taxol cells than paclitaxel 10 2.76, 1H, m 33.2 (IC = 1.80 μM) (Table 5). 11 211.7 These active compounds belong to cucurbitane-type 12α 3.11, d (14.9) 48.7 triterpenoids (1, 3, and 4) and a 2-(2-phenylethyl)chr- 12β 2.68, d (14.9) omone (6). This result agrees with those reported in the 13 50.4 literature that cucurbitane-type triterpenoids were found 14 48.0 to be the main constituents contributing to the cytotoxic 15α 1.46, 1H, m 45.5 activities in A. sinensis fruits and peels [14, 15]. 15β 1.88, 1H, dd (13.2, 9.8) Both cucurbotacin E (3) and 23,24-dihydrocucurbita- 16 4.36, 1H, m 71.3 cin E (2) have a four-ringed core structure in the cucur- 17 2.45, 1H, br d (7.1) 58.2 bitane skeleton, except for the side chain with an olefinic 18 0.98, 3H, s 19.9 bond at C-23 in compound 3. Cucurbitacin E showed 19 1.05, 3H, s 18.5 significant cytotoxic activities against human lung can - 20 78.1 cer SPC-A-1, NCI-H520, A549, and A549/Taxol cell lines 21 1.42, 3H, s 23.9 with IC values less than or equal to 0.02 μM. However, 22 202.4 23,24-dihydrocucurbitacin E (2) was inactive. This indi - 23 6.44, 1H, d (15.7) 120.2 cated that the side chain with the olefinic bond at C-23 24 7.05, 1H, d (15.7) 152.1 seems to be key to the cytotoxic activity of this type of 25 79.4 compound. 26 1.54, 3H, s 26.5 The inhibitory activities of cucurbitacins E (3) and B 27 1.57, 3H, overlapped 25.9 (4) were close to each other, with IC values less than 28 1.53, 3H, s 30.6 or equal to 0.03  μM. However, the inhibitory activity 29 1.51, 3H, s 31.5 of 1 was significantly weaker than that of compounds 3 30 1.33, 3H, s 18.4 and 4. The difference between 1 and 3 and 4 is ring A. 16‑ OH 1.75, 1H, d (6.7) This indicates that the structure of ring A is also key to 20‑ OH 4.28, 1H, s the cytotoxic activity of this type of compound. The cyto - 25‑ OAc 170.3 toxic potency of cucurbitacins in A549 cells was related 2.02, 3H, s 22.0 to multivariate factors, among which the electrophilicity of molecules played a pivotal role, according to the mul- tivariate structure–activity relationship (SAR) and quan- titative structure–activity relationship modeling (QSAQ) analyses of cucurbitacin derivatives [16]. The known compounds were identified as 23,24-dihy - drocucurbitacin E (2) [10], cucurbitacin E (3) [11], cucurbitacin B (4) [10], (−)-(2S)-5,4ʹ-dihydroxy-7- 3 Experimental section methoxyflavanone (5) [12], and 7-hydroxy-6-methoxy- 3.1 General experimental procedures 2-[2-(4-methoxyphenyl)ethyl]-4H-1-benzopyran-4-one The reagents and instrumentation utilized for extraction, (6) [13] by comparison of the obtained spectroscopic isolation, and structure characterization throughout this data with those published in the literature. study are described in Additional file 1. Yang et al. Natural Products and Bioprospecting (2022) 12:11 Page 8 of 9 3.2 C ollection of plant samples 60% MeOH-eluted portion (Fr. B-1–7) was purified by The flowers of Aquilaria sinensis were collected from column chromatography (silica gel; petroleum ether/ Menghai County, Yunnan Province, China, in 2019. A EtOAc, 5:1 → 0:1, v/v) to yield six further fractions voucher specimen (No. KIB001-003) was identified by (B-1-7-1–B-1-7-6). Fr. B-1-7-5 and Fr. B-1–7-6 were Ms. Jun Yang at Kunming Institute of Botany, Chinese recrystallized from MeOH to yield 3 (18.5 mg). Academy of Sciences. Aquilarolide A (1). White amorphous powder; [α] − 7.1 (c 0.10, MeOH); ECD (c 0.056, MeOH) λ (Δε) 334 (− 0.22), 298 (+ 2.68), 219 (− 1.67), 199 3.3 P reparation of extractions and fractions and isolation max (+ 5.67) nm; UV (MeOH) λ 282 (2.70), 229 (3.84) nm; of compounds max 1 13 H and C NMR data, see Table  6; ESI–MS m/z 567 Air-dried, powdered flowers (50.0  g) of A. sinensis were + + [M + Na] ; HRESIMS m/z 