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

Bioassay-guided fractionation of the EtOH extract from the flowers of Aquilaria sinensis (Lour.) Spreng. (Thymelaeaceae) led to the isolation of a new cucurbitane-type triterpenoid, aquilarolide A (1), along with five known compounds (2–6). The structure of 1 was elucidated by extensive 1D and 2D nuclear magnetic resonance (NMR) experiments and mass spectrometry (MS) data and theoretical calculations of its electronic circular dichroism (ECD) spectra. Aquilarolide A, cucurbitacin E (3), cucurbitacin B (4), and 7-hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]-4H-1-benzopyran-4-one (6) showed significant cytotoxicity against human lung adenocarcinoma SPC-A-1, human lung squamous cell carcinoma NCI-H520, human lung adenocarcinoma A549, and paclitaxel-resistant A549 (A549/Taxol) cell lines. All four active compounds, with IC50 values ranging from 0.002 to 0.91 μM, had better inhibitory activities against A549/Taxol cells than paclitaxel (IC50 = 1.80 μM). Among them, cucurbitacin E (IC50 = 0.002 μM) is the most active. Further studies are needed to evaluate their in vivo antitumor activities and to clarify their mechanisms. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-022-00334-3.


Introduction
Cancer is a major public health problem and one of the leading causes of mortality and morbidity worldwide [1]. Chemical drugs, such as paclitaxel, are useful to treat cancers. However, resistance to paclitaxel reduces the efficacy of chemotherapy and limits its clinical application [2]. Therefore, it is necessary to develop novel and effective therapeutic medicines or adjuvants for cancer. Natural plant resources are a rich source of anticancer agents. The discovery of new effective cancer drugs and understanding their underlying mechanism is one of the most studied topics among biologists and chemists.
Aquilaria sinensis (Lour.) Spreng. (Thymelaeaceae) is widely distributed in Hainan, Fujian, Yunnan, Guangdong, and Taiwan in China [3]. It has a particular economic interest because it is the principal source of agarwood (chen-xiang in Chinese), namely, the resinous heartwood of the plant. As a traditional Chinese medicine, agarwood has been widely investigated [4]. However, there have been only a few studies on the chemical constituents and bioactivities of A. sinensis flowers. The volatile constituents from flowers of A. sinensis have been analyzed by GC-MS [5]. Flavonoids and their glycosides are found in its flowers [6]. Benzophenone glycosides have been isolated from the flower buds of A. sinensis and two compounds, aquilasides B and C, displayed moderate cytotoxicity against SK-MEL cells with IC 50 values of 17.0 and 12.0 μM, respectively [7].

Structural elucidation of isolates 1-6
In total, six secondary metabolites (Fig. 2), including a new metabolite (1), were isolated from the cytotoxically active fractions of A. sinensis flowers as a result of chromatographic separations. The chemical structure of the new compound was elucidated by 1D and 2D nuclear magnetic resonance (NMR) experiments as well as high-resolution electron ionization mass spectrometry (HRESIMS) and electronic circular dichroism (ECD) calculations.
Compound 1 was isolated as a white amorphous powder and exhibited a quasi-molecular ion peak at m/z 567. These data showed a similar signal pattern with those of a lactone-type norcucurbitacin, neocucurbitacin E, except for the double bond at Δ 23 in 1 [8].
Based on the 1 H-1 H COSY correlations (Fig. 3 16) was correlated to the carbon atoms at δ C = 172.0 (C-2), 136.7 (C-5), and 47.7 (C-9) ppm, as well as H 3 -28 and H 3 -29 to C-5 (Fig. 3) and H-6 to C-4. According to the HMBC correlations from H 3 -19 to C-8, C-10, and C-11, from H 3 -30 to C-8, C-13, and C-15, and from H 3 -18 to C-12, C-14, and C-17, rings B-D were deduced. Based on the HMBC correlations from H-16 to C-20, from H 3 -21 and 20-OH to C-17 and C-22, from H-23 to C-25, from H-24 to C-22, and from H 3 -26 and H 3 -27 to C-24, the side chain was confirmed and was located at C-17 of ring D. The acetyl group was located at C-25 (δ C 79.4) by comparing the chemical shift of C-25 in 25-OH analogs (δ C is approximately 71 ppm) and 25-OAc analogs (δ C is approximately 79 ppm) [9]. Thus, the planar structure of 1 was determined as shown in Fig. 3. The relative configuration of 1 was deduced by ROESY correlations (Fig. 3)
These active compounds belong to cucurbitane-type triterpenoids (1, 3, and 4) and a 2-(2-phenylethyl)chromone (6). This result agrees with those reported in the literature that cucurbitane-type triterpenoids were found to be the main constituents contributing to the cytotoxic activities in A. sinensis fruits and peels [14,15].
Both cucurbotacin E (3) and 23,24-dihydrocucurbitacin E (2) have a four-ringed core structure in the cucurbitane skeleton, except for the side chain with an olefinic bond at C-23 in compound 3. Cucurbitacin E showed significant cytotoxic activities against human lung cancer SPC-A-1, NCI-H520, A549, and A549/Taxol cell lines with IC 50 values less than or equal to 0.02 μM. However, 23,24-dihydrocucurbitacin E (2) was inactive. This indicated that the side chain with the olefinic bond at C-23 seems to be key to the cytotoxic activity of this type of compound.
The inhibitory activities of cucurbitacins E (3) and B (4) were close to each other, with IC 50 values less than or equal to 0.03 μM. However, the inhibitory activity of 1 was significantly weaker than that of compounds 3 and 4. The difference between 1 and 3 and 4 is ring A. This indicates that the structure of ring A is also key to the cytotoxic activity of this type of compound. The cytotoxic potency of cucurbitacins in A549 cells was related to multivariate factors, among which the electrophilicity of molecules played a pivotal role, according to the multivariate structure-activity relationship (SAR) and quantitative structure-activity relationship modeling (QSAQ) analyses of cucurbitacin derivatives [16].

General experimental procedures
The reagents and instrumentation utilized for extraction, isolation, and structure characterization throughout this study are described in Additional file 1.

Preparation of extractions and fractions and isolation of compounds
Air-dried, powdered flowers (50.0 g) of A. sinensis were extracted under ultrasound with H 2 O (500 mL × 3) at 60 ℃ for 30 min. The remaining residue was further extracted with 90% EtOH (500 mL × 3) at 60 ℃ for 30 min and the solvent was removed to yield crude extract PXS65 (2.4 g).

MTS assay for cytotoxicity
The cytotoxicity activities were evaluated by MTS assay as previously described [17].

Computational methods
The absolute configuration of the new compound was determined by time-dependent density functional theory (TDDFT) calculations of ECD spectra according to our previously published paper [18].