567.2937 [M + Na] (calcd. for extracted under ultrasound with H O (500  mL × 3) C H NaO , 567.2934) (Additional file 1). at 60 ℃ for 30  min. The remaining residue was fur - 31 44 8 ther extracted with 90% EtOH (500  mL × 3) at 60 ℃ for 30  min and the solvent was removed to yield crude 3.4 MTS assay for cytotoxicity extract PXS65 (2.4 g). The cytotoxicity activities were evaluated by MTS assay Air-dried, powdered flowers (1.2  kg) of A. sinensis as previously described [17]. were extracted under ultrasound with 90% EtOH (2 L × 4) at 60 ℃ for 30 min and the solvent was removed to yield crude extract PXS66 (174.2  g). PXS66 was 3.5 Computational methods suspended in water (500  mL) and then partitioned The absolute configuration of the new compound was in turn with petroleum ether (500  mL × 4), EtOAc determined by time-dependent density functional theory (500  mL × 4), and n-BuOH (500  mL × 4) to yield (TDDFT) calculations of ECD spectra according to our three fractions, PXS66-1 (21.1  g), PXS66-2 (23.0  g), previously published paper [18]. and PXS66-3 (54.0  g), respectively. The solvent in the remaining water phase was removed to yield PXS66-4 (54.9 g).4 Conclusion PXS66-2 (23.0  g) was subjected to column chroma In this study, bioassay-guided fractionation and purifica - tion were used to isolate the cytotoxic compounds of the tography (silica gel; CH Cl /MeOH, 1:0 → 0:1, v/v) 2 2 extract from A. sinensis flowers. First, the crude extract to yield six further fractions (B-1–B-6). Fr. B-1 was showed significant inhibitory activities against 16 cancer separated on an RP C silica gel column eluted with cell lines with the most significant activities against the MeOH/H O (5% → 100%) to yield twelve further frac- lung cancer SPC-A-1, NCI-H520, and A549 cell lines. tions (B-1-1–B-1-12). The 50% MeOH-eluted portion Second, all fractions, subfractions, and pure compounds (Fr. B-1–6) was purified by column chromatography were screened for their cytotoxic activity against lung (silica gel; petroleum ether/EtOAc, 5:1 → 0:1, v/v) to cancer SPC-A-1, NCI-H520, A549, and A549/Taxol cell yield six further fractions (B-1-6-1–B-1-6-6). Fr. B-1- lines and normal human bronchial epithelial BEAS-2B 6-1 was recrystallized from MeOH to yield 5 (92.2 mg). Fr. B-1-6-3 was purified by Sephadex LH-20 column cells. From the active fraction, six compounds, includ- chromatography (MeOH) and recrystallized from ing a new cucurbitane-type triterpenoid, aquilarolide A MeOH to yield 2 (11.0  mg). Fr. B-1-6-4 was recrystal (1), five known compounds, namely, 23,24-dihydrocu - curbitacin E (2), cucurbitacin E (3), cucurbitacin B (4), lized from MeOH to yield 3 (8.9  mg), and the remain- (−)-(2S)-5,4ʹ-dihydroxy-7-methoxyflavanone (5), and ing mother liquor was subjected to Sephadex LH-20 7-hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]- column chromatography (MeOH) to yield four fur- 4H-1-benzopyran-4-one (6), were identified. Compounds ther fractions (B-1-6-4-1–B-1-6-4-4). Fr. B-1-6-4-1 1, 3, 4, and 6 showed significant cytotoxicity activities (33.5  mg) was purified by semipreparative high-per - against these four human lung cancer cell lines. All four formance liquid chromatography (HPLC) (Welch active compounds, with activity strengths of 3 > 4 > 1 > 6, Ultimate AQ-C , 7.8 × 250  mm, MeOH/H O, 20:70, 18 2 had better inhibitory activities against A549/Taxol cells v = 2  mL/min) to yield 1 (3.1  mg, t = 24.543  min) and than paclitaxel. Further studies are needed to evaluate 4 (16.6  mg, t = 28.464  min). Fr. B-1–6-4–2 (72.6  mg) in vivo antitumor activities and clarify the mechanisms of was purified by semipreparative HPLC (Welch Ulti - these active compounds. mate AQ-C , 7.8 × 250  mm, MeOH/H O, 15:85, 18 2 v = 2 mL/min) to yield 6 (2.0 mg, t = 27.467  min). The R Y ang et al. Natural Products and Bioprospecting (2022) 12:11 Page 9 of 9 11. Maatooq G, El‑Sharkawy S, Afifi MS, Rosazza JPN. Microbial transformation Supplementary Information of cucurbitacin E 2‑O‑β‑D ‑ glucopyranoside. J Nat Prod. 1995;58:165–71. The online version contains supplementary material available at https:// doi. 12. Valdés E, González C, Díaz K, Vásquez‑Martínez Y, Mascayano C, Torrent C, org/ 10. 1007/ s13659‑ 022‑ 00334‑3. Cabezas F, Mejias S, Montoya M, Martín C‑S, Muñoz MA, Joseph‑Nathan P, Osorio M, Taborga L. Biological properties and absolute configura‑ tion of flavanones from Calceolaria thyrsiflora Graham. Front Pharmacol. Additional file 1. General experimental procedures, computational meth‑ 2020;11:1125. ods for the ECD of compound 1, and NMR, HRESIMS, and ECD spectra of 13. Wu B, Kwon SW, Hwang GS, Park JH. Eight new 2‑(2‑phenylethyl)chr ‑ compound 1. omone (= 2‑(2‑phenylethyl)‑4H‑1‑benzopyran‑4‑ one) derivatives from Aquilaria malaccensis agarwood. Helv Chim Acta. 2012;95:1657–65. 14. Mei W‑L, Lin F, Zuo W ‑ J, Wang H, Dai H‑F. Cucurbitacins from fruits of Acknowledgements Aquilaria sinensis. Chin J Nat Med. 2012;10:234–7. This study was supported by Beijing Sino‑Science Aquilaria Technology Co., 15. Zhang X, Tao M‑H, Chen Y ‑ C, Gao X‑ X, Tan Y‑Z, Zhang W ‑M. Five cucurbi‑ Ltd., Beijing, China (Grant No. KET202101). tacins from Aquilaria sinensis peels and their cytotoxic activities. Nat Prod Res Dev. 2014;26:354–7. Author contributions 16. Silva IT, Carvalho A, Lang KL, Dudek SE, Masemann D, Duran FJ, Caro MSB, All authors read and approved the final manuscript. Rapp UR, Wixler V, Schenkel EP, Simões CMO, Ludwig S. In vitro and in vivo antitumor activity of a novel semisynthetic derivative of cucurbitacin B. Funding PLoS ONE. 2015;10:e0117794. Funding was provided by Beijing Sino‑Science Aquilaria Technology Co., Ltd., 17. Yang J, Su Y, Luo J‑F, Gu W, Niu H‑M, Li Y, Wang Y ‑H, Long C‑L. New amide Beijing, China (Grant No. KET202101). alkaloids from Piper longum fruits. Nat Prod Bioprospect. 2013;3:277–81. 18. Wei S‑ Y, Hu D‑B, Xia M ‑ Y, Luo J‑F, Yan H, Yang J‑H, Wang Y ‑S, Wang Declarations Y‑H. Sesquiterpenoids and 2‑(2‑phenylethyl)chromone derivatives from the resinous heartwood of Aquilaria sinensis. Nat Prod Bioprosp. Competing interests 2021;11:545–55. The authors declare that there are no conflicts of interest associated with this work. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Author details lished maps and institutional affiliations. Key Laboratory of Economic Plants and Biotechnology and Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China. School of Chemical Biology and Environment, Yuxi Normal University, Yuxi 653100, People’s Republic of China. College of Science, Yunnan Agricultural University, Kunming 650201, People’s Republic of China. Received: 9 February 2022 Accepted: 17 March 2022 References 1. El‑Hussein A, Manoto SL, Ombinda‑Lemboumba S, Alrowaili ZA, Mthunzi‑ Kufa P. A review of chemotherapy and photodynamic therapy for lung cancer treatment. Anti‑ Cancer Agents Med Chem. 2021;21:149–61. 2. Du J, Li J, Gao M, Guan Q, Liu T, Wu Y, Li Z, Zuo D, Zhang W, Wu Y. MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/Taxol cells and inhibits epithelial‑mesenchymal transition in A549/Taxol cells. Chem Biol Interact. 2020;323:109074. 3. Wang Y, Gilbert MG, Mathew B, Brickell CD, Nevling LI. 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Natural Products and BioprospectingSpringer Journals

Published: Dec 1, 2022

Keywords: Thymelaeaceae; Aquilaria sinensis; Paclitaxel-resistant lung cancer cells; Cucurbitane-type triterpenoids; 2-(2-Phenylethyl)chromones

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